Molecular and morphological systematics of Dolabrifera Gray, 1847 (Mollusca: Gastropoda: Heterobranchia: Aplysiomorpha)

Molecular and morphological systematics of Dolabrifera Gray, 1847 (Mollusca: Gastropoda:... Abstract Molecular and morphological data from newly collected specimens and a review of the literature and type material indicate that the widespread tropical sea hare Dolabrifera dolabrifera is a species complex of five genetically distinct taxa. The name Dolabrifera dolabrifera is retained for a widespread species in the Indo-Pacific tropics. Dolabrifera nicaraguana is endemic to the eastern Pacific. Dolabrifera ascifera, D. virens and a new species described herein are restricted to the tropical Atlantic, with partially overlapping ranges in the Caribbean region and St. Helena. The temperate Pacific species Dolabrifera brazieri is also distinct and endemic to temperate southeastern Australia and New Zealand. These species of Dolabrifera constitute highly divergent lineages and most contain unique internal anatomical characteristics, particularly in the male reproductive morphology and shell shape, making them relatively easy to identify upon dissection. However, externally all these species are extremely variable in colour pattern and morphology and are virtually indistinguishable. This is particularly problematic for identification in the Atlantic Ocean where three species co-occur in the Caribbean region. new species, phylogenetics, species delimitation, taxonomy INTRODUCTION Dolabrifera dolabrifera (Rang, 1828) has been considered a pantropical species of sea hare (Aplysiidae), typically found under rocks in the upper intertidal zone (Kay, 1964; Bebbington, 1974) and on mudflat pools (Bebbington, 1977). Numerous species names have been introduced for specimens from different geographical locations over the years, but nearly all of them have been synonymized with D. dolabrifera (Engel & Hummelinck, 1936; Eales, 1944). Dolabrifera dolabrifera displays a considerable range of variation in colour, external morphology and anatomy (Marcus, 1972), which suggests it could constitute a species complex. Recent molecular studies have shown that other tropical and subtropical widespread heterobranch sea slugs resulted to be species complexes with marked geographical structure (Ornelas-Gatdula et al., 2012; Alexander & Valdés, 2013; Krug et al., 2013; Carmona et al., 2014a, b; Goodheart et al., 2015), and D. dolabrifera could be a similar case. Adding to the taxonomy complexity in Dolabrifera, the closely related temperate Australian species, Dolabrifera brazieri G. B. Sowerby II, 1870, is considered distinct by some authors (Klussman-Kolb, 2004; Burn, 2006), but the relationships between the two species is unclear. Specimens of Dolabrifera contain secondary metabolites with potential medical applications. For example, the propionate-derived compound Dolabriferol with antibiotic properties (Kigoshi & Kita, 2015) was originally characterized from specimens collected in the Caribbean region (Ciavatta et al., 1996). Subsequent studies on other Caribbean animals revealed the presence of chemically related, but distinct compounds (Jiménez-Romero, González & Rodríguez, 2012). Further research on specimens from the Eastern Pacific resulted in the isolation of 5α,8α-epidioxycholest-6-en-3β-ol, a compound with antileishmanial properties (Clark et al., 2013). This chemical diversity may be suggestive of a more complex taxonomy than previously recognized. The recent discovery of cryptic species complexes in other biomedically important groups of sea slugs (Krug et al., 2013; Lindsay & Valdés, 2016) highlights the need for modern approaches to investigate the biodiversity of such groups. In this article, we examine the systematics of Dolabrifera dolabrifera and D. brazieri using a combination of molecular and anatomical data to determine the taxonomic status of these two nominal species and unveil possible cryptic diversity in this group. MATERIAL AND METHODS Source of specimens Specimens were collected from localities covering mostly the known range of Dolabrifera (Fig. 1), mainly during low tides or snorkelling, under rocks. Specimens were photographed alive, preserved in 90–99% ethanol and deposited at the California State Polytechnic University Invertebrate Collection (CPIC), the University Museum of Bergen (ZMBN), the Zoologische Staatssammlung München (ZSM) and the Colección Nacional de Moluscos, Instituto de Biología, Universidad Nacional Autónoma de México (CNMO). Additional specimens, photographs and/or tissue samples were obtained from museum collections, including the California Academy of Sciences, Invertebrate Zoology (CASIZ), the Natural History Museum of Los Angeles County (LACM), the Natural History Museum, London (NHMUK), the Australian Museum (AM), the Academy of Natural Sciences of Drexel University, Philadelphia (ANSP) and the Yale Peabody Museum of Natural History (YPM). Figure 1. View largeDownload slide Distribution of Dolabrifera dolabrifera. A, map indicating the approximate location of the type localities of all nominal species synonymized with D. dolabrifera with their geographical region colour coded. B, localities of specimens examined morphologically and/or sequenced in this study. An asterisk denotes localities for which only GenBank sequence data were available. Localities in close proximity within countries are merged into single points. Localities are colour coded by biogeographical region. Figure 1. View largeDownload slide Distribution of Dolabrifera dolabrifera. A, map indicating the approximate location of the type localities of all nominal species synonymized with D. dolabrifera with their geographical region colour coded. B, localities of specimens examined morphologically and/or sequenced in this study. An asterisk denotes localities for which only GenBank sequence data were available. Localities in close proximity within countries are merged into single points. Localities are colour coded by biogeographical region. Morphology At least three specimens (if available) of each species identified with molecular data were examined morphologically. Based on the studies by Eales (1944) and Bebbington (1974, 1977), diagnostic traits for species in Dolabrifera include the radula, jaws, shell, gizzard plates and male reproductive anatomy. These structures/organs were used here as the main anatomical traits for species identification and characterization. The anterior portion of the digestive system was removed via a dorsal incision on the anterior end of the body. The buccal mass, oesophagus and gizzard were observed under a Nikon SMZ100 dissecting microscope and drawn with the aid of a camera lucida. Subsequently, these organs were placed in a sodium hydroxide 10% solution until the surrounding tissues were softened. The radulae, jaws and post-radular armature were removed from the partially dissolved tissue, rinsed in distilled water, mounted on a stub and sputter coated for examination under a Jeol JSM-6010 variable pressure scanning electron microscope (SEM). The gizzard plates were also removed from the partially dissolved tissue, rinsed in distilled water, transferred to ethanol 99% for 3 min and soaked in 2 mL of hexamethyldisilazane (HMDS) until all the liquid evaporated; then, they were mounted on a stub and coated for SEM examination. The male reproductive organs were dissected through the same incision used to extract the digestive organs, once the buccal mass was removed. The male reproductive organs were submerged in ethanol 70% and photographed under a Leica EZ4D microscope with a built-in digital camera; subsequently, the penial sheath of each specimen was carefully opened to expose the penial papilla, transferred to ethanol 99% for 3 min, soaked in 2 mL of HMDS until all the liquid evaporated and mounted for sputter coating and SEM examination. The shells along with the surrounding tissue were dissected from the posterior end of the animal and completely submerged in sodium hydroxide 10% until the shells were easily detachable from the tissue. The shells were rinsed in distilled water, photographed dorsally and ventrally under a Leica EZ4D microscope, mounted on a stub and sputter coated for examination under the SEM. DNA extraction, amplification and sequencing A total of 63 specimens of Dolabrifera were sequenced for this study (Table 1). Specimens were collected from localities spanning most of the range of all nominal species of Dolabrifera. GenBank sequences of two additional specimens of Dolabrifera and two outgroup taxa were also included in the analyses (Table 1). DNA extractions were performed using a hot Chelex extraction protocol with minor modifications and c. 1–3 mg of foot tissue. Tissue samples were placed into 1.7-mL tubes containing 1.0-mL TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 7.8) and allowed to incubate at room temperature on a rotator overnight to rehydrate the tissue and allow cells to begin dissociating. Samples were then vortexed and centrifuged for 3 min at 23897 × g. Then, 975 µL of the original 1 mL of TE buffer in each tube was carefully removed without disturbing the pellet of tissue. The Chelex solution was then added (175 µL), and samples were heated in a 56 °C water bath for 20 min, then immediately placed in a 100 °C heating block for 8 min. The supernatant was the final product used for polymerase chain reaction (PCR). Table 1. Specimens sequenced for this study, including museum voucher number, isolate number, collection locality and date and GenBank accession numbers Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  For countries with coastlines in two ocean basins, the ocean in which the specimens were collected is indicated. Museum abbreviations: AM, Australian Museum; CASIZ, California Academy of Sciences, Invertebrate Zoology; CNMO, Colección Nacional de Moluscos, Universidad Nacional Autónoma de México; CPIC, California State Polytechnic University Invertebrate Collection; LACM, Natural History Museum of Los Angeles County Malacology Collection; MZUCT, Museo de Zoología, Universidad de Costa Rica; ZSM, Zoologische Staatssammlung München; ZMBN, University Museum of Bergen Systematic Invertebrate Collection. Other abbreviations: ‘*’, outgroup taxa; ‘–’, missing data; ‘#’, tissue sample only; Pac, Pacific Ocean; Car, Caribbean Sea. View Large Table 1. Specimens sequenced for this study, including museum voucher number, isolate number, collection locality and date and GenBank accession numbers Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  For countries with coastlines in two ocean basins, the ocean in which the specimens were collected is indicated. Museum abbreviations: AM, Australian Museum; CASIZ, California Academy of Sciences, Invertebrate Zoology; CNMO, Colección Nacional de Moluscos, Universidad Nacional Autónoma de México; CPIC, California State Polytechnic University Invertebrate Collection; LACM, Natural History Museum of Los Angeles County Malacology Collection; MZUCT, Museo de Zoología, Universidad de Costa Rica; ZSM, Zoologische Staatssammlung München; ZMBN, University Museum of Bergen Systematic Invertebrate Collection. Other abbreviations: ‘*’, outgroup taxa; ‘–’, missing data; ‘#’, tissue sample only; Pac, Pacific Ocean; Car, Caribbean Sea. View Large PCR was used to amplify portions of two mitochondrial genes, cytochrome c oxidase 1 (COI) and16S ribosomal RNA (16S), as well as the nuclear gene histone H3 (H3). The following universal primers were used to amplify the regions of interest for all three genes: COI (LCO1490 5′-GGTCAACAAATCATAAAGATATTGG-3′, HCO2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ developed by Folmer et al., 1994), 16S rRNA (16S ar-L 5′-CGCCTGTTTATCAAAAACAT-3′, 16S br-H 5′-CCGGTCTGAACTCAGATCACGT-3′ developed by Palumbi, 1996) and H3 (H3 AF 5′-ATGGCTCGTACCAAGCAGACGGC-3′, H3 AR 5′-ATATCCTTGGGCATGATGGTGAC-3′ developed by Colgan, Ponder & Eggler, 2000). Agarose gel electrophoresis with ethidium bromide was used to detect the presence of DNA. Sequencing was outsourced to Source Bioscience Inc. (Santa Fe Springs, CA, USA). Sequences were assembled and edited using Geneious Pro R8 (Kearse et al., 2012). Phylogenetic analyses The species Akera bullata Müller, 1776 and Dolabella auricularia (Lightfoot, 1786) were selected as outgroups based on Medina & Walsh’s (2000) molecular phylogeny of the Aplysiomorpha (=Anaspidea), which placed Akera at the base of this group and Dolabella in the same clade as Dolabrifera, sister to Aplysia. Phylogenetic analyses were conducted for all genes concatenated and for each gene individually. The best-fit models of evolution (GTR + G for 16S, GTR + I for COI codon positions 1 + 2, HKY + I for COI codon position 3, HKY for H3 codon positions 1 + 2, HKY + G for H3 codon position 3 and GTR + I for the entire concatenated data set) were determined using the Akaike information criterion (Akaike, 1974) implemented in JModelTest 2.1.10 (Darriba et al., 2012). Bayesian analyses were conducted using MrBayes 3.2.6 (Ronquist et al., 2012), partitioned by gene and codon position (unlinked). The Markov chain Monte Carlo analysis was run with two runs of six chains for 50 million generations, with sampling every 1000 generations. Convergence was assessed with Tracer 1.6 (Rambaut & Drummond, 2013). A 25% burn-in was applied before constructing the majority-rule consensus tree. Maximum likelihood analyses were conducted for the entire concatenated alignment with raxmlGUI 1.0 (Silvestro & Michalak, 2012) using the bootstrap + consensus option (10000 replicates) and the GTR + I model. Species delimitation analysis To provide evidence supporting species status for clades recovered in the phylogenetic analyses, a species delimitation analysis based on the barcode gap was performed with ABGD (Puillandre et al., 2012) using the COI sequence alignment. The analysis was run twice using Kimura 2-parameter and Tamura-Nei distance matrices generated in MEGA 7.0.16 (Kumar, Stecher & Tamura, 2016). The matrices were uploaded into the online ABGD web tool (http://wwwabi.snv. jussieu.fr/public/abgd/abgdweb.html) and analysed. The default relative gap width (x) of 1.5 and a range of prior values for maximum divergence of intraspecific diversity (P) from 0.001 to 0.1 were implemented. RESULTS Bayesian and maximum likelihood analyses of the three concatenated genes (16S + COI + H3) produced trees with the same overall topology (Fig. 2). In both trees, Dolabrifera is monophyletic and well supported (posterior probability [PP] = 1; Bootstrap support [BS] = 97). Although the base of the Dolabrifera clade was recovered as an unresolved polytomy (Fig. 2), this group contains six distinct, well-supported clades. The geographical composition of clades and support values are as follows: (1) specimens from the Eastern Pacific Ocean (PP = 1; BS = 100); (2) specimens from the Eastern Atlantic Ocean, the South Atlantic and the Gulf of Mexico (PP = 0.99; BS = 91); (3) specimens from the tropical Indo-Pacific – from Madagascar to the Hawaiian Islands (PP = 0.99; BS = 92); (4) specimens from the Gulf of Mexico and Caribbean – Mexico, Jamaica and Venezuela (PP = 0.9; BS = 97); (5) specimens from temperate New Zealand – originally identified as D. brazieri (PP = 1; BS = 100) and (6) specimens from the Caribbean – Panama and Jamaica – as well as from the South Atlantic Ocean – St. Helena (PP = 1; BS = 98). Clades 4 and 5 are sisters (PP = 1; BS = 84), but this is the only sister relationship recovered among Dolabrifera clades. Individual gene trees also generally agree with the consensus trees with some differences (Supporting Information, Fig. S1). The main differences are the lack of support for the D. dolabrifera clade in the 16S analyses and for one of the Atlantic clades in the H3 analyses. Figure 2. View largeDownload slide Bayesian consensus tree (16S + COI + H3) for 65 specimens of Dolabrifera + outgroup taxa, including posterior probabilities (above nodes) and bootstrap support values from the maximum likelihood analysis (below nodes). For each specimen, the geographical origin and isolate number are indicated (see Table 1). The general geographical region of origin for all specimens is colour coded as in Figure 1. Figure 2. View largeDownload slide Bayesian consensus tree (16S + COI + H3) for 65 specimens of Dolabrifera + outgroup taxa, including posterior probabilities (above nodes) and bootstrap support values from the maximum likelihood analysis (below nodes). For each specimen, the geographical origin and isolate number are indicated (see Table 1). The general geographical region of origin for all specimens is colour coded as in Figure 1. The species delimitation analysis confirmed that these clades constitute distinct candidate species, as they were recovered in different groups in ABGD. Examination of external and internal anatomical traits also revealed the existence of consistent differences between the candidate species recovered with molecular data, thus becoming confirmed candidate species (Vieites et al., 2009). The taxonomy, nomenclatural history (if any) and diagnostic traits for all species are described and discussed in the Systematics section. Also, a formal description is provided for a confirmed candidate species that was unnamed. SYSTEMATICS Genus dolabrifera Gray, 1847 Dolabrifera Gray, 1847: 162. Type species: Dolabrifera dolabrifera (Rang, 1828), by original designation. Diagnosis Body oval to elongate, wider posteriorly, narrowing gradually towards head. Oral tentacles, rhinophores slit, flattened, wider distally. Oral tentacles emerging anteriorly from the head, clearly differentiated. Rhinophores dorsal, typically smooth. Parapodial lobes reduced, located behind body midline, fused posteriorly, overlapping partially in middle. Dorsum typically covered with tubercles of different sizes. Shell solid, calcified, reduced, flat, elongate, subtriangular, wider posteriorly, tapering anteriorly into a conspicuous nucleus. Jaws reduced to small section of the labial cuticle with simple denticles. Radula with large, subtriangular rachidian teeth, with several denticles on each side of the cusp; lateral teeth with long, coarsely denticulate cusps. The posterior end of buccal bulb, at insertion of the oesophagus, with two elongate structures (post-radular armature), of numerous elongate denticles. Gizzard muscular, with numerous irregular plates. Male reproductive system with distinct penial canal and penial sheath, where penial papilla is typically located. Remarks Since its original introduction by Gray (1847), several nominal species have been described in or transferred to Dolabrifera, many of them based on shell material or preserved specimens. Nearly all of those species were confirmed to be members of Dolabrifera, but later synonymized with D. dolabrifera (see Engel & Hummelinck, 1936; Eales, 1944) or D. brazieri (see Hedley, 1917). However, the taxonomic assignment of two of those species is questionable. The first one, Dolabrifera holboelli Bergh, 1872, was described from a single specimen collected in ‘Mare grönland’ (=Greenland Sea). Bergh (1872: pl. 5, figs 1–24) provided illustrations of the preserved animal, shell, radula, jaw, gill and other anatomical details (some of them reproduced in Supporting Information, Fig. S2S–U). All descriptions are consistent with the characteristics of a member of Dolabrifera, and examination of the holotype (ZMUC GAS-2163) confirms the generic placement of this species in Dolabrifera. However, there are no confirmed reports of Dolabrifera from Greenland, nor from other temperate or cold-water regions in the North Atlantic; thus, the most likely explanation for this record is that the specimen was mislabelled. Based on the radular and shell morphology, D. holboelli resembles the tropical Indo-Pacific species D. dolabrifera because the lateral teeth have bifurcated apices with two equally large denticles, and the shell widens abruptly near the apical end, but none of these traits are conclusive. In the absence of molecular data or information on the geographical origin of the holotype, D. holboelli is here treated as an uncertain species. The second problematic species, Dolabrifera pelsartensis O’Donoghue, 1924, was described from ‘Pelsart Island’ (Pelsaert Island), Western Australia, based on two preserved specimens. O’Donoghue (1924: pl. 27, fig. 3, pl. 29, figs 26–28) illustrated a preserved animal (Supporting Information, Fig. S2R) and a radula, which shows mid-lateral teeth with two to three denticles and simple cusps. No illustrations of the shell or other anatomical features were provided. With the available information, it is difficult to compare D. pelsartensis with the species here described. The radular morphology is most similar to that of D. brazieri, which appears to be endemic to temperate Eastern Australia and New Zealand. Examination of material from Western Australia is necessary to clarify the status of this species. Another species, Dolabrifera triangularis Watson, 1884, has almost certainly been erroneously assigned to Dolabrifera. Watson (1884) described D. triangularis based on a shell collected from Simon’s Bay, South Africa. Although Watson (1884) described the shell in detail, provided no illustrations and the live animal is unknown. Years later, Watson (1886: 673–674, pl. 50, fig. 7; Supporting Information, Fig. S2V, W) redescribed and illustrated this shell, which is nearly triangular, short, with a large nucleus. The only remaining syntype (NHMUK 1887.2.9.2296) here examined, matches this description, but is much shorter and thicker that other shells of Dolabrifera spp. In addition, no specimens of Dolabrifera have been documented in False Bay and surrounding areas despite the comprehensive studies (Gosliner, 1987; Zsilavecz, 2007). Based on the available evidence, we cannot identify D. triangularis and therefore is here regarded as a nomen dubium. Dolabrifera dolabrifera (Rang, 1828) (Figs 3A–G, 4–6) ‘Dolabella dolabrifera’ Cuvier, 1817: 398. Type locality: ‘Isle-de-France’ (=Mauritius) (nomen nudum). ‘Dolabella dolabrifera’ de Férussac, 1822: table xxx (nomen nudum). Aplysia dolabrifera Rang, 1828: 51, pl. 4, figs 1–6. Type locality: ‘Ile Bourbon’ (=Réunion). Aplysia oahouensis Souleyet, 1852: 461–462, pl. 25, figs 10–13. Type locality: ‘Oahou’ (=O‘ahu, Hawaiian Islands). Dolabrifera cuvieri H. Adams & A. Adams, 1854: 33; 1858: pl. 59, figs 4, 4a. Unjustified emendation for Dolabella dolabrifera Cuvier, 1817. Dolabrifera olivaceaPease, 1860: 22–23. Type locality: Sandwich Islands (=Hawaiian Islands). Dolabrifera tahitensisPease, 1861: 245–246. Type locality: Tahiti. Dolabrifera maillardi Deshayes, 1863: 53–54, pl. 7, figs 20–22. Type locality: Réunion. Dolabrifera fusca Pease, 1868: 76, pl. 8, fig. 4. Type locality: Polynesia. Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868: pl. 1, fig. 1a, b. Type locality: ‘Narai’, Fiji. Dolabrifera pacifica Pease in Reeve, 1868: pl. 1, fig. 3. Type locality: Islands of the Pacific. Dolabrifera marmorea Pease in Reeve, 1868: pl. 1, fig. 5a, b. Type locality: Sandwich Islands (=Hawaiian Islands). Type material Aplysia dolabrifera Rang, 1828 not found at MNHN (Valdés & Héros, 1998). Aplysia oahouensis Souleyet, 1852, one syntype at MNHN (Valdés & Héros, 1998). Dolabrifera olivacea Pease, 1860, lectotype at NHMUK 1964376. Dolabrifera maillardi Deshayes, 1863, three syntypes at MNHN. Dolabrifera tahitensis Pease, 1861, type material untraceable, not at NHMUK. Dolabrifera fusca Pease, 1868, one syntype at ANSP 20710. Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868, two syntypes at NHMUK 1856.9.24.202. Dolabrifera marmorea Pease in Reeve, 1868, one syntype at NHMUK 20160139. Dolabrifera pacifica Pease in Reeve, 1868, two syntypes at NHMUK 20160140. Material examined Hekili Point, Maui, Hawaiian Islands, 1 m depth, 21 June 2016, one specimen 19 mm preserved length (CPIC 01654). Maliko Bay, Maui, Hawaiian Islands, 1 m depth, 22 June 2016, five specimens 5–14 mm preserved length (CPIC 01657). Mama’s Fish House Reef, Maui, Hawaiian Islands, 1 m depth, 25 June 2016, five specimens 9–12 mm preserved length (CPIC 01696); 27 June 2016, one specimen 4 mm preserved length (CPIC 01718); 28 June 2016, two specimens 2–3 mm preserved length (CPIC 01752). Sharks Cove, O‘ahu, Hawaiian Islands, 1 m depth, 5 July 2016, one specimen 10 mm preserved length (CPIC 01770). Kaloko, Kona, Hawai‘i, Hawaiian Islands, 26 July 2014, two specimens 16–19 mm preserved length, leg. S. Medrano (CPIC 01130). Onekahakaha, Hilo, Hawai‘i, Hawaiian Islands, 26 July 2014, two specimens 12–20 mm preserved length, leg. S. Medrano (CPIC 01132). Merizo, Guam, intertidal, 16 June 2009, one specimen 12 mm preserved length, leg. E. Ornelas-Gatdula (CPIC 00413). In front of Bali Sol Hotel, Nusa Dua, Bali, Indonesia (8°48.0′S, 115°14.0′E), intertidal, 22 May 1986, two specimens 35–40 mm preserved length, leg. J. McLean (LACM 1986-164.16). Cement Mixer Reef, Madang, Papua New Guinea, 4 December 2012, one specimen 14 mm preserved length, leg. J. Goodheart (CPIC 00820). Rempi, Madang, Papua New Guinea, 21 November 2012, one specimen 10 mm preserved length, leg. Papua New Guinea Biodiversity Expedition 2012 (CASIZ 191380). Kranket Island, Madang, Papua New Guinea (5°11′30.9″S, 145°49′6.7″E), 6 November 2012, one specimen 6 mm preserved length, leg. Papua New Guinea Biodiversity Expedition 2012 (CASIZ 191000). Platier Flacourt, Taolagnaro, Madagascar (25°1.7′S, 47°0.1′E), 13 May 2010, two specimens 24–25 mm preserved length, leg. Atimo Vatae Expedition (CASIZ 194443). Plage Monseigneur, Taolagnaro, Madagascar (25°2.1′S, 46°59.9′E), 15 May 2010, two specimens 18–26 preserved length, leg. Atimo Vatae Expedition (CASIZ 194428). Shib Ammar, Saudi Arabia (19°34.232′N, 40°0.519′E), 20 m depth, 3 March 2013, tissue sample, leg. A. Anker (CASIZ 192302). Description External morphology Body up to 100 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with small, rounded, scattered tubercles, each with retractile, highly ramified, papilla on apex; tubercles typically tightly arranged, giving animal bumpy (Fig. 3A, G) or rough appearance (Fig. 3C–F), in some specimens surrounded by large areas of smooth tissue (Fig. 3B). Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. Figure 3. View largeDownload slide Live animals of the Pacific Ocean species of Dolabrifera. A–G, Dolabrifera dolabrifera (Rang, 1828). A, Guam (CPIC 00413); B, Anilao, Philippines (CASIZ 158347); C, Maui, Hawaiian Islands (CPIC 01654); D, Maui, Hawaiian Islands (CPIC 01696); E, Maui, Hawaiian Islands (CPIC 01718); F, O‘ahu, Hawaiian Islands (CPIC 01770); G, Marsa Alam, Egypt (ZSM Mol 20170610). H, Dolabrifera brazieri G. B. Sowerby II, 1870 (photograph: G. Cobb). I, J, Dolabrifera nicaraguana Pilsbry, 1896. I, Pacific coast of Panama (photograph: J. Goodheart); J. Puerto Vallarta, Mexico (CPIC 00547). Figure 3. View largeDownload slide Live animals of the Pacific Ocean species of Dolabrifera. A–G, Dolabrifera dolabrifera (Rang, 1828). A, Guam (CPIC 00413); B, Anilao, Philippines (CASIZ 158347); C, Maui, Hawaiian Islands (CPIC 01654); D, Maui, Hawaiian Islands (CPIC 01696); E, Maui, Hawaiian Islands (CPIC 01718); F, O‘ahu, Hawaiian Islands (CPIC 01770); G, Marsa Alam, Egypt (ZSM Mol 20170610). H, Dolabrifera brazieri G. B. Sowerby II, 1870 (photograph: G. Cobb). I, J, Dolabrifera nicaraguana Pilsbry, 1896. I, Pacific coast of Panama (photograph: J. Goodheart); J. Puerto Vallarta, Mexico (CPIC 00547). Colour Background colour variable, pink, purple, dark brown, or red, with spots and/or irregular patches of different colours. Mantle margin with same general colour as rest of body. Ventral side typically brown with numerous white, green or brown spots. Digestive system Radular formulae: 38 × 78.1.41 (CPIC 00336), 37 × 62.1.56 (CPIC 00336), 25 × 58.1.70 (LACM 1986-164.1), 28 × 56.1.46 (CPIC 00820); radulae typically asymmetrical, with more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 4A, D); teeth wider at base, both upper and lower ends of teeth convex. Lateral teeth elongate (Fig. 4B, E), with long bases; each cusp with basal, blunt, inner denticle, apex bifurcated into two blunt denticles equal in size. Outer teeth very elongate (Fig. 4C, F), with or without a secondary denticle, apex bifurcate or simple. Jaws with simple, irregular denticles (Fig. 4J). Two elongate structures (post-radular armature) located posterior to radula, at oesophagus insertion point, with numerous spines with elongate bases, triangular cusps (Fig. 4K). Gizzard plates variable in shape, typically irregular, with some striations more visible near centre (Fig. 4G–I). Figure 4. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera dolabrifera (Rang, 1828). A–C, radular teeth of a specimen from Maui, Hawaiian Islands (CPIC 00336). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bali, Indonesia (LACM 1986-164.16). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Maui, Hawaiian Islands (CPIC 00336). I, gizzard plate of a specimen from Madang, Papua New Guinea (CPIC 00820). J, jaw elements of a specimen from Madang, Papua New Guinea (CPIC 00820). K, post-radular armature of a specimen from Maui, Hawaiian Islands (CPIC 00336). L, M, dorsal and ventral views of the shell apex of a specimen from Madang, Papua New Guinea (CPIC 00820). N, O, dorsal and ventral views of the shell apex of a specimen from Bali, Indonesia (LACM 1986-164.16). Figure 4. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera dolabrifera (Rang, 1828). A–C, radular teeth of a specimen from Maui, Hawaiian Islands (CPIC 00336). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bali, Indonesia (LACM 1986-164.16). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Maui, Hawaiian Islands (CPIC 00336). I, gizzard plate of a specimen from Madang, Papua New Guinea (CPIC 00820). J, jaw elements of a specimen from Madang, Papua New Guinea (CPIC 00820). K, post-radular armature of a specimen from Maui, Hawaiian Islands (CPIC 00336). L, M, dorsal and ventral views of the shell apex of a specimen from Madang, Papua New Guinea (CPIC 00820). N, O, dorsal and ventral views of the shell apex of a specimen from Bali, Indonesia (LACM 1986-164.16). Shell Shell flat, well calcified, oval (Fig. 5); nucleus conspicuous, formed by single lobe, occasionally two (Fig. 4L–O). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, typically widening abruptly near apical end, but more gradually in some specimens. Widest portion of shell mid-length forward, generally close to nucleus. Sculpture of conspicuous growth lines. Figure 5. View largeDownload slide Shells of Dolabrifera dolabrifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from the Hawaiian Islands (CPIC 00336), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Guam (CPIC 00413), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Indonesia (LACM 1986-164.16), scale bar = 1 mm. G, H, dorsal and ventral view of a syntype of Dolabrifera marmorea Pease in Reeve, 1868 (NHMUK 20160139), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a syntype of Dolabrifera fusca Pease, 1868 (ANSP 20710), scale bar = 5 mm (photographs: ©ANSP). K, L, dorsal and ventral view of a syntype of Dolabrifera pacifica Pease in Reeve, 1868 (NHMUK 20160140), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). M, N, dorsal and ventral view of a specimen from Papua New Guinea (CPIC 00820), scale bar = 1 mm. O, P, dorsal and ventral view of the lectotype of Dolabrifera olivacea Pease, 1860 (NHMUK 1964376), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Q, R, two syntypes of Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868 mounted in dorsal and ventral positions on a plate (NHMUK 1856.9.24.202), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Figure 5. View largeDownload slide Shells of Dolabrifera dolabrifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from the Hawaiian Islands (CPIC 00336), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Guam (CPIC 00413), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Indonesia (LACM 1986-164.16), scale bar = 1 mm. G, H, dorsal and ventral view of a syntype of Dolabrifera marmorea Pease in Reeve, 1868 (NHMUK 20160139), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a syntype of Dolabrifera fusca Pease, 1868 (ANSP 20710), scale bar = 5 mm (photographs: ©ANSP). K, L, dorsal and ventral view of a syntype of Dolabrifera pacifica Pease in Reeve, 1868 (NHMUK 20160140), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). M, N, dorsal and ventral view of a specimen from Papua New Guinea (CPIC 00820), scale bar = 1 mm. O, P, dorsal and ventral view of the lectotype of Dolabrifera olivacea Pease, 1860 (NHMUK 1964376), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Q, R, two syntypes of Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868 mounted in dorsal and ventral positions on a plate (NHMUK 1856.9.24.202), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Male reproductive system Penial canal large, muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 6C, D). Penial canal narrows and expands abruptly into oval penial sheath containing penial papilla. Oval section connects to large, strong retractor muscle connecting to ventral side of body wall internally. Penial papilla elongate, with curved cusp and longitudinal groove (Fig. 6A, B). Figure 6. View largeDownload slide Penial morphology of Dolabrifera dolabrifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bali, Indonesia (LACM 1986-164.16). B, SEM micrograph of a partially dissected penial papilla of a specimen from Maui, Hawaiian Is. (CPIC 01654). C, male copulatory organs of a specimen from Maui, Hawaiian Is. (CPIC 00336). D, male copulatory organs of a specimen from Madang, Papua New Guinea (CPIC 00820). E, male copulatory organs of a specimen from Guam (CPIC 00413). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Figure 6. View largeDownload slide Penial morphology of Dolabrifera dolabrifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bali, Indonesia (LACM 1986-164.16). B, SEM micrograph of a partially dissected penial papilla of a specimen from Maui, Hawaiian Is. (CPIC 01654). C, male copulatory organs of a specimen from Maui, Hawaiian Is. (CPIC 00336). D, male copulatory organs of a specimen from Madang, Papua New Guinea (CPIC 00820). E, male copulatory organs of a specimen from Guam (CPIC 00413). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Range Widespread in the tropical Indo-Pacific region, from East Africa to the Hawaiian Islands (see Remarks below). Remarks Extensive anatomical and sequence data obtained from specimens of Dolabrifera collected throughout the Indo-Pacific tropics, from the Hawaiian Islands to the Red Sea, revealed the presence of a single species in this vast biogeographical region, including samples from Saudi Arabia, Madagascar, Guam, Papua New Guinea, Indonesia, Fiji, Samoa and Hawai‘i. Only specimens from temperate regions in Australia and New Zealand are distinct and consequently assigned to a different species, D. brazieri (see below). Tropical Indo-Pacific specimens are characterized by having a large and muscular penial canal, lacking tubercles, and an oval penial sheath containing an elongate penial papilla with a curved cusp and a longitudinal groove. The radulae of Indo-Pacific specimens contain lateral teeth with the apex bifurcated into two blunt denticles equal in size. To determine the valid name for the tropical Indo-Pacific species and its synonyms, a review of the literature is discussed in the next paragraphs. Cuvier (1817) introduced for the first time the name ‘Dolabella dolabrifera’ based on a specimen collected in ‘Isle-de-France’ (=Mauritius). The name was mentioned in a footnote with no description and therefore is a nomen nudum. Subsequently, de Férussac (1822) mentioned the name ‘Dolabella dolabrifera’ and attributed it to Cuvier in a systematic list and classification of species of Mollusca, again with no description. Rang (1828) formally described and illustrated for the first time specimens assigned to Aplysia dolabrifera and collected from ‘Île Bourbon’ (=Réunion). The description included illustrations of the live animal and shell (Supporting Information, Fig. S3A–D), which are sufficient to corroborate they correspond to the modern usage of the name Dolabrifera. In this study, a single species of Dolabrifera has been detected among numerous samples from the Indo-Pacific region, ranging the Hawaiian Island to the Red Sea. Consequently, the name A. dolabrifera, which the oldest available name introduced from the Indo-Pacific region, is assigned to this species. Several decades later, Adams & Adams (1854) introduced the new species name Dolabrifera cuvieri for ‘A. dolabrifera Cuv’. The description consists of a short description of the genus; it was accompanied by a figure of a live animal reproduced from Rang’s (1828) description of A. dolabrifera (pl. 4, fig. 9) and the illustration of a shell, both published in Volume 3 of the same work (Adams & Adams, 1858: pl. 59, fig. 4, 4a). According to Pilsbry (1896: 119), D. cuvieri was ‘proposed as a substitute name for D. dolabrifera Cuv., evidently to avoid the duplication of names consequent upon the recognition of Dolabrifera as a genus’. Pilsbry (1896) indicated differences in the shell morphology between the ‘type of D. dolabrifera’ [referring to the specimen illustrated by Rang (1828: pl. 4, fig. 6)], which is elongate, and the more square shell illustrated by Adams & Adams (1858: pl. 59, fig. 4a). Based on the original description and subsequent comments by Pilsbry (1896), it is clear that Adams & Adams (1854) intended to intentionally change the original name of A. dolabrifera and therefore Dolabrifera cuvieri constitutes an unjustified emendation (ICZN, 1999: Article 33.2). Souleyet (1852) described Aplysia oahouensis from O‘ahu, Hawaiian Islands, with a short description and an illustration previously published by Eydoux & Souleyet (1846–1849). The illustration and description of the live animal and shell (Supporting Information, Fig. S3E–H) also correspond to the modern usage of Dolabrifera and fit within the variability of D. dolabrifera. Specimens of Dolabrifera from O‘ahu sequenced in this study are conspecific with specimens from the rest of the Indo-Pacific confirming that A. oahouensis is a synonym of D. dolabrifera. Deshayes (1863) introduced Dolabrifera maillardi based on a shell collected from Réunion. The illustrations of the shell in different views (Supporting Information, Fig. S3K–L) clearly represent a specimen of Dolabrifera. Again, due to the geographical origin of this shell, and its morphology, D. maillardi is here confirmed as a synonym of D. dolabrifera. Other similar nominal species described from various localities throughout Polynesia include Dolabrifera tahitensis Pease, 1861, type locality Tahiti, Dolabrifera fusca Pease, 1868, with no type locality specified (Pease, 1868), Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868, type locality ‘Narai’, Fiji (Reeve, 1868), Dolabrifera pacifica Pease in Reeve, 1868, again with no type locality specified (Reeve, 1868) and Dolabrifera marmorea Pease in Reeve, 1868, type locality Sandwich Islands (=Hawaiian Islands). The original description of D. tahitensis consists of a short text, but illustrations of the live animal were later published by Pease (1868: pl. 8, fig. 5; Supporting Information, Fig. S3O–P) and clearly represent a species of Dolabrifera. The original description of D. fusca included drawings of the live animal (Pease, 1868: pl. 8, fig. 4; Supporting Information, Fig. S3M, N), which match the characteristics of D. dolabrifera. In addition, the shell morphology of a remaining syntype (Fig. 5I, J) is consistent with that of other specimens of D. dolabrifera here examined and are regarded as synonyms. The original descriptions of D. vitraea, D. pacifica and D. marmorea included only illustrations of a shell each (Reeve, 1868: pl. 1, fig. 1a, b; pl. 1, fig. 3; pl. 1, fig. 5a, b) all of them reprinted here (Supporting Information, Fig. S3Q–U); photographs of the syntypes (shells) of all three species (Fig. 5Q–R, K–L, G–H) match the illustrations in the original descriptions. As in the previous cases, because of the geographical origin of the specimens and the characteristics of the shells, D. tahitensis, D. vitraea, D. pacifica and D. marmorea are most likely synonyms of D. dolabrifera and are here considered as such. Dolabrifera brazieri G. B. Sowerby II, 1870 (Figs 3H, 7–9) Dolabrifera brazieri G. B. Sowerby II, 1870: 250. Type locality: Northhead, Botany Bay, Australia. ?Dolabrifera jacksoniensis Pilsbry, 1896: 120–121, pl. 44, figs 38–41. Type locality: Port Jackson, Australia. Type material Dolabrifera brazieri G. B. Sowerby II, 1870, possibly syntype at NHMUK 1877.5.12.91. Dolabrifera jacksoniensis Pilsbry, 1896, syntypes at ANSP 64931 (dry) and A7040 (wet). Material examined Lord Howe Island, New South Wales, Australia (31°32.94′S 159°03.72′E), 1887, six specimens, 32–45 mm preserved length (AM C.54298). Boat Cove, Raoul Island, Kermadec Islands, New Zealand (29°16.79898′S, 177°53.66268′W), 14 May 2011, 0.25 m depth, one specimen, 39 mm preserved length (AM C.475882). NW corner of North Meyer Island, Kermadec Islands, New Zealand (29°14.4984′S, 177°52.6734′W), 5–15 m depth, 13 May 2011, one specimen 45 mm preserved length (AM C.475783). Description External morphology Body up to 150 mm long, oval to elongate, flattened. Dorsum covered with large, blunt, rounded to semi-conical tubercles, each with retractile, ramified, papilla on apex; large tubercles typically surrounded by smaller rounded tubercles (Fig. 3H). Rhinophores enrolled, elongated. Oral tentacles wide, flattened, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, typically brown or greenish-brown, sometimes pale grey or nearly black, with irregular darker, lighter areas. Dorsum often covered with dark brown network of anastomosing lines surrounding tubercles, which do not penetrate mantle margin, rhinophores. Mantle margin typically green, with whitish spots. Digestive system Radular formulae: 33 × 92.1.80 (AM C.54298); radulae asymmetrical, with more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 7A); teeth wider at base, both upper and lower ends of teeth convex. Lateral teeth elongate (Fig. 7B), with long bases; each cusp with proximal and distal elongate denticle on inner side, apex blunt, simple. Outer teeth very elongate (Fig. 7C), with bifurcate apices. Jaws with simple, irregular denticles (Fig. 7F). Post-radular armature with numerous spines with elongate bases, triangular cusps (Fig. 7G). Gizzard plates variable in shape, typically irregular, with some striations more visible near the centre (Fig. 7D). Figure 7. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A–C, radular teeth. A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D, E, gizzard plates. F, jaw elements. G, post-radular armature. H, I, dorsal and ventral views of the shell apex. Figure 7. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A–C, radular teeth. A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D, E, gizzard plates. F, jaw elements. G, post-radular armature. H, I, dorsal and ventral views of the shell apex. Shell Shell flat, well calcified, oval (Fig. 8); nucleus conspicuous, formed by single lobe connected to rest of shell by narrow area (Fig. 7H, I). On apertural view with nucleus on upper end, left side of shell straight, sometimes slightly concave or convex. Right side of shell convex, narrower near nucleus, widening gradually near mid-length. Widest portion of shell mid-length or slightly forward. Sculpture of conspicuous growth lines. Figure 8. View largeDownload slide Shells of Dolabrifera brazieri G. B. Sowerby II, 1870. A, B, dorsal and ventral view of specimen from Lord Howe Island, Australia (AM C.54298), scale bar = 1 mm. C, D, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.67), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). E, F, dorsal and ventral view of a syntype of Dolabrifera jacksoniensis Pilsbry, 1896 (ANSP 64931), scale bar = 1 mm (photographs: ©ANSP). G, H, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.68), scale bar = 1 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a possible syntype of Dolabrifera brazieri G. B. Sowerby II, 1870 (NHMUK 1877.5.12.91), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of specimen from Raoul Island, New Zealand (AM A.475882), scale bar = 1 mm. Figure 8. View largeDownload slide Shells of Dolabrifera brazieri G. B. Sowerby II, 1870. A, B, dorsal and ventral view of specimen from Lord Howe Island, Australia (AM C.54298), scale bar = 1 mm. C, D, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.67), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). E, F, dorsal and ventral view of a syntype of Dolabrifera jacksoniensis Pilsbry, 1896 (ANSP 64931), scale bar = 1 mm (photographs: ©ANSP). G, H, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.68), scale bar = 1 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a possible syntype of Dolabrifera brazieri G. B. Sowerby II, 1870 (NHMUK 1877.5.12.91), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of specimen from Raoul Island, New Zealand (AM A.475882), scale bar = 1 mm. Male reproductive system Penial canal large, muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 9B, C). Penial canal narrows and expands abruptly into elongate penial sheath section containing penial papilla. This section ends in large, strong retractor muscle connecting to ventral side of body wall internally. Penial papilla elongate, with curved cusp and longitudinal groove (Fig. 9A). Figure 9. View largeDownload slide Penial morphology of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A, SEM micrograph of the penial papilla. B, C, male copulatory organs. Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Figure 9. View largeDownload slide Penial morphology of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A, SEM micrograph of the penial papilla. B, C, male copulatory organs. Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Range Temperate regions in eastern Australia (mainly New South Wales, including Lord Howe Island) and northern New Zealand (Rudman, 2003), including the Kermadec Islands (Morley & Hayward, 2015). According to Morley & Hayward (2015), the presence of this species in New Zealand is the result of a human-mediated introduction. Remarks Dolabrifera brazieri was described by Sowerby (1870) with a short Latin description of the shell, based on two specimens (shells) collected in Northhead, Botany Bay, Australia. Sowerby (1870: 250) indicated that ‘No note seems to have been made of the character of the animal; but the shell, which is large, differs from that of other known species’. This species has been considered a synonym of D. dolabrifera by several authors (Eales, 1944; Bebbington, 1974, 1977), whereas others suggested that is an anatomically distinct, valid species, endemic to temperate regions in eastern Australia and New Zealand (Hedley, 1917; Willan & Morton, 1984; Rudman, 2003; Burn, 2006). Differences between D. brazieri and D. dolabrifera include the much larger size of D. brazieri (up to 150 mm long) and the presence of the large smooth conical tubercles all over the body (Rudman, 2003). In this study, we examined specimens identified as D. brazieri from Lord Howe Island, Australia and the Kermadec Islands, New Zealand, which are genetically distinct from D. dolabrifera collected in other areas of the Indo-Pacific region. These animals also differed anatomically from specimens of D. dolabrifera by the presence of a much more elongate penial sheath, lateral radular teeth with a proximal and distal elongate denticle and a simple, blunt apex and a shell with a very elongate apex with a conspicuous nucleus, formed by a single lobe, typically connected to the rest of the shell by a narrow area. Examination of a possible syntype of D. brazieri (NHMUK 1877.5.12.91) reveals that the shell has an unusually elongated apex with a wide, round nucleus (Fig. 8I, J), similar to the characteristics of the other specimens collected in Australia and New Zealand here examined. Therefore, this evidence supports the maintenance of D. brazieri as a valid species of Dolabrifera. Dolabrifera jacksoniensis was described by Pilsbry (1896) from Port Jackson (Sydney Harbour), Australia, just north of Botany Bay. The illustrations of a preserved specimen and a shell by Pilsbry (1896: pl. 44, figs 38–41) confirm it is a species of Dolabrifera. However, the morphology of the shell does not correspond to that of other specimens of D. brazieri here examined, as it has a much shorter apex (Supporting Information, Fig. S2B, C). Photographs of a syntype shell specimen (ANSP 64931) (Fig. 8E, F) show the shell apex is indeed not as elongate as in other specimens of D. brazieri here examined, but it has a conspicuous nucleus, formed by a single lobe, connected to the rest of the shell by a narrow area. Thus, the shell characteristics of D. jacksoniensis are largely consistent with those of D. brazieri. Other authors have considered D. jacksoniensis as a synonym of D. brazieri (see Hedley, 1917), and this synonymy is here confirmed with the available evidence. Dolabrifera ascifera (Rang, 1828) (Figs 10A–D, 11–13) Aplysia ascifera Rang, 1828: 51–52, pl. 4, figs 7–9. Type locality: ‘Saint-Jean de Cayenne’ (=Cayenne), French Guiana. Dolabrifera swiftii Pilsbry, 1896: 125, pl. 67, figs 19–20. Type locality: West Indies. Dolabrifera sowerbyi Guilding in Reeve, 1868: pl. 1, fig. 2a, b. Type locality: ‘St. Vincent’s W.I’. Type material Aplysia ascifera Rang, 1828, two syntypes at MNHN (Valdés & Héros, 1998). Dolabrifera swiftii Pilsbry, 1896, one syntype at ANSP 67519. Dolabrifera sowerbyi Guilding in Reeve, 1868, two syntypes at NHMUK 1839.9.15.45. Material examined Lower Matecumbe Key, Florida, USA, intertidal, 22 September 1950, four specimens 21–34 mm preserved length (LACM 1955-59.4). Arrecife Gallega, Veracruz, Veracruz, Mexico, 27 June 2014, one specimen 47 mm preserved length (CNMO 6611). Veracruz, Mexico, 16 June 1973, six specimens 30–45 mm preserved length, leg. J.W. Tunnell (LACM 179119). Arrecife Isla Lobos, Veracruz, Mexico, intertidal, 3 June 1973, three specimens 18–44 mm preserved length, leg. J.W. Tunnell (LACM 179120). Urchin Cove, Jamaica, 24 May 2006, one specimen 27 mm preserved length, leg. Ann DuPont (LACM 173238). East side of Playa El Yaque’s Lagoon, Isla Tortuga, Venezuela, 18 March 2010, 0.5 m depth, one specimen 55 mm preserved length (ZMUB 84907.1); one specimen 54 mm preserved length (ZMUB 84907.2). Bocas del Drago, Bocas del Toro, Panama, 28 July 2015, one specimen 22 mm preserved length (CPIC 01563). Description External morphology Body up to 90 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with numerous tubercles of different sizes, each with retractile, simple papilla on apex; larger tubercles surrounded by smaller ones (Fig. 10A–D), tightly arranged, giving animal bumpy appearance. Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude; edge of parapodia tuberculate. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. Figure 10. View largeDownload slide Live animals of the Atlantic Ocean species of Dolabrifera. A–D, Dolabrifera ascifera Rang, 1828. A, Venezuela (ZMBN 84918); B, Jamaica (LACM 173238); C, Veracruz, Mexico (CNMO 6611); D, Bocas del Toro, Panama (CPIC 01563). E, F, Dolabrifera edmundsi sp. nov., Madeira, Portugal, photograph: P. Wirtz (ZSM 20160193, ZSM 20160194). G, H, Dolabrifera virens Verrill, 1901. G, Jamaica (LACM 173265); H, St. Helena (ZSM Mol 20170625). Figure 10. View largeDownload slide Live animals of the Atlantic Ocean species of Dolabrifera. A–D, Dolabrifera ascifera Rang, 1828. A, Venezuela (ZMBN 84918); B, Jamaica (LACM 173238); C, Veracruz, Mexico (CNMO 6611); D, Bocas del Toro, Panama (CPIC 01563). E, F, Dolabrifera edmundsi sp. nov., Madeira, Portugal, photograph: P. Wirtz (ZSM 20160193, ZSM 20160194). G, H, Dolabrifera virens Verrill, 1901. G, Jamaica (LACM 173265); H, St. Helena (ZSM Mol 20170625). External coloration Background colour variable, brown or green, with spots and/or irregular patches of different colours. Mantle margin semi-translucent, with same general colour as rest of the body and light spots. Ventral side typically same colour as rest of the body with numerous white spots. Digestive system Radular formulae: 34 × 90.1.77 (CPIC 01563); 35 × 90.1.95 (LACM 173238); radulae typically asymmetrical, with more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 11A, D); teeth wider at base, both upper, lower ends of teeth convex. Some rachidian teeth with sharp conical extensions on one of the apical ends (typically right one). Lateral teeth elongate, with long bases (Fig. 11B, E); each cusp with basal, blunt, inner denticle, apex bifurcated into two blunt denticles, one clearly larger. Outer teeth very elongate, with or without secondary denticle, apex bifurcate or simple (Fig. 11C, F). Jaws with simple, irregular denticles (Fig. 11J). Post-radular armature with numerous spines with elongate bases, triangular cusps (Fig. 11K). Gizzard plates variable in shape, typically irregular, with some striations more visible near centre (Fig. 11G–I). Figure 11. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera ascifera (Rang, 1828). A–C, radular teeth of a specimen from Bocas del Toro, Panama (CPIC 01563). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173238). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Venezuela (ZMBN 84907). I, gizzard plate of a specimen from Bocas del Toro, Panama (CPIC 01563). J, jaw elements of a specimen from Venezuela (ZMBN 84907). K, post-radular armature of a specimen from Venezuela (ZMBN 84907). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173238). N, O, dorsal and ventral views of the shell apex of a specimen from Venezuela (ZMBN 84907). Figure 11. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera ascifera (Rang, 1828). A–C, radular teeth of a specimen from Bocas del Toro, Panama (CPIC 01563). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173238). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Venezuela (ZMBN 84907). I, gizzard plate of a specimen from Bocas del Toro, Panama (CPIC 01563). J, jaw elements of a specimen from Venezuela (ZMBN 84907). K, post-radular armature of a specimen from Venezuela (ZMBN 84907). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173238). N, O, dorsal and ventral views of the shell apex of a specimen from Venezuela (ZMBN 84907). Shell Shell flat, well calcified, oval (Fig. 12); nucleus conspicuous, formed by single lobe (Fig. 12A–D, I, J), occasionally two (Fig. 12E–H, K, L). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, typically widening abruptly near mid-length, more gradually in some specimens. Widest portion of shell mid-length or slightly forward. Sculpture of conspicuous growth lines. Figure 12. View largeDownload slide Shells of Dolabrifera ascifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bocas del Toro, Panama (CPIC 01563), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Jamaica (LACM 173238). I, J, dorsal and ventral view of a syntype of Dolabrifera sowerbyi Guilding in Reeve, 1868 (NHMUK 1839.9.15.45), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of a syntype of Dolabrifera swiftii Pilsbry, 1896 (ANSP 67519), scale bar = 5 mm (photographs: ©ANSP), scale bar = 1 mm. Figure 12. View largeDownload slide Shells of Dolabrifera ascifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bocas del Toro, Panama (CPIC 01563), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Jamaica (LACM 173238). I, J, dorsal and ventral view of a syntype of Dolabrifera sowerbyi Guilding in Reeve, 1868 (NHMUK 1839.9.15.45), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of a syntype of Dolabrifera swiftii Pilsbry, 1896 (ANSP 67519), scale bar = 5 mm (photographs: ©ANSP), scale bar = 1 mm. Male reproductive system Penial canal long and muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 13B–D). Penial canal ends in long retractor muscle connecting to ventral side of body wall internally. No distinguishable penial sheath. Penial papilla very long, occupies almost entire penial canal (Fig. 13A). Figure 13. View largeDownload slide Penial morphology of Dolabrifera ascifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Venezuela (ZMBN 84907). B, male copulatory organs of a specimen from Veracruz, Mexico (CNMO 6611). C, male copulatory organs of a specimen from Jamaica (LACM 173238). D, male copulatory organs of a specimen from Venezuela (ZMBN 84907). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Figure 13. View largeDownload slide Penial morphology of Dolabrifera ascifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Venezuela (ZMBN 84907). B, male copulatory organs of a specimen from Veracruz, Mexico (CNMO 6611). C, male copulatory organs of a specimen from Jamaica (LACM 173238). D, male copulatory organs of a specimen from Venezuela (ZMBN 84907). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Range Western Atlantic, from Florida to possibly Brazil. Confirmed records in this study based on molecular data include Mexico, Jamaica, Panama and Venezuela. Confirmed records based on morphology include Florida, Mexico, the US Virgin Islands and French Guiana. Remarks Some specimens of Dolabrifera collected in several localities in the Western Atlantic (Veracruz, Mexico, Jamaica, Bocas del Toro, Panama and Venezuela) are genetically and morphologically similar to each other and distinct from other specimens Dolabrifera here sequenced. Species delimitation analysis confirmed these specimens constitute a distinct species. The main distinctive characteristics of these animals are in the internal anatomy and include the male reproductive system, with a long and muscular penial canal lacking tubercles, no distinguishable penial sheath and a very long penial papilla occupying almost the entire penial canal. The rachidian radular teeth have characteristic sharp conical extensions on one of the apical ends, the lateral radular teeth have a bifurcated apex with two blunt denticles, one clearly larger than the other. The shells are wide and short, typically narrower near the nucleus, often widening abruptly near mid-length; the nucleus is conspicuous, formed by a single lobe, occasionally two. To determine the valid name and synonyms for this species, a review of the literature was conducted and discussed below. The oldest name available for Western Atlantic Dolabrifera is Aplysia ascifera Rang, 1828, described from ‘Saint-Jean de Cayenne’ (=Cayenne), French Guiana. Rang (1828: pl. 4, figs 7–9) illustrated the external morphology of a live animal and the shell here reproduced in Supporting Information (Fig. S2D–F). The characteristics of the live animal and particularly of the shell match those of some specimens here described; the live animal is a greenish-grey Dolabrifera with numerous ramified tubercles on the dorsum, similar to specimens from Jamaica and Mexico illustrated herein (Fig. 10B, C); the shell is short and wide (c. 1:1.5–2 width/length ratio), narrower near the nucleus, widening near mid-length, the nucleus is conspicuous. Other Western Atlantic specimens here examined morphologically, which cluster together in the same clade, also have short and wide shells with a similar overall morphology (Fig. 12). Because of these similarities, we propose using the name Dolabrifera ascifera for this species. Pilsbry (1896) described Dolabrifera swiftii based on specimens collected from the West Indies. The original description included illustrations of a shell (Pilsbry, 1896: pl. 67, figs 19, 20; Supporting Information, Fig. S2M, N), which also resembles the characteristics of the original description of D. ascifera as well as those of other Western Atlantic specimens here examined and assigned to this species. These characteristics (wide and short shell, narrower near nucleus, widening near mid-length, nucleus conspicuous) were confirmed with the examination of photographs of a syntype (Fig. 12K, L). Thus, we consider D. swiftii a synonym of D. ascifera. Two additional species described from the Caribbean region are Dolabrifera virens Verrill, 1901, type locality Hungry Bay, Bermuda and Dolabrifera sowerbyi Guilding in Reeve, 1868, type locality St. Vincent, US Virgin Islands. The shells of both species were illustrated in the original descriptions (Verrill, 1901: pl. 2, figs 4, 5; Reeve, 1868: pl. 1, fig. 2a, b) and reproduced here (Supporting Information, Fig. S2G, I, K, L). Examination of the syntype of D. sowerbyi (Fig. 12I, J) reveals that this shell is similar to those of other shells of D. ascifera. Thus, D. sowerbyi is also regarded as a synonym of D. ascifera. On the contrary, the shell of a syntype of D. virens is much longer and narrower and probably belongs to a different species described below. Dolabrifera nicaraguana Pilsbry, 1896 (Figs 3I–J, 14–16) Dolabrifera nicaraguana Pilsbry, 1896: 124, pl. 63, figs 12–16. Type locality: San Juan del Sur, Nicaragua (Pacific coast). Type material Three syntypes at ANSP 67517 (dry) and ANSP A7048 (wet). Material examined Puerto Vallarta, Mexico, intertidal, 2 May 2002, one specimen 15 mm preserved length (CPIC 00547). El Salvador, no locality data or date, two shells, leg. A.J. Ferreira (LACM 10383–10384). Costa Rica, no locality data or date, two shells, leg. A.J. Ferreira (LACM 10381–10382). North side of San Juan del Sur Bay, Nicaragua (11°15′N, 85°52′W), 25 Jan 1974, six specimens 10–13 mm preserved length, one shell removed (CASIZ 001449). Bahía Salinas, Guanacaste, Costa Rica (11°02′N, 83°42′W), intertidal, 10 February 1935, two specimens 30–35 mm preserved length (LACM 1935-107.25); one specimen 35 mm preserved length (LACM 1935-107.25). Puerto Parker, Guanacaste, Costa Rica (10°56′N, 85°48′47″W), intertidal, 9 February 1935, one specimen 23 mm preserved length (LACM 1935-115.17). South of Punta Mala, Puerto Culebra, Guanacaste, Costa Rica (10°36′30″N, 85°42′15″W), intertidal, 24 February 1934, three specimens 25–35 mm preserved length (LACM 1934-130.21); eight specimens 20–30 mm preserved length (LACM 1934-130.21). Bahía San Josecito, Provincia Puntarenas, Costa Rica, 14 January 2003, two specimens 24–28 mm preserved length (CPIC 00551). North side of Isla del Caño, Costa Rica (8°43′15″N, 83°53′07″W), intertidal, 18–19 March 1972, four specimens 10–15 mm preserved length, leg. J.H. McLean (LACM 1972-68.45). Bahía Honda, Panama (7°43′30″N, 81°32′40″W), intertidal, 28 March 1939, seven specimens 22–30 mm preserved length (LACM 1939–52.1). South of Punta Marzo, Bahía Octavia, Choco, Colombia (6°49.8′N, 77°49.6′W, intertidal, 27 January 1935, one specimen 21 mm preserved length (LACM 1935–73.9). Bahía Cupica, Choco, Colombia (6°39.2′N, 77°30.7′W, intertidal, 26 January 1935, one specimen 22 mm preserved length (LACM 1935–73.9). Puerto Utria, Choco, Colombia (5°59.2′N, 77°21.3′W, intertidal, 23 January 1935, one specimen 30 mm preserved length (LACM 1935–52.25); 25 February 1938, three specimens 23–31 mm preserved length (LACM 1938-126.18). Isla Gorgona, Cauca, Colombia (3°0.5′N, 78°11.8′W), intertidal, 12 February 1934, one specimen 24 mm preserved length (LACM 1934–92.17). Cabo de San Francisco, Ecuador (0°39′30″N, 80°06′30″W), intertidal, two specimens 32–37 mm (LACM 1938-116.11). Sombrero Chino, Isla Santiago, Islas Galápagos, Ecuador (0°22′20″S, 90°34′30″W), intertidal, 22 March 1971, six specimens 15–22 mm preserved length (LACM 1971-64.6). Punta Alfaro, Isla Isabela, Islas Galápagos, Ecuador (0°25′20″S, 90°57′10″W), intertidal, 25 March 1971, three specimens 8–12 mm preserved length (LACM 1971-70.10). Isla Pinzón, Islas Galápagos, Ecuador (0°35′50″S, 90°39′15″W), intertidal, 25–26 March 1971, five specimens 16–22 mm preserved length (LACM 1971-72.16). Darwin Research Station, Academy Bay, Isla Santa Cruz, Islas Galápagos, Ecuador (0°45′06″S, 90°15′38″W), 1 m, March 1971, one specimen 23 mm preserved length (LACM 1971-45.20); intertidal, March 1971, one specimen 21 mm preserved length (LACM 1971-44.17). Flamingo Cove, near Post Office Bay, Isla Floreana, Islas Galápagos, Ecuador (1°14′S, 90°27′30″W), intertidal, 15 March 1971, two specimens 13–24 mm preserved length (LACM 1971-52.13). Isla de la Plata, Ecuador (1°16′S, 81°05′10″W), intertidal, 22 January 1933, one specimen 45 mm preserved length (LACM 1933-22.21). Salinas, Santa Elena, Ecuador (2°12′S, 81°58′W), 5–6 March 1970, one specimens 28 mm preserved length, leg. J.H. McLean (LACM 1970–9.29). South Point of Santa Elena, Ecuador (2°12′23″S, 81°00’05″W), 8 February 1934, three specimens 40 mm preserved length (LACM 1934–81.15). Balneario Punta Sal, Tumbes, Peru, intertidal, 9 August 2005, one specimen 16 mm preserved length, leg. K. Nakamura (CPIC 00194). Description External morphology Body up to 50 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with densely packed tubercles of different sizes, each with retractile, simple, short papilla on apex; larger tubercles surrounded by smaller ones (Fig. 3I, J). Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude; edge of parapodia tuberculate. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, typically brown, greyish-green, or red; tubercles often lighter than background coloration, sometimes with papillae of different colours (pink). Mantle margin with same general colour as rest of body with light spots. Ventral side typically same colour as rest of body with numerous white spots. Digestive system Radular formulae: 25 × 86.1.84 (CPIC 00547); 43 × 111.1.95 (CPIC 00551); radulae slightly asymmetrical, with few more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 14A, D), two innermost denticles much larger than two outermost; teeth wider at base, both upper and lower ends of teeth convex. Some rachidian teeth with small sharp conical extensions on one of apical ends (typically right one). Lateral teeth elongate, with long bases (Fig. 14B, E); each cusp with basal, narrow, inner denticle, apex bifurcated into two blunt denticles, one slightly larger. Outer teeth very elongate, with or without secondary denticle, apex bifurcate or simple (Fig. 14C, F). Jaws with simple, irregular denticles (Fig. 14J). Post-radular armature with numerous spines with elongate bases and triangular cusps (Fig. 14K). Gizzard plates variable in shape, typically irregular, with numerous striations (Fig. 14G–I). Figure 14. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera nicaraguana Pilsbry, 1896. A–C, radular teeth of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551) – D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, gizzard plate of a specimen from Puerto Vallarta, Mexico (CPIC 00547). H, I, gizzard plates of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). J, jaw elements of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). K, post-radular armature of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). L, M, dorsal and ventral views of the shell apex of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). N, O, dorsal and ventral views of the shell apex of a specimen from Puerto Vallarta, Mexico (CPIC 00547). Figure 14. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera nicaraguana Pilsbry, 1896. A–C, radular teeth of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551) – D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, gizzard plate of a specimen from Puerto Vallarta, Mexico (CPIC 00547). H, I, gizzard plates of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). J, jaw elements of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). K, post-radular armature of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). L, M, dorsal and ventral views of the shell apex of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). N, O, dorsal and ventral views of the shell apex of a specimen from Puerto Vallarta, Mexico (CPIC 00547). Shell Shell flat, well calcified, oval to elongate (Fig. 15); nucleus conspicuous, formed by single lobe (Fig. 14L–P). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, typically widening slightly towards mid-length, abruptly in some specimens; some shells very narrow across entire length. Widest portion of shell mid-length backward. Sculpture of conspicuous growth lines. Figure 15. View largeDownload slide Shells of Dolabrifera nicaraguana Pilsbry, 1896. A, B, dorsal and ventral view of a syntype of Dolabrifera nicaraguana Pilsbry, 1896 (ANSP 67517), scale bar = 1 mm (photographs: ©ANSP). C, D, dorsal and ventral view of a specimen from Puerto Culebra, Costa Rica (LACM 256-34), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Puerto Vallarta, Mexico (CPIC 00547), scale bar = 1 mm. I, J, dorsal and ventral view of a specimen from Bahía Salinas, Costa Rica (LACM 474-35), scale bar = 1 mm. K, L, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. M, N, dorsal and ventral view of a specimen from Tumbes, Peru (CPIC 00194), scale bar = 1 mm. O, P, dorsal and ventral view of a specimen from Santa Elena, Ecuador (LACM 1934–81.15), scale bar = 1 mm. Figure 15. View largeDownload slide Shells of Dolabrifera nicaraguana Pilsbry, 1896. A, B, dorsal and ventral view of a syntype of Dolabrifera nicaraguana Pilsbry, 1896 (ANSP 67517), scale bar = 1 mm (photographs: ©ANSP). C, D, dorsal and ventral view of a specimen from Puerto Culebra, Costa Rica (LACM 256-34), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Puerto Vallarta, Mexico (CPIC 00547), scale bar = 1 mm. I, J, dorsal and ventral view of a specimen from Bahía Salinas, Costa Rica (LACM 474-35), scale bar = 1 mm. K, L, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. M, N, dorsal and ventral view of a specimen from Tumbes, Peru (CPIC 00194), scale bar = 1 mm. O, P, dorsal and ventral view of a specimen from Santa Elena, Ecuador (LACM 1934–81.15), scale bar = 1 mm. Male reproductive system Penial canal large and muscular, with a series of tubercles and two proximal retractor muscles, on each side of the opening into the body wall (Fig. 16B, C). The penial canal widens gradually into the distal end, right before narrowing abruptly and widening again into a small, vesicular-like penial sheath, which contains the penial papilla. The distal end of the penial sheath connects to a long and strong retractor muscle attaching to the ventral side of the body wall internally. The penial papilla is small, triangular, with a longitudinal groove (Fig. 16A). Figure 16. View largeDownload slide Penial morphology of Dolabrifera nicaraguana Pilsbry, 1896. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). B, male copulatory organs of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). C, male copulatory organs of a specimen from Santa Elena, Ecuador (LACM 1934-81.15). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Figure 16. View largeDownload slide Penial morphology of Dolabrifera nicaraguana Pilsbry, 1896. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). B, male copulatory organs of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). C, male copulatory organs of a specimen from Santa Elena, Ecuador (LACM 1934-81.15). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Range Eastern Pacific Ocean, from Bahía Las Cruces, Baja California Sur, Mexico (Bertsch, 1970) to Tumbes, Peru (Nakamura, 2007; Uribe et al., 2013). Remarks Anatomical and sequence data reveal that all specimens here studied from the Eastern Pacific belong to the same species. All of them have a large and muscular penial canal, with a series of tubercles, as well as a very small, vesicular-like penial sheath containing a small penial papilla; the shells tend to be narrower than in Dolabrifera species from other regions and the lateral radular teeth have the apex bifurcated into two blunt denticles, one slightly larger than the other. The ABGD analysis reveals that Eastern Pacific specimens group together in the same species, this includes specimens ranging from Mexico to Peru, which constitutes the known range of Dolabrifera in this region. Moreover, specimens from the Eastern Pacific are genetically and morphologically distinct from other Dolabrifera specimens here examined, confirming the Eastern Pacific animals represent a different species. A review of the literature reveals that there is only one name available for Eastern Pacific Dolabrifera, D. nicaraguana Pilsbry, 1896, described from San Juan del Sur, Nicaragua. The original description included illustrations of a preserved specimen and a shell (Pilsbry, 1896: pl. 63, figs 12–16; Supporting Information, Fig. S2O–Q). The shell of the syntype is straight, very narrow and elongate (Fig. 15A, B), which is different from other shells from the Eastern Pacific here examined (Fig. 15C–H, K, P), but similar to others (Fig. 15I, J), underscoring the variability in shell morphology within this species. Here, we examined specimens from San Juan del Sur, Nicaragua and adjacent localities in Costa Rica, all of which share similar shell, penial and radular morphology with other specimens from the Eastern Pacific. Thus, we confidently assign the name D. nicaraguana to the Eastern Pacific species of Dolabrifera. Dolabrifera virens Verrill, 1901 (Figs 10G–H, 17–19) Dolabrifera virens Verrill, 1901: 24–25, pl. 2, fig. 4, 5, pl. 4, fig. 11. Type locality: Hungry Bay, Bermuda. Type material Dolabrifera virens Verrill, 1901, two syntypes at YPM IZ 048817 (wet) and YPM IZ 029268 (dry). Material examined Urchin Cove, St Ann’s Bay, Jamaica (18°27.20′N, 77°13.55′W), 0.3–0.6 m depth, 25 May 2006, two specimens 10–15 mm preserved length, leg. A. DuPont (LACM 173265). Lauderdale-by-the-Sea, Florida, USA, 8 m depth, 18 June 2014, one specimen 21 mm preserved length, leg. A. Dimitris (CPIC 01134). Vista Alegre, Curaçao (12°0.50′N, 68°53.30′W), intertidal, 23 April 1939, nine specimens 14–40 mm preserved length (LACM 1939-215.1). Description External morphology Body up to 80 mm long, oval, flattened, broader posteriorly, narrowing slightly towards head. Dorsum covered with densely packed tubercles of different sizes, each with elongate papilla on apex, some ramified at tips; larger tubercles surrounded by smaller ones, each bearing papilla, giving animal appearance of being densely covered with papillae (Fig. 10G–H). Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude; edge of parapodia tuberculate. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, red, brown or green, sometimes white, with spots and/or irregular patches of different colours. Mantle margin semi-translucent, with same general colour as rest of body and few small white dots. Ventral side typically same colour as rest of the body with numerous white spots. Digestive system Radular formulae: 32 × 61.1.60 (LACM 173265A); 27 × 50.1.57 (LACM 173265B); radulae nearly symmetrical, with about same number of teeth on each row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 17A, D); teeth wider at base, both upper and lower ends of teeth convex. Lateral teeth elongate, with long bases (Fig. 17B, E); each cusp with two to three basal, blunt, inner denticles, apex bifurcated into two blunt denticles equal in size. Outer teeth very elongate, with secondary denticle, apex bifurcate (Fig. 17C, F). Jaws with simple, irregular denticles (Fig. 17J). Post-radular armature with numerous spines with elongate bases and low cusps (Fig. 17K). Gizzard plates variable in shape, typically short, wide, with some striations more visible near one end (Fig. 17G–I). Figure 17. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera virens Verrill, 1901. A–C, radular teeth of a specimen from Jamaica (LACM 173265A). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173265B). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Jamaica (LACM 173265A). J, jaw elements of a specimen from Jamaica (LACM 173265B). K, post-radular armature of a specimen from Jamaica (LACM 173265A). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265B). N, O, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265A). Figure 17. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera virens Verrill, 1901. A–C, radular teeth of a specimen from Jamaica (LACM 173265A). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173265B). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Jamaica (LACM 173265A). J, jaw elements of a specimen from Jamaica (LACM 173265B). K, post-radular armature of a specimen from Jamaica (LACM 173265A). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265B). N, O, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265A). Shell Shell flat, well calcified, elongate, width/length ratio greater than 1:2 (Fig. 18); nucleus inconspicuous, short, formed by single lobe. On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, widening gradually towards mid-length. Widest portion of shell mid-length or slightly backward. Sculpture of conspicuous growth lines. Figure 18. View largeDownload slide Shells of Dolabrifera virens Verrill, 1901. A, B, dorsal and ventral view of a specimen from Jamaica (LACM 173265A), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134), scale bar = 1 mm. E, F, dorsal and ventral view of a syntype from Bermuda (YPM IZ 029268), scale bar = 1 mm. Figure 18. View largeDownload slide Shells of Dolabrifera virens Verrill, 1901. A, B, dorsal and ventral view of a specimen from Jamaica (LACM 173265A), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134), scale bar = 1 mm. E, F, dorsal and ventral view of a syntype from Bermuda (YPM IZ 029268), scale bar = 1 mm. Male reproductive system Penial canal long, muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 19B, C). Penial canal ends in long retractor muscle connecting to ventral side of body wall internally. No distinguishable penial sheath. Penial papilla very long, occupies almost entire penial canal (Fig. 19A). Figure 19. View largeDownload slide Penial morphology of Dolabrifera virens Verrill, 1901. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Jamaica (LACM 173265A). B, male copulatory organs of a specimen from Jamaica (LACM 173265A). C, male copulatory organs of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Figure 19. View largeDownload slide Penial morphology of Dolabrifera virens Verrill, 1901. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Jamaica (LACM 173265A). B, male copulatory organs of a specimen from Jamaica (LACM 173265A). C, male copulatory organs of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Range Western and South Atlantic from Jamaica to St. Helena. Confirmed records based on molecular data include Panama, Jamaica and St. Helena. Confirmed records based on morphology include Florida, Bermuda and Curaçao. Remarks The phylogenetic analyses recovered a clade including specimens from Bocas del Toro, Panama, Jamaica and St. Helena in the South Atlantic, distinct from the clade containing other Western Atlantic specimens, to which the name D. ascifera was assigned herein. The species delimitation analysis confirmed this clade is a distinct species. These animals have a male reproductive anatomy similar to that of D. ascifera, but they differ in other regards. For example, the rachidian radular teeth lack sharp conical extensions on the apical ends, the lateral radular teeth have a bifurcated apex with two blunt denticles equal in size, the shells are narrower and proportionally longer, narrowing gradually towards the nucleus; the nucleus is inconspicuous, formed by a single lobe. Because these animals are genetically and morphologically distinct from D. ascifera, it is necessary to determine whether an available name exists for this taxon. Dolabrifera virens Verrill, 1901 was described from Hungry Bay, Bermuda, based on several specimens, two of which remain at the YPM collections. One of the syntypes was photographed alive (Verrill, 1901: text fig. 1, pl. 4, fig. 11) and is covered by numerous tubercles with elongate, branched papillae (Supporting Information, Fig. S2J). This is similar to the animals here described, which contrary to D. ascifera appear to be covered by densely arranged, ramified papillae. The shells of several specimens were illustrated in the original description (Verrill, 1901: pl. 2, figs 4, 5), and photographs of one of the syntypes are reproduced here (Fig. 18E, F). The syntype shell (YPM IZ 029268) is very narrow and elongate, width/length ratio greater than 1:2, very similar to other shells dissected from specimens clustering into the unnamed Western Atlantic clade (Fig. 18). These shells are consistently longer and narrower than all the shells of D. ascifera here examined (Fig. 12). Because specimens in this unnamed Western Atlantic clade match the original description of D. virens, this name is assigned to it. Dolabrifera virens and D. ascifera are externally undistinguishable and sympatric in large portions of the Western Atlantic, making the identification of these species problematic and requiring anatomical examination and/or DNA sequence data. Dolabrifera edmundsi sp. nov. (Figs 10E–F, 20–22) Type material Holotype at ZSM Mol 20160193, Porto Moniz, Madeira, September 2014, 18 mm preserved length. Material examined Porto Moniz, Madeira, September 2014, one specimen 17 mm preserved length, leg. P. Wirtz (ZSM Mol 20160194), one specimen 19 mm preserved length, leg. P. Wirtz (ZSM Mol 20160195), one specimen 20 mm preserved length, leg. P. Wirtz (ZSM Mol 20160196), one specimen 20 mm preserved length, leg. P. Wirtz (ZSM Mol 20160197). Ilhéu Bombom, Ilha do Príncipe, São Tomé and Príncipe, 20 January 2009, one specimen 11 mm preserved length (CASIZ 179395). South of Terrebonne Bay, LA, USA (28°5.57′N, 91°1.34′W), 56–58 m depth, 4 June 2005, four specimens 12–14 mm preserved length, leg. E. Garcia (LACM 179615). Marsh Island, LA, USA (27°98.34′N, 92°22.42′W), 68–86 m depth, 22 June 2005, two specimens 11–16 mm preserved length, leg. E. Garcia (LACM 179619). Description External morphology Body up to 80 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with small, rounded, scattered tubercles, each with retractile, highly ramified, papilla on apex (Fig. 10E, F); tubercles typically surrounded by large areas of smooth tissue. Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, typically greenish-grey or brown, sometimes red, with spots and/or irregular patches of different colours. Mantle margin with same general colour as rest of body. Digestive system Radular formula: 40 × 70.1.71 (ZSM Mol 20160195); 38 × 71.1.72 (ZSM Mol 20160196); radulae symmetrical, with just about same number teeth in both half-rows. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 20A, D), all similar in size; cusp with or without denticles on both sides; rachidian teeth wider at base, both upper and lower ends of teeth convex. Some rachidian teeth with small extensions on apical ends. Lateral teeth elongate, with long bases (Fig. 20B, E); each cusp with basal, narrow, inner denticle, apex bifurcated into two blunt denticles equal in length. Outer teeth very elongate, with or without secondary denticle, apex bifurcated (Fig. 20C, F). Jaws with simple denticles (Fig. 20J). Post-radular armature with numerous spines with elongate bases and triangular cusps (Fig. 20K). Gizzard plates variable in shape, typically irregular, with numerous striations (Fig. 20G–I). Figure 20. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera edmundsi sp. nov. A–C, radular teeth of a specimen from Madeira (ZSM Mol 20160195). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Maderia (ZSM Mol 20160196). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Madeira (ZSM Mol 20160195). J, jaw elements of a specimen from Madeira (ZSM Mol 20160196). K, post-radular armature of a specimen from Madeira (ZSM Mol 20160196). L, M, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160195). N, O, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160196). Figure 20. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera edmundsi sp. nov. A–C, radular teeth of a specimen from Madeira (ZSM Mol 20160195). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Maderia (ZSM Mol 20160196). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Madeira (ZSM Mol 20160195). J, jaw elements of a specimen from Madeira (ZSM Mol 20160196). K, post-radular armature of a specimen from Madeira (ZSM Mol 20160196). L, M, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160195). N, O, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160196). Shell Shell flat, well calcified, oval to elongate (Fig. 21); nucleus conspicuous, formed by single lobe (Fig. 20L–O). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, widening slightly towards mid-length. Widest portion of shell mid-length. Sculpture of conspicuous growth lines. Figure 21. View largeDownload slide Shells of Dolabrifera edmundsi sp. nov. A, B, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160195), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160196), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160197), scale bar = 1 mm. Figure 21. View largeDownload slide Shells of Dolabrifera edmundsi sp. nov. A, B, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160195), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160196), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160197), scale bar = 1 mm. Male reproductive system Penial canal large, muscular, with two proximal retractor muscles, on each side of opening into body wall (Fig. 22B–D). Penial canal widens gradually into oval penial sheath, containing penial papilla. Distal end of penial sheath connects to thick and strong retractor muscle attaching to ventral side of body wall internally. Penial papilla elongate, triangular, with longitudinal groove (Fig. 22A). Figure 22. View largeDownload slide Penial morphology of Dolabrifera edmundsi sp. nov. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Madeira (ZSM Mol 20160196). B, male copulatory organs of a specimen from Madeira (ZSM Mol 20160195). C, male copulatory organs of a specimen from Madeira (ZSM Mol 20160196). D, male copulatory organs of a specimen from Louisiana, USA (LACM 176306). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Figure 22. View largeDownload slide Penial morphology of Dolabrifera edmundsi sp. nov. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Madeira (ZSM Mol 20160196). B, male copulatory organs of a specimen from Madeira (ZSM Mol 20160195). C, male copulatory organs of a specimen from Madeira (ZSM Mol 20160196). D, male copulatory organs of a specimen from Louisiana, USA (LACM 176306). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Range Amphiatlantic. Primarily found in the eastern Atlantic, confirmed records include the Canary Islands, Madeira, Cape Verde, and São Tomé and Príncipe. Also present in St. Helena (South Atlantic) and the Gulf of Mexico (Western Atlantic). Etymology Named in memory of our colleague Dr Malcolm Edmunds to recognize his seminal contribution to the study of sea slugs, including the discovery of many tropical Atlantic species and his insights on their biogeographical patterns. Remarks Specimens from the Eastern Atlantic and some specimens from the South Atlantic (St. Helena) and deep waters in the Gulf of Mexico are genetically distinct from other species of Dolabrifera. These animals have tubercles with long, ramified papillae. Their shells are elongate, wider near mid-length and much narrower posteriorly and anteriorly, and the male reproductive organs contain a well-developed penial papillae. These characteristics are very different from the other two Atlantic species here recognized. Dolabrifera virens is the only Atlantic species with ramified papillae, but they are shorter. A review of the literature reveals that there is no name available for this taxon, and therefore, it is here described as a new species. DISCUSSION Species identification and characterization The genus Dolabrifera, which was often considered to include the single species D. dolabrifera (see Engel & Hummelinck, 1936; Eales, 1944), now includes five different tropical species, in addition to the temperate species D. brazieri, which was already considered distinct by some authors (Klussman-Kolb, 2004; Burn, 2006). These results are hardly surprising, considering that other species of sea hares and heterobranch sea slugs with broad tropical ranges studied to date have been found to constitute species complexes (e.g. Ornelas-Gatdula et al., 2012; Alexander & Valdés, 2013; Krug et al., 2013; Carmona et al., 2014a, b; Goodheart et al., 2015; Kienberger et al., 2016). However, the case of Dolabrifera is unique in several respects. For example, in most other cases, the species complexes are composed of primarily allopatric taxa, often found in different ocean basins (Ornelas-Gatdula et al., 2012; Alexander & Valdés, 2013; Carmona et al., 2014b) or with multiple sympatric species in the Indo-Pacific tropics (Krug et al., 2013; Carmona et al., 2014a). On the contrary, in Dolabrifera, there is a single tropical Indo-Pacific species ranging from the western Indian Ocean to the Hawaiian Islands and displaying virtually no geographical structure; at the same time, there are three partially sympatric species in the Atlantic Ocean, two of them widely distributed across the ocean basin. Another difference is that in most other species complexes identified to date, newly characterized species have been found to be pseudocryptic, meaning that they can be distinguished based on external morphological traits, but those differences only became evident once molecular data were available (Sáez & Lozano, 2005). In the case of Dolabrifera, we have been unable to identify reliable external traits for species identification; this is due to the high degree of intraspecific variation in both colour and papillae morphology in all species and the lack of unique external characteristics in individual species. Reliable differences found among species are in the morphology of the male reproductive organs and the shells (Table 2), but these differences are not always consistent. For instance, shell morphology is variable in some species (i.e. D. dolabrifera, D. ascifera, D. nicaraguana) and there is a certain degree of overlap among species, for example some shells of D. dolabrifera (Fig. 5M, N) are similar to shells of Dolabrifera edmundsi (Fig. 16). Regarding the penial morphology, there is also some overlap, for example between D. ascifera and D. virens and between D. edmundsi and D. brazieri. The lack of reliable morphological traits for species identification and characterization is particularly problematic in the Atlantic Ocean where three species co-occur. Based on the information collected in this study, species of Dolabrifera appear to be completely cryptic externally and only molecular data, and to some extent internal morphological data, can be used to reliably identify specimens. Table 2. Summary of the principal morphological characteristics that differentiate species of Dolabrifera Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  View Large Table 2. Summary of the principal morphological characteristics that differentiate species of Dolabrifera Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  View Large Biogeography Dolabrifera displays an unusual biogeographical pattern, with the lowest species diversity in the Indo-Pacific tropics and the highest species diversity in the Atlantic region. A single species, D. dolabrifera, occurs from the Hawaiian Islands to the eastern Indian Ocean, the longest stretch of uninterrupted tropical ocean in the planet, which contains the highest species diversity for most groups of marine organisms (Briggs, 1999). In addition, the phylogenetic analyses recovered no geographical structure and very short branches in D. dolabrifera, suggesting this species is probably panmictic across its range. Further research using population genetics approaches is necessary to verify this hypothesis, but it is clear that D. dolabrifera must have a great dispersal potential. Contrary to expectations, the Atlantic Ocean, a much smaller body of water with comparatively lower levels of marine biodiversity, contains three species of Dolabrifera, and all three overlap in range in the Caribbean region. Although D. edmundsi is primarily found in the eastern Atlantic, several specimens collected in deep water from the Gulf of Mexico share the same penial morphology as other material here examined; one of them was successfully sequenced confirming that it belongs to D. edmundsi. The other two Atlantic species, D. virens and D. ascifera, are sympatric in the Caribbean region, and specimens collected in the same localities in Jamaica and Panama were confirmed to belong to these two species. Also, D. virens and D. edmundsi are sympatric in St. Helena. The patterns of cladogenesis and speciation that produced this unique pattern are unclear. Further research on other groups of heterobranch sea slugs and other marine invertebrates is necessary to elucidate the unusual biogeographical pattern and complex evolutionary history of species of Dolabrifera. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Individual Bayesian consensus trees of specimens of Dolabrifera + outgroup taxa for each gene analysed individually (16S, COI, H3), including posterior probabilities (above nodes) and bootstrap support values from the maximum likelihood analysis (below nodes). Figure S2. Illustrations of specimens and/or shells in the original descriptions of various species of Dolabrifera. A–C, Dolabrifera jacksoniensis by Pilsbry (1896); D–F, Aplysia ascifera by Rang (1828); G–J, Dolabrifera virens by Verrill (1901); K, L, Dolabrifera sowerbyi by Guilding inReeve (1868); M, N, Dolabrifera swiftii by Pilsbry (1896); O–Q, Dolabrifera nicaraguana by Pilsbry (1896); R, Dolabrifera pelsartensis by O’Donoghue (1924); S–U, Dolabrifera holboelli by Bergh (1872); V–W, Dolabrifera triangularis by Watson (1884). Figure S3. Illustrations of specimens and/or shells in the original descriptions of species of Dolabrifera from the tropical Indo-Pacific region (excluding temperate South Africa and Australia). A–D, Aplysia dolabrifera by Rang (1828); E–H, Aplysia oahouensis by Eydoux & Souleyet (1846–49); I–J, Dolabrifera cuvieri by H. Adams & A. Adams (1854); K, L, Dolabrifera maillardi by Deshayes (1863); M, N, Dolabrifera fusca by Pease (1868); O, P, Dolabrifera tahitensis by Pease (1868); Q, R, Dolabrifera vitraea by G. B. Sowerby II inReeve (1868); S, Dolabrifera pacifica by Pease in Reeve (1868); T–U, Dolabrifera marmorea by Pease in Reeve (1868). [Version of Record, published online 29 December 2017; http://zoobank.org/urn:lsid:zoobank.org:pub:1BDCBB96-B722-4095-9D6C-1E418A2D961E] ACKNOWLEDGEMENTS Several individuals provided access to museum specimens, photographs and data: Lindsey Groves (LACM), Janet Waterhouse (AM), Mandy Reid (AM), Wilma Blom (AM), Elizabeth Kools (CASIZ), Andreia Salvador (NHMUK), Juliana Bahia (ZSM), Kevin Webb (NHMUK), Edna Naranjo-García (CNMO), Paul Callomon (ANSP), Rachele Trevisi (ZSM) and Eric Lazo-Wasem (YPM). Judith Brown (Ascension Government) and Peter Wirtz made available important specimens from St. Helena and Madeira, respectively. Gary Cobb provided a photograph of a live animal of Dolabrifera brazieri. The SEM was conducted at the California State Polytechnic University SEM Laboratory supported by the US National Science Foundation (NSF) grant DMR-1429674. 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Molecular and morphological systematics of Dolabrifera Gray, 1847 (Mollusca: Gastropoda: Heterobranchia: Aplysiomorpha)

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Abstract

Abstract Molecular and morphological data from newly collected specimens and a review of the literature and type material indicate that the widespread tropical sea hare Dolabrifera dolabrifera is a species complex of five genetically distinct taxa. The name Dolabrifera dolabrifera is retained for a widespread species in the Indo-Pacific tropics. Dolabrifera nicaraguana is endemic to the eastern Pacific. Dolabrifera ascifera, D. virens and a new species described herein are restricted to the tropical Atlantic, with partially overlapping ranges in the Caribbean region and St. Helena. The temperate Pacific species Dolabrifera brazieri is also distinct and endemic to temperate southeastern Australia and New Zealand. These species of Dolabrifera constitute highly divergent lineages and most contain unique internal anatomical characteristics, particularly in the male reproductive morphology and shell shape, making them relatively easy to identify upon dissection. However, externally all these species are extremely variable in colour pattern and morphology and are virtually indistinguishable. This is particularly problematic for identification in the Atlantic Ocean where three species co-occur in the Caribbean region. new species, phylogenetics, species delimitation, taxonomy INTRODUCTION Dolabrifera dolabrifera (Rang, 1828) has been considered a pantropical species of sea hare (Aplysiidae), typically found under rocks in the upper intertidal zone (Kay, 1964; Bebbington, 1974) and on mudflat pools (Bebbington, 1977). Numerous species names have been introduced for specimens from different geographical locations over the years, but nearly all of them have been synonymized with D. dolabrifera (Engel & Hummelinck, 1936; Eales, 1944). Dolabrifera dolabrifera displays a considerable range of variation in colour, external morphology and anatomy (Marcus, 1972), which suggests it could constitute a species complex. Recent molecular studies have shown that other tropical and subtropical widespread heterobranch sea slugs resulted to be species complexes with marked geographical structure (Ornelas-Gatdula et al., 2012; Alexander & Valdés, 2013; Krug et al., 2013; Carmona et al., 2014a, b; Goodheart et al., 2015), and D. dolabrifera could be a similar case. Adding to the taxonomy complexity in Dolabrifera, the closely related temperate Australian species, Dolabrifera brazieri G. B. Sowerby II, 1870, is considered distinct by some authors (Klussman-Kolb, 2004; Burn, 2006), but the relationships between the two species is unclear. Specimens of Dolabrifera contain secondary metabolites with potential medical applications. For example, the propionate-derived compound Dolabriferol with antibiotic properties (Kigoshi & Kita, 2015) was originally characterized from specimens collected in the Caribbean region (Ciavatta et al., 1996). Subsequent studies on other Caribbean animals revealed the presence of chemically related, but distinct compounds (Jiménez-Romero, González & Rodríguez, 2012). Further research on specimens from the Eastern Pacific resulted in the isolation of 5α,8α-epidioxycholest-6-en-3β-ol, a compound with antileishmanial properties (Clark et al., 2013). This chemical diversity may be suggestive of a more complex taxonomy than previously recognized. The recent discovery of cryptic species complexes in other biomedically important groups of sea slugs (Krug et al., 2013; Lindsay & Valdés, 2016) highlights the need for modern approaches to investigate the biodiversity of such groups. In this article, we examine the systematics of Dolabrifera dolabrifera and D. brazieri using a combination of molecular and anatomical data to determine the taxonomic status of these two nominal species and unveil possible cryptic diversity in this group. MATERIAL AND METHODS Source of specimens Specimens were collected from localities covering mostly the known range of Dolabrifera (Fig. 1), mainly during low tides or snorkelling, under rocks. Specimens were photographed alive, preserved in 90–99% ethanol and deposited at the California State Polytechnic University Invertebrate Collection (CPIC), the University Museum of Bergen (ZMBN), the Zoologische Staatssammlung München (ZSM) and the Colección Nacional de Moluscos, Instituto de Biología, Universidad Nacional Autónoma de México (CNMO). Additional specimens, photographs and/or tissue samples were obtained from museum collections, including the California Academy of Sciences, Invertebrate Zoology (CASIZ), the Natural History Museum of Los Angeles County (LACM), the Natural History Museum, London (NHMUK), the Australian Museum (AM), the Academy of Natural Sciences of Drexel University, Philadelphia (ANSP) and the Yale Peabody Museum of Natural History (YPM). Figure 1. View largeDownload slide Distribution of Dolabrifera dolabrifera. A, map indicating the approximate location of the type localities of all nominal species synonymized with D. dolabrifera with their geographical region colour coded. B, localities of specimens examined morphologically and/or sequenced in this study. An asterisk denotes localities for which only GenBank sequence data were available. Localities in close proximity within countries are merged into single points. Localities are colour coded by biogeographical region. Figure 1. View largeDownload slide Distribution of Dolabrifera dolabrifera. A, map indicating the approximate location of the type localities of all nominal species synonymized with D. dolabrifera with their geographical region colour coded. B, localities of specimens examined morphologically and/or sequenced in this study. An asterisk denotes localities for which only GenBank sequence data were available. Localities in close proximity within countries are merged into single points. Localities are colour coded by biogeographical region. Morphology At least three specimens (if available) of each species identified with molecular data were examined morphologically. Based on the studies by Eales (1944) and Bebbington (1974, 1977), diagnostic traits for species in Dolabrifera include the radula, jaws, shell, gizzard plates and male reproductive anatomy. These structures/organs were used here as the main anatomical traits for species identification and characterization. The anterior portion of the digestive system was removed via a dorsal incision on the anterior end of the body. The buccal mass, oesophagus and gizzard were observed under a Nikon SMZ100 dissecting microscope and drawn with the aid of a camera lucida. Subsequently, these organs were placed in a sodium hydroxide 10% solution until the surrounding tissues were softened. The radulae, jaws and post-radular armature were removed from the partially dissolved tissue, rinsed in distilled water, mounted on a stub and sputter coated for examination under a Jeol JSM-6010 variable pressure scanning electron microscope (SEM). The gizzard plates were also removed from the partially dissolved tissue, rinsed in distilled water, transferred to ethanol 99% for 3 min and soaked in 2 mL of hexamethyldisilazane (HMDS) until all the liquid evaporated; then, they were mounted on a stub and coated for SEM examination. The male reproductive organs were dissected through the same incision used to extract the digestive organs, once the buccal mass was removed. The male reproductive organs were submerged in ethanol 70% and photographed under a Leica EZ4D microscope with a built-in digital camera; subsequently, the penial sheath of each specimen was carefully opened to expose the penial papilla, transferred to ethanol 99% for 3 min, soaked in 2 mL of HMDS until all the liquid evaporated and mounted for sputter coating and SEM examination. The shells along with the surrounding tissue were dissected from the posterior end of the animal and completely submerged in sodium hydroxide 10% until the shells were easily detachable from the tissue. The shells were rinsed in distilled water, photographed dorsally and ventrally under a Leica EZ4D microscope, mounted on a stub and sputter coated for examination under the SEM. DNA extraction, amplification and sequencing A total of 63 specimens of Dolabrifera were sequenced for this study (Table 1). Specimens were collected from localities spanning most of the range of all nominal species of Dolabrifera. GenBank sequences of two additional specimens of Dolabrifera and two outgroup taxa were also included in the analyses (Table 1). DNA extractions were performed using a hot Chelex extraction protocol with minor modifications and c. 1–3 mg of foot tissue. Tissue samples were placed into 1.7-mL tubes containing 1.0-mL TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 7.8) and allowed to incubate at room temperature on a rotator overnight to rehydrate the tissue and allow cells to begin dissociating. Samples were then vortexed and centrifuged for 3 min at 23897 × g. Then, 975 µL of the original 1 mL of TE buffer in each tube was carefully removed without disturbing the pellet of tissue. The Chelex solution was then added (175 µL), and samples were heated in a 56 °C water bath for 20 min, then immediately placed in a 100 °C heating block for 8 min. The supernatant was the final product used for polymerase chain reaction (PCR). Table 1. Specimens sequenced for this study, including museum voucher number, isolate number, collection locality and date and GenBank accession numbers Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  For countries with coastlines in two ocean basins, the ocean in which the specimens were collected is indicated. Museum abbreviations: AM, Australian Museum; CASIZ, California Academy of Sciences, Invertebrate Zoology; CNMO, Colección Nacional de Moluscos, Universidad Nacional Autónoma de México; CPIC, California State Polytechnic University Invertebrate Collection; LACM, Natural History Museum of Los Angeles County Malacology Collection; MZUCT, Museo de Zoología, Universidad de Costa Rica; ZSM, Zoologische Staatssammlung München; ZMBN, University Museum of Bergen Systematic Invertebrate Collection. Other abbreviations: ‘*’, outgroup taxa; ‘–’, missing data; ‘#’, tissue sample only; Pac, Pacific Ocean; Car, Caribbean Sea. View Large Table 1. Specimens sequenced for this study, including museum voucher number, isolate number, collection locality and date and GenBank accession numbers Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  Species  Voucher number  Isolate  Locality  Date  GenBank Accession Numbers  COI  16S  H3  Akera bullata  –  –  –  –  AF156143  AF156127  EF133474  Dolabella auricularia*  CPIC 00327  JS157  Maui, Hawaiian Island  15 June 2011  MF669619  MF669573  MF669657  D. nicaraguana  CPIC 00194  JS015  Tumbes, Peru  –  –  MF669574  MF669658  D. nicaraguana  ZSM Mol 20100745a  VP012  Tumbes, Peru  11 April 2005  MF669620  MF669575  MF669659  D. nicaraguana  ZSM Mol 20100745b  VP013  Tumbes, Peru  12 April 2005  MF669621  MF669576  MF669660  D. nicaraguana  ZSM Mol 20100745c  VP014  Tumbes, Peru  13 April 2005  MF669622  MF669577  MF669661  D. nicaraguana  ZSM Mol 20100745d  VP015  Tumbes, Peru  14 April 2005  –  MF669578  MF669662  D. nicaraguana  ZSM Mol 20100745e  VP016  Tumbes, Peru  15 April 2005  MF669623  MF669579  –  D. nicaraguana  ZSM Mol 20100745f  VP017  Tumbes, Peru  2 July 2006  –  MF669580  MF669663  D. nicaraguana  #  EB053  Naos Island, Panama (Pac)  22 June 2015  MF669624  MF669581  MF669664  D. nicaraguana  #  EB054  Naos Island, Panama (Pac)  22 June 2015  MF669625  MF669582  MF669665  D. nicaraguana  #  EB055  Naos Island, Panama (Pac)  22 June 2015  MF669626  MF669583  MF669666  D. nicaraguana  –  YC001  Guanacaste, Costa Rica (Pac)  13 May 2005  MF669627  –  MF669667  D. nicaraguana  –  YC002  Bahía Ballena, Costa Rica (Pac)  5 January 2002  MF669628  –  MF669668  D. nicaraguana  MZUCR 6349  YC003  Isla del Caño, Costa Rica (Pac)  30 April 2006  MF669629  –  MF669669  D. nicaraguana  MZUCR 6208a  YC004  Isla de Coiba, Panama (Pac)  17 March 2006  MF669630  –  MF669670  D. nicaraguana  MZUCR 6208b  YC005  Isla de Coiba, Panama (Pac)  17 March 2006  MF669631  –  MF669671  D. edmundsi sp. nov.  ZSM Mol 20160193  VP001  Porto Moniz, Madeira  September 2014  MF669632  MF669584  MF669672  D. edmundsi sp. nov.  ZSM Mol 20160194  VP002  Porto Moniz, Madeira  September 2014  MF669633  MF669585  MF669673  D. edmundsi sp. nov.  ZSM Mol 20160195  VP003  Porto Moniz, Madeira  September 2014  –  MF669586  MF669674  D. edmundsi sp. nov.  ZSM Mol 20160196  VP004  Porto Moniz, Madeira  September 2014  MF669634  MF669587  MF669675  D. edmundsi sp. nov.  ZSM Mol 20160197  VP005  Porto Moniz, Madeira  September 2014  MF669635  MF669588  MF669676  D. edmundsi sp. nov.  CASIZ 179395  EB096  Principe Island, West Africa  20 January 2009  –  MF669589  MF669677  D. edmundsi sp. nov.  –  –  Cape Verde  –  AY345021  AY345021  –  D. edmundsi sp. nov.  ZSM Mol 20160204  MB09  St. Helena  25 January 2014  –  MF669590  –  D. edmundsi sp. nov.  ZSM Mol 20160203  MB10  St. Helena  25 January 2014  –  MF669591  –  D. edmundsi sp. nov.  LACM 179615  JS196  Louisiana  11 August 2011  –  –  MF669678  D. dolabrifera  –  –  Guam  –  AF156149  AF156133  –  D. dolabrifera  CPIC 00413  JS164  Guam  16 June 2009  –  –  MF669679  D. dolabrifera  ZSM Mol 20170608  VP006  Marsa Alam, Egypt  19 June 2014  –  –  MF669680  D. dolabrifera  ZSM Mol 20170609  VP007  Marsa Alam, Egypt  19 June 2014  –  MF669592  MF669681  D. dolabrifera  ZSM Mol 20170610  VP008  Marsa Alam, Egypt  19 June 2014  –  –  MF669682  D. dolabrifera  CPIC 01130  XV012  Kona, Hawaii, Hawaiian Island  26 July 2014  MF669636  MF669593  MF669683  D. dolabrifera  CPIC 01132  XV013  Hilo, Hawaii, Hawaiian Island  25 July 2014  –  MF669594  MF669684  D. dolabrifera  CPIC 00304  JS159  Maui, Hawaiian Island  13 June 2011  MF669637  MF669595  MF669685  D. dolabrifera  CPIC 00335  JS160  Maui, Hawaiian Island  16 June 2011  MF669638  MF669596  MF669686  D. dolabrifera  CPIC 00326  JS161  Maui, Hawaiian Island  15 June 2011  –  MF669597  MF669687  D. dolabrifera  ZSM Mol 20060207  VP011  Upolu Island, Samoa  13 August 2005  MF669639  –  –  D. dolabrifera  ZSM Mol 20061638  VP019  Laucala Bay, Fiji  29 August 2006  MF669640  MF669598  –  D. dolabrifera  ZSM Mol 20061880  VP020  Laucala Bay, Fiji  29 August 2006  –  MF669599  –  D. dolabrifera  CASIZ 158347  YC006  Anilao, Luzon Island, Philippines  6 May 2001  MF669641  –  MF669688  D. dolabrifera  CASIZ 171457  YC007  Panglao Island, Philippines  7 July 2004  –  –  MF669689  D. dolabrifera  CASIZ 176451  YC008  Panglao Island, Philippines  15 June 2004  MF669642  –  MF669690  D. dolabrifera  CASIZ 191308  EB094  Madang, Papua New Guinea  18 November 2012  MF669643  MF669600  MF669691  D. dolabrifera  CASIZ 191308  EB095  Madang, Papua New Guinea  18 November 2012  MF669644  MF669601  MF669692  D. dolabrifera  CASIZ 179129  YC009  Espiritu Santo Island, Vanuatu  3 October 2006  MF669645  –  MF669693  D. dolabrifera  CASIZ 176800  YC010  Espiritu Santo Island, Vanuatu  26 September 2006  MF669646  –  MF669694  D. dolabrifera  CASIZ 194426  EB097  Plage Monseigneur, Madagascar  15 May 2010  MF669647  MF669602  MF669695  D. dolabrifera  CASIZ 194428  EB098  Plage Monseigneur, Madagascar  15 May 2010  MF669648  MF669603  –  D. dolabrifera  CASIZ 194428  EB099  Plage Monseigneur, Madagascar  15 May 2010  –  MF669604  MF669696  D. dolabrifera  CASIZ 194443  EB100  Platier Flacourt, Madagascar  13 May 2010  MF669649  MF669605  MF669697  D. dolabrifera  CASIZ 194443  EB101  Platier Flacourt, Madagascar  13 May 2010  MF669650  MF669606  MF669698  D. ascifera  ZMBN 84907.1  TMM2  Isla Tortuga, Venezuela  18 March 2010  MF669651  MF669607  MF669699  D. ascifera  ZMBN 84907.2  TMM3  Isla Tortuga, Venezuela  18 March 2010  MF669652  MF669608  MF669700  D. ascifera  ZMBN 84918  TMM4  Isla Tortuga, Venezuela  16 March 2010  MF669653  MF669609  MF669701  D. ascifera  CNMO 6611  XV008  Veracruz, Mexico (Atl)  27 June 14  –  MF669610  MF669702  D. ascifera  LACM 173238  YC011  Urchin Cove, Jamaica  24 May 2006  –  –  MF669703  D. virens  ZSM Mol 20170611  VP010  Bocas del Toro, Panama (Car)  15 July 2014  –  MF669611  MF669704  D. virens  LACM 173265  JS046  Urchin Cove, Jamaica  25 May 2006  –  –  MF669705  D. virens  ZSM Mol 20170625  VP009  St. Helena  25 January 2014  –  MF669612  MF669706  D. virens  ZSM Mol 20160201  MB03  St. Helena  25 January 2014  MF669654  MF669613  –  D. virens  ZSM Mol 20160200  MB04  St. Helena  25 January 2014  MF669655  MF669614  –  D. virens  ZSM Mol 20160198  MB05  St. Helena  25 January 2014  –  MF669615  –  D. virens  ZSM Mol 20160199  MB06  St. Helena  25 January 2014  MF669656  –  MF669707  D. virens  ZSM Mol 20160202  MB07  St. Helena  25 January 2014  –  MF669616  –  D. brazieri  AM C.477722  EB118  Raoul Island, New Zealand  15 May 2011  –  MF669617  MF669708  D. brazieri  AM C.477723  EB119  Raoul Island, New Zealand  15 May 2011  –  MF669618  MF669709  For countries with coastlines in two ocean basins, the ocean in which the specimens were collected is indicated. Museum abbreviations: AM, Australian Museum; CASIZ, California Academy of Sciences, Invertebrate Zoology; CNMO, Colección Nacional de Moluscos, Universidad Nacional Autónoma de México; CPIC, California State Polytechnic University Invertebrate Collection; LACM, Natural History Museum of Los Angeles County Malacology Collection; MZUCT, Museo de Zoología, Universidad de Costa Rica; ZSM, Zoologische Staatssammlung München; ZMBN, University Museum of Bergen Systematic Invertebrate Collection. Other abbreviations: ‘*’, outgroup taxa; ‘–’, missing data; ‘#’, tissue sample only; Pac, Pacific Ocean; Car, Caribbean Sea. View Large PCR was used to amplify portions of two mitochondrial genes, cytochrome c oxidase 1 (COI) and16S ribosomal RNA (16S), as well as the nuclear gene histone H3 (H3). The following universal primers were used to amplify the regions of interest for all three genes: COI (LCO1490 5′-GGTCAACAAATCATAAAGATATTGG-3′, HCO2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ developed by Folmer et al., 1994), 16S rRNA (16S ar-L 5′-CGCCTGTTTATCAAAAACAT-3′, 16S br-H 5′-CCGGTCTGAACTCAGATCACGT-3′ developed by Palumbi, 1996) and H3 (H3 AF 5′-ATGGCTCGTACCAAGCAGACGGC-3′, H3 AR 5′-ATATCCTTGGGCATGATGGTGAC-3′ developed by Colgan, Ponder & Eggler, 2000). Agarose gel electrophoresis with ethidium bromide was used to detect the presence of DNA. Sequencing was outsourced to Source Bioscience Inc. (Santa Fe Springs, CA, USA). Sequences were assembled and edited using Geneious Pro R8 (Kearse et al., 2012). Phylogenetic analyses The species Akera bullata Müller, 1776 and Dolabella auricularia (Lightfoot, 1786) were selected as outgroups based on Medina & Walsh’s (2000) molecular phylogeny of the Aplysiomorpha (=Anaspidea), which placed Akera at the base of this group and Dolabella in the same clade as Dolabrifera, sister to Aplysia. Phylogenetic analyses were conducted for all genes concatenated and for each gene individually. The best-fit models of evolution (GTR + G for 16S, GTR + I for COI codon positions 1 + 2, HKY + I for COI codon position 3, HKY for H3 codon positions 1 + 2, HKY + G for H3 codon position 3 and GTR + I for the entire concatenated data set) were determined using the Akaike information criterion (Akaike, 1974) implemented in JModelTest 2.1.10 (Darriba et al., 2012). Bayesian analyses were conducted using MrBayes 3.2.6 (Ronquist et al., 2012), partitioned by gene and codon position (unlinked). The Markov chain Monte Carlo analysis was run with two runs of six chains for 50 million generations, with sampling every 1000 generations. Convergence was assessed with Tracer 1.6 (Rambaut & Drummond, 2013). A 25% burn-in was applied before constructing the majority-rule consensus tree. Maximum likelihood analyses were conducted for the entire concatenated alignment with raxmlGUI 1.0 (Silvestro & Michalak, 2012) using the bootstrap + consensus option (10000 replicates) and the GTR + I model. Species delimitation analysis To provide evidence supporting species status for clades recovered in the phylogenetic analyses, a species delimitation analysis based on the barcode gap was performed with ABGD (Puillandre et al., 2012) using the COI sequence alignment. The analysis was run twice using Kimura 2-parameter and Tamura-Nei distance matrices generated in MEGA 7.0.16 (Kumar, Stecher & Tamura, 2016). The matrices were uploaded into the online ABGD web tool (http://wwwabi.snv. jussieu.fr/public/abgd/abgdweb.html) and analysed. The default relative gap width (x) of 1.5 and a range of prior values for maximum divergence of intraspecific diversity (P) from 0.001 to 0.1 were implemented. RESULTS Bayesian and maximum likelihood analyses of the three concatenated genes (16S + COI + H3) produced trees with the same overall topology (Fig. 2). In both trees, Dolabrifera is monophyletic and well supported (posterior probability [PP] = 1; Bootstrap support [BS] = 97). Although the base of the Dolabrifera clade was recovered as an unresolved polytomy (Fig. 2), this group contains six distinct, well-supported clades. The geographical composition of clades and support values are as follows: (1) specimens from the Eastern Pacific Ocean (PP = 1; BS = 100); (2) specimens from the Eastern Atlantic Ocean, the South Atlantic and the Gulf of Mexico (PP = 0.99; BS = 91); (3) specimens from the tropical Indo-Pacific – from Madagascar to the Hawaiian Islands (PP = 0.99; BS = 92); (4) specimens from the Gulf of Mexico and Caribbean – Mexico, Jamaica and Venezuela (PP = 0.9; BS = 97); (5) specimens from temperate New Zealand – originally identified as D. brazieri (PP = 1; BS = 100) and (6) specimens from the Caribbean – Panama and Jamaica – as well as from the South Atlantic Ocean – St. Helena (PP = 1; BS = 98). Clades 4 and 5 are sisters (PP = 1; BS = 84), but this is the only sister relationship recovered among Dolabrifera clades. Individual gene trees also generally agree with the consensus trees with some differences (Supporting Information, Fig. S1). The main differences are the lack of support for the D. dolabrifera clade in the 16S analyses and for one of the Atlantic clades in the H3 analyses. Figure 2. View largeDownload slide Bayesian consensus tree (16S + COI + H3) for 65 specimens of Dolabrifera + outgroup taxa, including posterior probabilities (above nodes) and bootstrap support values from the maximum likelihood analysis (below nodes). For each specimen, the geographical origin and isolate number are indicated (see Table 1). The general geographical region of origin for all specimens is colour coded as in Figure 1. Figure 2. View largeDownload slide Bayesian consensus tree (16S + COI + H3) for 65 specimens of Dolabrifera + outgroup taxa, including posterior probabilities (above nodes) and bootstrap support values from the maximum likelihood analysis (below nodes). For each specimen, the geographical origin and isolate number are indicated (see Table 1). The general geographical region of origin for all specimens is colour coded as in Figure 1. The species delimitation analysis confirmed that these clades constitute distinct candidate species, as they were recovered in different groups in ABGD. Examination of external and internal anatomical traits also revealed the existence of consistent differences between the candidate species recovered with molecular data, thus becoming confirmed candidate species (Vieites et al., 2009). The taxonomy, nomenclatural history (if any) and diagnostic traits for all species are described and discussed in the Systematics section. Also, a formal description is provided for a confirmed candidate species that was unnamed. SYSTEMATICS Genus dolabrifera Gray, 1847 Dolabrifera Gray, 1847: 162. Type species: Dolabrifera dolabrifera (Rang, 1828), by original designation. Diagnosis Body oval to elongate, wider posteriorly, narrowing gradually towards head. Oral tentacles, rhinophores slit, flattened, wider distally. Oral tentacles emerging anteriorly from the head, clearly differentiated. Rhinophores dorsal, typically smooth. Parapodial lobes reduced, located behind body midline, fused posteriorly, overlapping partially in middle. Dorsum typically covered with tubercles of different sizes. Shell solid, calcified, reduced, flat, elongate, subtriangular, wider posteriorly, tapering anteriorly into a conspicuous nucleus. Jaws reduced to small section of the labial cuticle with simple denticles. Radula with large, subtriangular rachidian teeth, with several denticles on each side of the cusp; lateral teeth with long, coarsely denticulate cusps. The posterior end of buccal bulb, at insertion of the oesophagus, with two elongate structures (post-radular armature), of numerous elongate denticles. Gizzard muscular, with numerous irregular plates. Male reproductive system with distinct penial canal and penial sheath, where penial papilla is typically located. Remarks Since its original introduction by Gray (1847), several nominal species have been described in or transferred to Dolabrifera, many of them based on shell material or preserved specimens. Nearly all of those species were confirmed to be members of Dolabrifera, but later synonymized with D. dolabrifera (see Engel & Hummelinck, 1936; Eales, 1944) or D. brazieri (see Hedley, 1917). However, the taxonomic assignment of two of those species is questionable. The first one, Dolabrifera holboelli Bergh, 1872, was described from a single specimen collected in ‘Mare grönland’ (=Greenland Sea). Bergh (1872: pl. 5, figs 1–24) provided illustrations of the preserved animal, shell, radula, jaw, gill and other anatomical details (some of them reproduced in Supporting Information, Fig. S2S–U). All descriptions are consistent with the characteristics of a member of Dolabrifera, and examination of the holotype (ZMUC GAS-2163) confirms the generic placement of this species in Dolabrifera. However, there are no confirmed reports of Dolabrifera from Greenland, nor from other temperate or cold-water regions in the North Atlantic; thus, the most likely explanation for this record is that the specimen was mislabelled. Based on the radular and shell morphology, D. holboelli resembles the tropical Indo-Pacific species D. dolabrifera because the lateral teeth have bifurcated apices with two equally large denticles, and the shell widens abruptly near the apical end, but none of these traits are conclusive. In the absence of molecular data or information on the geographical origin of the holotype, D. holboelli is here treated as an uncertain species. The second problematic species, Dolabrifera pelsartensis O’Donoghue, 1924, was described from ‘Pelsart Island’ (Pelsaert Island), Western Australia, based on two preserved specimens. O’Donoghue (1924: pl. 27, fig. 3, pl. 29, figs 26–28) illustrated a preserved animal (Supporting Information, Fig. S2R) and a radula, which shows mid-lateral teeth with two to three denticles and simple cusps. No illustrations of the shell or other anatomical features were provided. With the available information, it is difficult to compare D. pelsartensis with the species here described. The radular morphology is most similar to that of D. brazieri, which appears to be endemic to temperate Eastern Australia and New Zealand. Examination of material from Western Australia is necessary to clarify the status of this species. Another species, Dolabrifera triangularis Watson, 1884, has almost certainly been erroneously assigned to Dolabrifera. Watson (1884) described D. triangularis based on a shell collected from Simon’s Bay, South Africa. Although Watson (1884) described the shell in detail, provided no illustrations and the live animal is unknown. Years later, Watson (1886: 673–674, pl. 50, fig. 7; Supporting Information, Fig. S2V, W) redescribed and illustrated this shell, which is nearly triangular, short, with a large nucleus. The only remaining syntype (NHMUK 1887.2.9.2296) here examined, matches this description, but is much shorter and thicker that other shells of Dolabrifera spp. In addition, no specimens of Dolabrifera have been documented in False Bay and surrounding areas despite the comprehensive studies (Gosliner, 1987; Zsilavecz, 2007). Based on the available evidence, we cannot identify D. triangularis and therefore is here regarded as a nomen dubium. Dolabrifera dolabrifera (Rang, 1828) (Figs 3A–G, 4–6) ‘Dolabella dolabrifera’ Cuvier, 1817: 398. Type locality: ‘Isle-de-France’ (=Mauritius) (nomen nudum). ‘Dolabella dolabrifera’ de Férussac, 1822: table xxx (nomen nudum). Aplysia dolabrifera Rang, 1828: 51, pl. 4, figs 1–6. Type locality: ‘Ile Bourbon’ (=Réunion). Aplysia oahouensis Souleyet, 1852: 461–462, pl. 25, figs 10–13. Type locality: ‘Oahou’ (=O‘ahu, Hawaiian Islands). Dolabrifera cuvieri H. Adams & A. Adams, 1854: 33; 1858: pl. 59, figs 4, 4a. Unjustified emendation for Dolabella dolabrifera Cuvier, 1817. Dolabrifera olivaceaPease, 1860: 22–23. Type locality: Sandwich Islands (=Hawaiian Islands). Dolabrifera tahitensisPease, 1861: 245–246. Type locality: Tahiti. Dolabrifera maillardi Deshayes, 1863: 53–54, pl. 7, figs 20–22. Type locality: Réunion. Dolabrifera fusca Pease, 1868: 76, pl. 8, fig. 4. Type locality: Polynesia. Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868: pl. 1, fig. 1a, b. Type locality: ‘Narai’, Fiji. Dolabrifera pacifica Pease in Reeve, 1868: pl. 1, fig. 3. Type locality: Islands of the Pacific. Dolabrifera marmorea Pease in Reeve, 1868: pl. 1, fig. 5a, b. Type locality: Sandwich Islands (=Hawaiian Islands). Type material Aplysia dolabrifera Rang, 1828 not found at MNHN (Valdés & Héros, 1998). Aplysia oahouensis Souleyet, 1852, one syntype at MNHN (Valdés & Héros, 1998). Dolabrifera olivacea Pease, 1860, lectotype at NHMUK 1964376. Dolabrifera maillardi Deshayes, 1863, three syntypes at MNHN. Dolabrifera tahitensis Pease, 1861, type material untraceable, not at NHMUK. Dolabrifera fusca Pease, 1868, one syntype at ANSP 20710. Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868, two syntypes at NHMUK 1856.9.24.202. Dolabrifera marmorea Pease in Reeve, 1868, one syntype at NHMUK 20160139. Dolabrifera pacifica Pease in Reeve, 1868, two syntypes at NHMUK 20160140. Material examined Hekili Point, Maui, Hawaiian Islands, 1 m depth, 21 June 2016, one specimen 19 mm preserved length (CPIC 01654). Maliko Bay, Maui, Hawaiian Islands, 1 m depth, 22 June 2016, five specimens 5–14 mm preserved length (CPIC 01657). Mama’s Fish House Reef, Maui, Hawaiian Islands, 1 m depth, 25 June 2016, five specimens 9–12 mm preserved length (CPIC 01696); 27 June 2016, one specimen 4 mm preserved length (CPIC 01718); 28 June 2016, two specimens 2–3 mm preserved length (CPIC 01752). Sharks Cove, O‘ahu, Hawaiian Islands, 1 m depth, 5 July 2016, one specimen 10 mm preserved length (CPIC 01770). Kaloko, Kona, Hawai‘i, Hawaiian Islands, 26 July 2014, two specimens 16–19 mm preserved length, leg. S. Medrano (CPIC 01130). Onekahakaha, Hilo, Hawai‘i, Hawaiian Islands, 26 July 2014, two specimens 12–20 mm preserved length, leg. S. Medrano (CPIC 01132). Merizo, Guam, intertidal, 16 June 2009, one specimen 12 mm preserved length, leg. E. Ornelas-Gatdula (CPIC 00413). In front of Bali Sol Hotel, Nusa Dua, Bali, Indonesia (8°48.0′S, 115°14.0′E), intertidal, 22 May 1986, two specimens 35–40 mm preserved length, leg. J. McLean (LACM 1986-164.16). Cement Mixer Reef, Madang, Papua New Guinea, 4 December 2012, one specimen 14 mm preserved length, leg. J. Goodheart (CPIC 00820). Rempi, Madang, Papua New Guinea, 21 November 2012, one specimen 10 mm preserved length, leg. Papua New Guinea Biodiversity Expedition 2012 (CASIZ 191380). Kranket Island, Madang, Papua New Guinea (5°11′30.9″S, 145°49′6.7″E), 6 November 2012, one specimen 6 mm preserved length, leg. Papua New Guinea Biodiversity Expedition 2012 (CASIZ 191000). Platier Flacourt, Taolagnaro, Madagascar (25°1.7′S, 47°0.1′E), 13 May 2010, two specimens 24–25 mm preserved length, leg. Atimo Vatae Expedition (CASIZ 194443). Plage Monseigneur, Taolagnaro, Madagascar (25°2.1′S, 46°59.9′E), 15 May 2010, two specimens 18–26 preserved length, leg. Atimo Vatae Expedition (CASIZ 194428). Shib Ammar, Saudi Arabia (19°34.232′N, 40°0.519′E), 20 m depth, 3 March 2013, tissue sample, leg. A. Anker (CASIZ 192302). Description External morphology Body up to 100 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with small, rounded, scattered tubercles, each with retractile, highly ramified, papilla on apex; tubercles typically tightly arranged, giving animal bumpy (Fig. 3A, G) or rough appearance (Fig. 3C–F), in some specimens surrounded by large areas of smooth tissue (Fig. 3B). Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. Figure 3. View largeDownload slide Live animals of the Pacific Ocean species of Dolabrifera. A–G, Dolabrifera dolabrifera (Rang, 1828). A, Guam (CPIC 00413); B, Anilao, Philippines (CASIZ 158347); C, Maui, Hawaiian Islands (CPIC 01654); D, Maui, Hawaiian Islands (CPIC 01696); E, Maui, Hawaiian Islands (CPIC 01718); F, O‘ahu, Hawaiian Islands (CPIC 01770); G, Marsa Alam, Egypt (ZSM Mol 20170610). H, Dolabrifera brazieri G. B. Sowerby II, 1870 (photograph: G. Cobb). I, J, Dolabrifera nicaraguana Pilsbry, 1896. I, Pacific coast of Panama (photograph: J. Goodheart); J. Puerto Vallarta, Mexico (CPIC 00547). Figure 3. View largeDownload slide Live animals of the Pacific Ocean species of Dolabrifera. A–G, Dolabrifera dolabrifera (Rang, 1828). A, Guam (CPIC 00413); B, Anilao, Philippines (CASIZ 158347); C, Maui, Hawaiian Islands (CPIC 01654); D, Maui, Hawaiian Islands (CPIC 01696); E, Maui, Hawaiian Islands (CPIC 01718); F, O‘ahu, Hawaiian Islands (CPIC 01770); G, Marsa Alam, Egypt (ZSM Mol 20170610). H, Dolabrifera brazieri G. B. Sowerby II, 1870 (photograph: G. Cobb). I, J, Dolabrifera nicaraguana Pilsbry, 1896. I, Pacific coast of Panama (photograph: J. Goodheart); J. Puerto Vallarta, Mexico (CPIC 00547). Colour Background colour variable, pink, purple, dark brown, or red, with spots and/or irregular patches of different colours. Mantle margin with same general colour as rest of body. Ventral side typically brown with numerous white, green or brown spots. Digestive system Radular formulae: 38 × 78.1.41 (CPIC 00336), 37 × 62.1.56 (CPIC 00336), 25 × 58.1.70 (LACM 1986-164.1), 28 × 56.1.46 (CPIC 00820); radulae typically asymmetrical, with more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 4A, D); teeth wider at base, both upper and lower ends of teeth convex. Lateral teeth elongate (Fig. 4B, E), with long bases; each cusp with basal, blunt, inner denticle, apex bifurcated into two blunt denticles equal in size. Outer teeth very elongate (Fig. 4C, F), with or without a secondary denticle, apex bifurcate or simple. Jaws with simple, irregular denticles (Fig. 4J). Two elongate structures (post-radular armature) located posterior to radula, at oesophagus insertion point, with numerous spines with elongate bases, triangular cusps (Fig. 4K). Gizzard plates variable in shape, typically irregular, with some striations more visible near centre (Fig. 4G–I). Figure 4. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera dolabrifera (Rang, 1828). A–C, radular teeth of a specimen from Maui, Hawaiian Islands (CPIC 00336). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bali, Indonesia (LACM 1986-164.16). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Maui, Hawaiian Islands (CPIC 00336). I, gizzard plate of a specimen from Madang, Papua New Guinea (CPIC 00820). J, jaw elements of a specimen from Madang, Papua New Guinea (CPIC 00820). K, post-radular armature of a specimen from Maui, Hawaiian Islands (CPIC 00336). L, M, dorsal and ventral views of the shell apex of a specimen from Madang, Papua New Guinea (CPIC 00820). N, O, dorsal and ventral views of the shell apex of a specimen from Bali, Indonesia (LACM 1986-164.16). Figure 4. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera dolabrifera (Rang, 1828). A–C, radular teeth of a specimen from Maui, Hawaiian Islands (CPIC 00336). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bali, Indonesia (LACM 1986-164.16). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Maui, Hawaiian Islands (CPIC 00336). I, gizzard plate of a specimen from Madang, Papua New Guinea (CPIC 00820). J, jaw elements of a specimen from Madang, Papua New Guinea (CPIC 00820). K, post-radular armature of a specimen from Maui, Hawaiian Islands (CPIC 00336). L, M, dorsal and ventral views of the shell apex of a specimen from Madang, Papua New Guinea (CPIC 00820). N, O, dorsal and ventral views of the shell apex of a specimen from Bali, Indonesia (LACM 1986-164.16). Shell Shell flat, well calcified, oval (Fig. 5); nucleus conspicuous, formed by single lobe, occasionally two (Fig. 4L–O). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, typically widening abruptly near apical end, but more gradually in some specimens. Widest portion of shell mid-length forward, generally close to nucleus. Sculpture of conspicuous growth lines. Figure 5. View largeDownload slide Shells of Dolabrifera dolabrifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from the Hawaiian Islands (CPIC 00336), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Guam (CPIC 00413), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Indonesia (LACM 1986-164.16), scale bar = 1 mm. G, H, dorsal and ventral view of a syntype of Dolabrifera marmorea Pease in Reeve, 1868 (NHMUK 20160139), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a syntype of Dolabrifera fusca Pease, 1868 (ANSP 20710), scale bar = 5 mm (photographs: ©ANSP). K, L, dorsal and ventral view of a syntype of Dolabrifera pacifica Pease in Reeve, 1868 (NHMUK 20160140), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). M, N, dorsal and ventral view of a specimen from Papua New Guinea (CPIC 00820), scale bar = 1 mm. O, P, dorsal and ventral view of the lectotype of Dolabrifera olivacea Pease, 1860 (NHMUK 1964376), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Q, R, two syntypes of Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868 mounted in dorsal and ventral positions on a plate (NHMUK 1856.9.24.202), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Figure 5. View largeDownload slide Shells of Dolabrifera dolabrifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from the Hawaiian Islands (CPIC 00336), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Guam (CPIC 00413), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Indonesia (LACM 1986-164.16), scale bar = 1 mm. G, H, dorsal and ventral view of a syntype of Dolabrifera marmorea Pease in Reeve, 1868 (NHMUK 20160139), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a syntype of Dolabrifera fusca Pease, 1868 (ANSP 20710), scale bar = 5 mm (photographs: ©ANSP). K, L, dorsal and ventral view of a syntype of Dolabrifera pacifica Pease in Reeve, 1868 (NHMUK 20160140), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). M, N, dorsal and ventral view of a specimen from Papua New Guinea (CPIC 00820), scale bar = 1 mm. O, P, dorsal and ventral view of the lectotype of Dolabrifera olivacea Pease, 1860 (NHMUK 1964376), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Q, R, two syntypes of Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868 mounted in dorsal and ventral positions on a plate (NHMUK 1856.9.24.202), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). Male reproductive system Penial canal large, muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 6C, D). Penial canal narrows and expands abruptly into oval penial sheath containing penial papilla. Oval section connects to large, strong retractor muscle connecting to ventral side of body wall internally. Penial papilla elongate, with curved cusp and longitudinal groove (Fig. 6A, B). Figure 6. View largeDownload slide Penial morphology of Dolabrifera dolabrifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bali, Indonesia (LACM 1986-164.16). B, SEM micrograph of a partially dissected penial papilla of a specimen from Maui, Hawaiian Is. (CPIC 01654). C, male copulatory organs of a specimen from Maui, Hawaiian Is. (CPIC 00336). D, male copulatory organs of a specimen from Madang, Papua New Guinea (CPIC 00820). E, male copulatory organs of a specimen from Guam (CPIC 00413). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Figure 6. View largeDownload slide Penial morphology of Dolabrifera dolabrifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bali, Indonesia (LACM 1986-164.16). B, SEM micrograph of a partially dissected penial papilla of a specimen from Maui, Hawaiian Is. (CPIC 01654). C, male copulatory organs of a specimen from Maui, Hawaiian Is. (CPIC 00336). D, male copulatory organs of a specimen from Madang, Papua New Guinea (CPIC 00820). E, male copulatory organs of a specimen from Guam (CPIC 00413). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Range Widespread in the tropical Indo-Pacific region, from East Africa to the Hawaiian Islands (see Remarks below). Remarks Extensive anatomical and sequence data obtained from specimens of Dolabrifera collected throughout the Indo-Pacific tropics, from the Hawaiian Islands to the Red Sea, revealed the presence of a single species in this vast biogeographical region, including samples from Saudi Arabia, Madagascar, Guam, Papua New Guinea, Indonesia, Fiji, Samoa and Hawai‘i. Only specimens from temperate regions in Australia and New Zealand are distinct and consequently assigned to a different species, D. brazieri (see below). Tropical Indo-Pacific specimens are characterized by having a large and muscular penial canal, lacking tubercles, and an oval penial sheath containing an elongate penial papilla with a curved cusp and a longitudinal groove. The radulae of Indo-Pacific specimens contain lateral teeth with the apex bifurcated into two blunt denticles equal in size. To determine the valid name for the tropical Indo-Pacific species and its synonyms, a review of the literature is discussed in the next paragraphs. Cuvier (1817) introduced for the first time the name ‘Dolabella dolabrifera’ based on a specimen collected in ‘Isle-de-France’ (=Mauritius). The name was mentioned in a footnote with no description and therefore is a nomen nudum. Subsequently, de Férussac (1822) mentioned the name ‘Dolabella dolabrifera’ and attributed it to Cuvier in a systematic list and classification of species of Mollusca, again with no description. Rang (1828) formally described and illustrated for the first time specimens assigned to Aplysia dolabrifera and collected from ‘Île Bourbon’ (=Réunion). The description included illustrations of the live animal and shell (Supporting Information, Fig. S3A–D), which are sufficient to corroborate they correspond to the modern usage of the name Dolabrifera. In this study, a single species of Dolabrifera has been detected among numerous samples from the Indo-Pacific region, ranging the Hawaiian Island to the Red Sea. Consequently, the name A. dolabrifera, which the oldest available name introduced from the Indo-Pacific region, is assigned to this species. Several decades later, Adams & Adams (1854) introduced the new species name Dolabrifera cuvieri for ‘A. dolabrifera Cuv’. The description consists of a short description of the genus; it was accompanied by a figure of a live animal reproduced from Rang’s (1828) description of A. dolabrifera (pl. 4, fig. 9) and the illustration of a shell, both published in Volume 3 of the same work (Adams & Adams, 1858: pl. 59, fig. 4, 4a). According to Pilsbry (1896: 119), D. cuvieri was ‘proposed as a substitute name for D. dolabrifera Cuv., evidently to avoid the duplication of names consequent upon the recognition of Dolabrifera as a genus’. Pilsbry (1896) indicated differences in the shell morphology between the ‘type of D. dolabrifera’ [referring to the specimen illustrated by Rang (1828: pl. 4, fig. 6)], which is elongate, and the more square shell illustrated by Adams & Adams (1858: pl. 59, fig. 4a). Based on the original description and subsequent comments by Pilsbry (1896), it is clear that Adams & Adams (1854) intended to intentionally change the original name of A. dolabrifera and therefore Dolabrifera cuvieri constitutes an unjustified emendation (ICZN, 1999: Article 33.2). Souleyet (1852) described Aplysia oahouensis from O‘ahu, Hawaiian Islands, with a short description and an illustration previously published by Eydoux & Souleyet (1846–1849). The illustration and description of the live animal and shell (Supporting Information, Fig. S3E–H) also correspond to the modern usage of Dolabrifera and fit within the variability of D. dolabrifera. Specimens of Dolabrifera from O‘ahu sequenced in this study are conspecific with specimens from the rest of the Indo-Pacific confirming that A. oahouensis is a synonym of D. dolabrifera. Deshayes (1863) introduced Dolabrifera maillardi based on a shell collected from Réunion. The illustrations of the shell in different views (Supporting Information, Fig. S3K–L) clearly represent a specimen of Dolabrifera. Again, due to the geographical origin of this shell, and its morphology, D. maillardi is here confirmed as a synonym of D. dolabrifera. Other similar nominal species described from various localities throughout Polynesia include Dolabrifera tahitensis Pease, 1861, type locality Tahiti, Dolabrifera fusca Pease, 1868, with no type locality specified (Pease, 1868), Dolabrifera vitraea G. B. Sowerby II in Reeve, 1868, type locality ‘Narai’, Fiji (Reeve, 1868), Dolabrifera pacifica Pease in Reeve, 1868, again with no type locality specified (Reeve, 1868) and Dolabrifera marmorea Pease in Reeve, 1868, type locality Sandwich Islands (=Hawaiian Islands). The original description of D. tahitensis consists of a short text, but illustrations of the live animal were later published by Pease (1868: pl. 8, fig. 5; Supporting Information, Fig. S3O–P) and clearly represent a species of Dolabrifera. The original description of D. fusca included drawings of the live animal (Pease, 1868: pl. 8, fig. 4; Supporting Information, Fig. S3M, N), which match the characteristics of D. dolabrifera. In addition, the shell morphology of a remaining syntype (Fig. 5I, J) is consistent with that of other specimens of D. dolabrifera here examined and are regarded as synonyms. The original descriptions of D. vitraea, D. pacifica and D. marmorea included only illustrations of a shell each (Reeve, 1868: pl. 1, fig. 1a, b; pl. 1, fig. 3; pl. 1, fig. 5a, b) all of them reprinted here (Supporting Information, Fig. S3Q–U); photographs of the syntypes (shells) of all three species (Fig. 5Q–R, K–L, G–H) match the illustrations in the original descriptions. As in the previous cases, because of the geographical origin of the specimens and the characteristics of the shells, D. tahitensis, D. vitraea, D. pacifica and D. marmorea are most likely synonyms of D. dolabrifera and are here considered as such. Dolabrifera brazieri G. B. Sowerby II, 1870 (Figs 3H, 7–9) Dolabrifera brazieri G. B. Sowerby II, 1870: 250. Type locality: Northhead, Botany Bay, Australia. ?Dolabrifera jacksoniensis Pilsbry, 1896: 120–121, pl. 44, figs 38–41. Type locality: Port Jackson, Australia. Type material Dolabrifera brazieri G. B. Sowerby II, 1870, possibly syntype at NHMUK 1877.5.12.91. Dolabrifera jacksoniensis Pilsbry, 1896, syntypes at ANSP 64931 (dry) and A7040 (wet). Material examined Lord Howe Island, New South Wales, Australia (31°32.94′S 159°03.72′E), 1887, six specimens, 32–45 mm preserved length (AM C.54298). Boat Cove, Raoul Island, Kermadec Islands, New Zealand (29°16.79898′S, 177°53.66268′W), 14 May 2011, 0.25 m depth, one specimen, 39 mm preserved length (AM C.475882). NW corner of North Meyer Island, Kermadec Islands, New Zealand (29°14.4984′S, 177°52.6734′W), 5–15 m depth, 13 May 2011, one specimen 45 mm preserved length (AM C.475783). Description External morphology Body up to 150 mm long, oval to elongate, flattened. Dorsum covered with large, blunt, rounded to semi-conical tubercles, each with retractile, ramified, papilla on apex; large tubercles typically surrounded by smaller rounded tubercles (Fig. 3H). Rhinophores enrolled, elongated. Oral tentacles wide, flattened, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, typically brown or greenish-brown, sometimes pale grey or nearly black, with irregular darker, lighter areas. Dorsum often covered with dark brown network of anastomosing lines surrounding tubercles, which do not penetrate mantle margin, rhinophores. Mantle margin typically green, with whitish spots. Digestive system Radular formulae: 33 × 92.1.80 (AM C.54298); radulae asymmetrical, with more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 7A); teeth wider at base, both upper and lower ends of teeth convex. Lateral teeth elongate (Fig. 7B), with long bases; each cusp with proximal and distal elongate denticle on inner side, apex blunt, simple. Outer teeth very elongate (Fig. 7C), with bifurcate apices. Jaws with simple, irregular denticles (Fig. 7F). Post-radular armature with numerous spines with elongate bases, triangular cusps (Fig. 7G). Gizzard plates variable in shape, typically irregular, with some striations more visible near the centre (Fig. 7D). Figure 7. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A–C, radular teeth. A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D, E, gizzard plates. F, jaw elements. G, post-radular armature. H, I, dorsal and ventral views of the shell apex. Figure 7. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A–C, radular teeth. A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D, E, gizzard plates. F, jaw elements. G, post-radular armature. H, I, dorsal and ventral views of the shell apex. Shell Shell flat, well calcified, oval (Fig. 8); nucleus conspicuous, formed by single lobe connected to rest of shell by narrow area (Fig. 7H, I). On apertural view with nucleus on upper end, left side of shell straight, sometimes slightly concave or convex. Right side of shell convex, narrower near nucleus, widening gradually near mid-length. Widest portion of shell mid-length or slightly forward. Sculpture of conspicuous growth lines. Figure 8. View largeDownload slide Shells of Dolabrifera brazieri G. B. Sowerby II, 1870. A, B, dorsal and ventral view of specimen from Lord Howe Island, Australia (AM C.54298), scale bar = 1 mm. C, D, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.67), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). E, F, dorsal and ventral view of a syntype of Dolabrifera jacksoniensis Pilsbry, 1896 (ANSP 64931), scale bar = 1 mm (photographs: ©ANSP). G, H, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.68), scale bar = 1 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a possible syntype of Dolabrifera brazieri G. B. Sowerby II, 1870 (NHMUK 1877.5.12.91), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of specimen from Raoul Island, New Zealand (AM A.475882), scale bar = 1 mm. Figure 8. View largeDownload slide Shells of Dolabrifera brazieri G. B. Sowerby II, 1870. A, B, dorsal and ventral view of specimen from Lord Howe Island, Australia (AM C.54298), scale bar = 1 mm. C, D, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.67), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). E, F, dorsal and ventral view of a syntype of Dolabrifera jacksoniensis Pilsbry, 1896 (ANSP 64931), scale bar = 1 mm (photographs: ©ANSP). G, H, dorsal and ventral view of specimen from Southern Australia (NHMUK 1880.12.11.68), scale bar = 1 mm (photographs: K. Webb, ©NHMUK). I, J, dorsal and ventral view of a possible syntype of Dolabrifera brazieri G. B. Sowerby II, 1870 (NHMUK 1877.5.12.91), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of specimen from Raoul Island, New Zealand (AM A.475882), scale bar = 1 mm. Male reproductive system Penial canal large, muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 9B, C). Penial canal narrows and expands abruptly into elongate penial sheath section containing penial papilla. This section ends in large, strong retractor muscle connecting to ventral side of body wall internally. Penial papilla elongate, with curved cusp and longitudinal groove (Fig. 9A). Figure 9. View largeDownload slide Penial morphology of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A, SEM micrograph of the penial papilla. B, C, male copulatory organs. Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Figure 9. View largeDownload slide Penial morphology of Dolabrifera brazieri G. B. Sowerby II, 1870, specimens from Lord Howe Island, Australia (AM C.54298). A, SEM micrograph of the penial papilla. B, C, male copulatory organs. Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle. Range Temperate regions in eastern Australia (mainly New South Wales, including Lord Howe Island) and northern New Zealand (Rudman, 2003), including the Kermadec Islands (Morley & Hayward, 2015). According to Morley & Hayward (2015), the presence of this species in New Zealand is the result of a human-mediated introduction. Remarks Dolabrifera brazieri was described by Sowerby (1870) with a short Latin description of the shell, based on two specimens (shells) collected in Northhead, Botany Bay, Australia. Sowerby (1870: 250) indicated that ‘No note seems to have been made of the character of the animal; but the shell, which is large, differs from that of other known species’. This species has been considered a synonym of D. dolabrifera by several authors (Eales, 1944; Bebbington, 1974, 1977), whereas others suggested that is an anatomically distinct, valid species, endemic to temperate regions in eastern Australia and New Zealand (Hedley, 1917; Willan & Morton, 1984; Rudman, 2003; Burn, 2006). Differences between D. brazieri and D. dolabrifera include the much larger size of D. brazieri (up to 150 mm long) and the presence of the large smooth conical tubercles all over the body (Rudman, 2003). In this study, we examined specimens identified as D. brazieri from Lord Howe Island, Australia and the Kermadec Islands, New Zealand, which are genetically distinct from D. dolabrifera collected in other areas of the Indo-Pacific region. These animals also differed anatomically from specimens of D. dolabrifera by the presence of a much more elongate penial sheath, lateral radular teeth with a proximal and distal elongate denticle and a simple, blunt apex and a shell with a very elongate apex with a conspicuous nucleus, formed by a single lobe, typically connected to the rest of the shell by a narrow area. Examination of a possible syntype of D. brazieri (NHMUK 1877.5.12.91) reveals that the shell has an unusually elongated apex with a wide, round nucleus (Fig. 8I, J), similar to the characteristics of the other specimens collected in Australia and New Zealand here examined. Therefore, this evidence supports the maintenance of D. brazieri as a valid species of Dolabrifera. Dolabrifera jacksoniensis was described by Pilsbry (1896) from Port Jackson (Sydney Harbour), Australia, just north of Botany Bay. The illustrations of a preserved specimen and a shell by Pilsbry (1896: pl. 44, figs 38–41) confirm it is a species of Dolabrifera. However, the morphology of the shell does not correspond to that of other specimens of D. brazieri here examined, as it has a much shorter apex (Supporting Information, Fig. S2B, C). Photographs of a syntype shell specimen (ANSP 64931) (Fig. 8E, F) show the shell apex is indeed not as elongate as in other specimens of D. brazieri here examined, but it has a conspicuous nucleus, formed by a single lobe, connected to the rest of the shell by a narrow area. Thus, the shell characteristics of D. jacksoniensis are largely consistent with those of D. brazieri. Other authors have considered D. jacksoniensis as a synonym of D. brazieri (see Hedley, 1917), and this synonymy is here confirmed with the available evidence. Dolabrifera ascifera (Rang, 1828) (Figs 10A–D, 11–13) Aplysia ascifera Rang, 1828: 51–52, pl. 4, figs 7–9. Type locality: ‘Saint-Jean de Cayenne’ (=Cayenne), French Guiana. Dolabrifera swiftii Pilsbry, 1896: 125, pl. 67, figs 19–20. Type locality: West Indies. Dolabrifera sowerbyi Guilding in Reeve, 1868: pl. 1, fig. 2a, b. Type locality: ‘St. Vincent’s W.I’. Type material Aplysia ascifera Rang, 1828, two syntypes at MNHN (Valdés & Héros, 1998). Dolabrifera swiftii Pilsbry, 1896, one syntype at ANSP 67519. Dolabrifera sowerbyi Guilding in Reeve, 1868, two syntypes at NHMUK 1839.9.15.45. Material examined Lower Matecumbe Key, Florida, USA, intertidal, 22 September 1950, four specimens 21–34 mm preserved length (LACM 1955-59.4). Arrecife Gallega, Veracruz, Veracruz, Mexico, 27 June 2014, one specimen 47 mm preserved length (CNMO 6611). Veracruz, Mexico, 16 June 1973, six specimens 30–45 mm preserved length, leg. J.W. Tunnell (LACM 179119). Arrecife Isla Lobos, Veracruz, Mexico, intertidal, 3 June 1973, three specimens 18–44 mm preserved length, leg. J.W. Tunnell (LACM 179120). Urchin Cove, Jamaica, 24 May 2006, one specimen 27 mm preserved length, leg. Ann DuPont (LACM 173238). East side of Playa El Yaque’s Lagoon, Isla Tortuga, Venezuela, 18 March 2010, 0.5 m depth, one specimen 55 mm preserved length (ZMUB 84907.1); one specimen 54 mm preserved length (ZMUB 84907.2). Bocas del Drago, Bocas del Toro, Panama, 28 July 2015, one specimen 22 mm preserved length (CPIC 01563). Description External morphology Body up to 90 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with numerous tubercles of different sizes, each with retractile, simple papilla on apex; larger tubercles surrounded by smaller ones (Fig. 10A–D), tightly arranged, giving animal bumpy appearance. Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude; edge of parapodia tuberculate. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. Figure 10. View largeDownload slide Live animals of the Atlantic Ocean species of Dolabrifera. A–D, Dolabrifera ascifera Rang, 1828. A, Venezuela (ZMBN 84918); B, Jamaica (LACM 173238); C, Veracruz, Mexico (CNMO 6611); D, Bocas del Toro, Panama (CPIC 01563). E, F, Dolabrifera edmundsi sp. nov., Madeira, Portugal, photograph: P. Wirtz (ZSM 20160193, ZSM 20160194). G, H, Dolabrifera virens Verrill, 1901. G, Jamaica (LACM 173265); H, St. Helena (ZSM Mol 20170625). Figure 10. View largeDownload slide Live animals of the Atlantic Ocean species of Dolabrifera. A–D, Dolabrifera ascifera Rang, 1828. A, Venezuela (ZMBN 84918); B, Jamaica (LACM 173238); C, Veracruz, Mexico (CNMO 6611); D, Bocas del Toro, Panama (CPIC 01563). E, F, Dolabrifera edmundsi sp. nov., Madeira, Portugal, photograph: P. Wirtz (ZSM 20160193, ZSM 20160194). G, H, Dolabrifera virens Verrill, 1901. G, Jamaica (LACM 173265); H, St. Helena (ZSM Mol 20170625). External coloration Background colour variable, brown or green, with spots and/or irregular patches of different colours. Mantle margin semi-translucent, with same general colour as rest of the body and light spots. Ventral side typically same colour as rest of the body with numerous white spots. Digestive system Radular formulae: 34 × 90.1.77 (CPIC 01563); 35 × 90.1.95 (LACM 173238); radulae typically asymmetrical, with more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 11A, D); teeth wider at base, both upper, lower ends of teeth convex. Some rachidian teeth with sharp conical extensions on one of the apical ends (typically right one). Lateral teeth elongate, with long bases (Fig. 11B, E); each cusp with basal, blunt, inner denticle, apex bifurcated into two blunt denticles, one clearly larger. Outer teeth very elongate, with or without secondary denticle, apex bifurcate or simple (Fig. 11C, F). Jaws with simple, irregular denticles (Fig. 11J). Post-radular armature with numerous spines with elongate bases, triangular cusps (Fig. 11K). Gizzard plates variable in shape, typically irregular, with some striations more visible near centre (Fig. 11G–I). Figure 11. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera ascifera (Rang, 1828). A–C, radular teeth of a specimen from Bocas del Toro, Panama (CPIC 01563). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173238). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Venezuela (ZMBN 84907). I, gizzard plate of a specimen from Bocas del Toro, Panama (CPIC 01563). J, jaw elements of a specimen from Venezuela (ZMBN 84907). K, post-radular armature of a specimen from Venezuela (ZMBN 84907). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173238). N, O, dorsal and ventral views of the shell apex of a specimen from Venezuela (ZMBN 84907). Figure 11. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera ascifera (Rang, 1828). A–C, radular teeth of a specimen from Bocas del Toro, Panama (CPIC 01563). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173238). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, H, gizzard plates of a specimen from Venezuela (ZMBN 84907). I, gizzard plate of a specimen from Bocas del Toro, Panama (CPIC 01563). J, jaw elements of a specimen from Venezuela (ZMBN 84907). K, post-radular armature of a specimen from Venezuela (ZMBN 84907). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173238). N, O, dorsal and ventral views of the shell apex of a specimen from Venezuela (ZMBN 84907). Shell Shell flat, well calcified, oval (Fig. 12); nucleus conspicuous, formed by single lobe (Fig. 12A–D, I, J), occasionally two (Fig. 12E–H, K, L). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, typically widening abruptly near mid-length, more gradually in some specimens. Widest portion of shell mid-length or slightly forward. Sculpture of conspicuous growth lines. Figure 12. View largeDownload slide Shells of Dolabrifera ascifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bocas del Toro, Panama (CPIC 01563), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Jamaica (LACM 173238). I, J, dorsal and ventral view of a syntype of Dolabrifera sowerbyi Guilding in Reeve, 1868 (NHMUK 1839.9.15.45), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of a syntype of Dolabrifera swiftii Pilsbry, 1896 (ANSP 67519), scale bar = 5 mm (photographs: ©ANSP), scale bar = 1 mm. Figure 12. View largeDownload slide Shells of Dolabrifera ascifera (Rang, 1828) and synonyms. A, B, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Venezuela (ZMBN 84907), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bocas del Toro, Panama (CPIC 01563), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Jamaica (LACM 173238). I, J, dorsal and ventral view of a syntype of Dolabrifera sowerbyi Guilding in Reeve, 1868 (NHMUK 1839.9.15.45), scale bar = 5 mm (photographs: K. Webb, ©NHMUK). K, L, dorsal and ventral view of a syntype of Dolabrifera swiftii Pilsbry, 1896 (ANSP 67519), scale bar = 5 mm (photographs: ©ANSP), scale bar = 1 mm. Male reproductive system Penial canal long and muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 13B–D). Penial canal ends in long retractor muscle connecting to ventral side of body wall internally. No distinguishable penial sheath. Penial papilla very long, occupies almost entire penial canal (Fig. 13A). Figure 13. View largeDownload slide Penial morphology of Dolabrifera ascifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Venezuela (ZMBN 84907). B, male copulatory organs of a specimen from Veracruz, Mexico (CNMO 6611). C, male copulatory organs of a specimen from Jamaica (LACM 173238). D, male copulatory organs of a specimen from Venezuela (ZMBN 84907). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Figure 13. View largeDownload slide Penial morphology of Dolabrifera ascifera (Rang, 1828). A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Venezuela (ZMBN 84907). B, male copulatory organs of a specimen from Veracruz, Mexico (CNMO 6611). C, male copulatory organs of a specimen from Jamaica (LACM 173238). D, male copulatory organs of a specimen from Venezuela (ZMBN 84907). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Range Western Atlantic, from Florida to possibly Brazil. Confirmed records in this study based on molecular data include Mexico, Jamaica, Panama and Venezuela. Confirmed records based on morphology include Florida, Mexico, the US Virgin Islands and French Guiana. Remarks Some specimens of Dolabrifera collected in several localities in the Western Atlantic (Veracruz, Mexico, Jamaica, Bocas del Toro, Panama and Venezuela) are genetically and morphologically similar to each other and distinct from other specimens Dolabrifera here sequenced. Species delimitation analysis confirmed these specimens constitute a distinct species. The main distinctive characteristics of these animals are in the internal anatomy and include the male reproductive system, with a long and muscular penial canal lacking tubercles, no distinguishable penial sheath and a very long penial papilla occupying almost the entire penial canal. The rachidian radular teeth have characteristic sharp conical extensions on one of the apical ends, the lateral radular teeth have a bifurcated apex with two blunt denticles, one clearly larger than the other. The shells are wide and short, typically narrower near the nucleus, often widening abruptly near mid-length; the nucleus is conspicuous, formed by a single lobe, occasionally two. To determine the valid name and synonyms for this species, a review of the literature was conducted and discussed below. The oldest name available for Western Atlantic Dolabrifera is Aplysia ascifera Rang, 1828, described from ‘Saint-Jean de Cayenne’ (=Cayenne), French Guiana. Rang (1828: pl. 4, figs 7–9) illustrated the external morphology of a live animal and the shell here reproduced in Supporting Information (Fig. S2D–F). The characteristics of the live animal and particularly of the shell match those of some specimens here described; the live animal is a greenish-grey Dolabrifera with numerous ramified tubercles on the dorsum, similar to specimens from Jamaica and Mexico illustrated herein (Fig. 10B, C); the shell is short and wide (c. 1:1.5–2 width/length ratio), narrower near the nucleus, widening near mid-length, the nucleus is conspicuous. Other Western Atlantic specimens here examined morphologically, which cluster together in the same clade, also have short and wide shells with a similar overall morphology (Fig. 12). Because of these similarities, we propose using the name Dolabrifera ascifera for this species. Pilsbry (1896) described Dolabrifera swiftii based on specimens collected from the West Indies. The original description included illustrations of a shell (Pilsbry, 1896: pl. 67, figs 19, 20; Supporting Information, Fig. S2M, N), which also resembles the characteristics of the original description of D. ascifera as well as those of other Western Atlantic specimens here examined and assigned to this species. These characteristics (wide and short shell, narrower near nucleus, widening near mid-length, nucleus conspicuous) were confirmed with the examination of photographs of a syntype (Fig. 12K, L). Thus, we consider D. swiftii a synonym of D. ascifera. Two additional species described from the Caribbean region are Dolabrifera virens Verrill, 1901, type locality Hungry Bay, Bermuda and Dolabrifera sowerbyi Guilding in Reeve, 1868, type locality St. Vincent, US Virgin Islands. The shells of both species were illustrated in the original descriptions (Verrill, 1901: pl. 2, figs 4, 5; Reeve, 1868: pl. 1, fig. 2a, b) and reproduced here (Supporting Information, Fig. S2G, I, K, L). Examination of the syntype of D. sowerbyi (Fig. 12I, J) reveals that this shell is similar to those of other shells of D. ascifera. Thus, D. sowerbyi is also regarded as a synonym of D. ascifera. On the contrary, the shell of a syntype of D. virens is much longer and narrower and probably belongs to a different species described below. Dolabrifera nicaraguana Pilsbry, 1896 (Figs 3I–J, 14–16) Dolabrifera nicaraguana Pilsbry, 1896: 124, pl. 63, figs 12–16. Type locality: San Juan del Sur, Nicaragua (Pacific coast). Type material Three syntypes at ANSP 67517 (dry) and ANSP A7048 (wet). Material examined Puerto Vallarta, Mexico, intertidal, 2 May 2002, one specimen 15 mm preserved length (CPIC 00547). El Salvador, no locality data or date, two shells, leg. A.J. Ferreira (LACM 10383–10384). Costa Rica, no locality data or date, two shells, leg. A.J. Ferreira (LACM 10381–10382). North side of San Juan del Sur Bay, Nicaragua (11°15′N, 85°52′W), 25 Jan 1974, six specimens 10–13 mm preserved length, one shell removed (CASIZ 001449). Bahía Salinas, Guanacaste, Costa Rica (11°02′N, 83°42′W), intertidal, 10 February 1935, two specimens 30–35 mm preserved length (LACM 1935-107.25); one specimen 35 mm preserved length (LACM 1935-107.25). Puerto Parker, Guanacaste, Costa Rica (10°56′N, 85°48′47″W), intertidal, 9 February 1935, one specimen 23 mm preserved length (LACM 1935-115.17). South of Punta Mala, Puerto Culebra, Guanacaste, Costa Rica (10°36′30″N, 85°42′15″W), intertidal, 24 February 1934, three specimens 25–35 mm preserved length (LACM 1934-130.21); eight specimens 20–30 mm preserved length (LACM 1934-130.21). Bahía San Josecito, Provincia Puntarenas, Costa Rica, 14 January 2003, two specimens 24–28 mm preserved length (CPIC 00551). North side of Isla del Caño, Costa Rica (8°43′15″N, 83°53′07″W), intertidal, 18–19 March 1972, four specimens 10–15 mm preserved length, leg. J.H. McLean (LACM 1972-68.45). Bahía Honda, Panama (7°43′30″N, 81°32′40″W), intertidal, 28 March 1939, seven specimens 22–30 mm preserved length (LACM 1939–52.1). South of Punta Marzo, Bahía Octavia, Choco, Colombia (6°49.8′N, 77°49.6′W, intertidal, 27 January 1935, one specimen 21 mm preserved length (LACM 1935–73.9). Bahía Cupica, Choco, Colombia (6°39.2′N, 77°30.7′W, intertidal, 26 January 1935, one specimen 22 mm preserved length (LACM 1935–73.9). Puerto Utria, Choco, Colombia (5°59.2′N, 77°21.3′W, intertidal, 23 January 1935, one specimen 30 mm preserved length (LACM 1935–52.25); 25 February 1938, three specimens 23–31 mm preserved length (LACM 1938-126.18). Isla Gorgona, Cauca, Colombia (3°0.5′N, 78°11.8′W), intertidal, 12 February 1934, one specimen 24 mm preserved length (LACM 1934–92.17). Cabo de San Francisco, Ecuador (0°39′30″N, 80°06′30″W), intertidal, two specimens 32–37 mm (LACM 1938-116.11). Sombrero Chino, Isla Santiago, Islas Galápagos, Ecuador (0°22′20″S, 90°34′30″W), intertidal, 22 March 1971, six specimens 15–22 mm preserved length (LACM 1971-64.6). Punta Alfaro, Isla Isabela, Islas Galápagos, Ecuador (0°25′20″S, 90°57′10″W), intertidal, 25 March 1971, three specimens 8–12 mm preserved length (LACM 1971-70.10). Isla Pinzón, Islas Galápagos, Ecuador (0°35′50″S, 90°39′15″W), intertidal, 25–26 March 1971, five specimens 16–22 mm preserved length (LACM 1971-72.16). Darwin Research Station, Academy Bay, Isla Santa Cruz, Islas Galápagos, Ecuador (0°45′06″S, 90°15′38″W), 1 m, March 1971, one specimen 23 mm preserved length (LACM 1971-45.20); intertidal, March 1971, one specimen 21 mm preserved length (LACM 1971-44.17). Flamingo Cove, near Post Office Bay, Isla Floreana, Islas Galápagos, Ecuador (1°14′S, 90°27′30″W), intertidal, 15 March 1971, two specimens 13–24 mm preserved length (LACM 1971-52.13). Isla de la Plata, Ecuador (1°16′S, 81°05′10″W), intertidal, 22 January 1933, one specimen 45 mm preserved length (LACM 1933-22.21). Salinas, Santa Elena, Ecuador (2°12′S, 81°58′W), 5–6 March 1970, one specimens 28 mm preserved length, leg. J.H. McLean (LACM 1970–9.29). South Point of Santa Elena, Ecuador (2°12′23″S, 81°00’05″W), 8 February 1934, three specimens 40 mm preserved length (LACM 1934–81.15). Balneario Punta Sal, Tumbes, Peru, intertidal, 9 August 2005, one specimen 16 mm preserved length, leg. K. Nakamura (CPIC 00194). Description External morphology Body up to 50 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with densely packed tubercles of different sizes, each with retractile, simple, short papilla on apex; larger tubercles surrounded by smaller ones (Fig. 3I, J). Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude; edge of parapodia tuberculate. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, typically brown, greyish-green, or red; tubercles often lighter than background coloration, sometimes with papillae of different colours (pink). Mantle margin with same general colour as rest of body with light spots. Ventral side typically same colour as rest of body with numerous white spots. Digestive system Radular formulae: 25 × 86.1.84 (CPIC 00547); 43 × 111.1.95 (CPIC 00551); radulae slightly asymmetrical, with few more teeth in one half-row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 14A, D), two innermost denticles much larger than two outermost; teeth wider at base, both upper and lower ends of teeth convex. Some rachidian teeth with small sharp conical extensions on one of apical ends (typically right one). Lateral teeth elongate, with long bases (Fig. 14B, E); each cusp with basal, narrow, inner denticle, apex bifurcated into two blunt denticles, one slightly larger. Outer teeth very elongate, with or without secondary denticle, apex bifurcate or simple (Fig. 14C, F). Jaws with simple, irregular denticles (Fig. 14J). Post-radular armature with numerous spines with elongate bases and triangular cusps (Fig. 14K). Gizzard plates variable in shape, typically irregular, with numerous striations (Fig. 14G–I). Figure 14. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera nicaraguana Pilsbry, 1896. A–C, radular teeth of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551) – D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, gizzard plate of a specimen from Puerto Vallarta, Mexico (CPIC 00547). H, I, gizzard plates of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). J, jaw elements of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). K, post-radular armature of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). L, M, dorsal and ventral views of the shell apex of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). N, O, dorsal and ventral views of the shell apex of a specimen from Puerto Vallarta, Mexico (CPIC 00547). Figure 14. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera nicaraguana Pilsbry, 1896. A–C, radular teeth of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551) – D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G, gizzard plate of a specimen from Puerto Vallarta, Mexico (CPIC 00547). H, I, gizzard plates of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). J, jaw elements of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). K, post-radular armature of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). L, M, dorsal and ventral views of the shell apex of a specimen from Santa Elena, Ecuador (LACM 1934–81.15). N, O, dorsal and ventral views of the shell apex of a specimen from Puerto Vallarta, Mexico (CPIC 00547). Shell Shell flat, well calcified, oval to elongate (Fig. 15); nucleus conspicuous, formed by single lobe (Fig. 14L–P). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, typically widening slightly towards mid-length, abruptly in some specimens; some shells very narrow across entire length. Widest portion of shell mid-length backward. Sculpture of conspicuous growth lines. Figure 15. View largeDownload slide Shells of Dolabrifera nicaraguana Pilsbry, 1896. A, B, dorsal and ventral view of a syntype of Dolabrifera nicaraguana Pilsbry, 1896 (ANSP 67517), scale bar = 1 mm (photographs: ©ANSP). C, D, dorsal and ventral view of a specimen from Puerto Culebra, Costa Rica (LACM 256-34), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Puerto Vallarta, Mexico (CPIC 00547), scale bar = 1 mm. I, J, dorsal and ventral view of a specimen from Bahía Salinas, Costa Rica (LACM 474-35), scale bar = 1 mm. K, L, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. M, N, dorsal and ventral view of a specimen from Tumbes, Peru (CPIC 00194), scale bar = 1 mm. O, P, dorsal and ventral view of a specimen from Santa Elena, Ecuador (LACM 1934–81.15), scale bar = 1 mm. Figure 15. View largeDownload slide Shells of Dolabrifera nicaraguana Pilsbry, 1896. A, B, dorsal and ventral view of a syntype of Dolabrifera nicaraguana Pilsbry, 1896 (ANSP 67517), scale bar = 1 mm (photographs: ©ANSP). C, D, dorsal and ventral view of a specimen from Puerto Culebra, Costa Rica (LACM 256-34), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. G, H, dorsal and ventral view of a specimen from Puerto Vallarta, Mexico (CPIC 00547), scale bar = 1 mm. I, J, dorsal and ventral view of a specimen from Bahía Salinas, Costa Rica (LACM 474-35), scale bar = 1 mm. K, L, dorsal and ventral view of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551), scale bar = 1 mm. M, N, dorsal and ventral view of a specimen from Tumbes, Peru (CPIC 00194), scale bar = 1 mm. O, P, dorsal and ventral view of a specimen from Santa Elena, Ecuador (LACM 1934–81.15), scale bar = 1 mm. Male reproductive system Penial canal large and muscular, with a series of tubercles and two proximal retractor muscles, on each side of the opening into the body wall (Fig. 16B, C). The penial canal widens gradually into the distal end, right before narrowing abruptly and widening again into a small, vesicular-like penial sheath, which contains the penial papilla. The distal end of the penial sheath connects to a long and strong retractor muscle attaching to the ventral side of the body wall internally. The penial papilla is small, triangular, with a longitudinal groove (Fig. 16A). Figure 16. View largeDownload slide Penial morphology of Dolabrifera nicaraguana Pilsbry, 1896. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). B, male copulatory organs of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). C, male copulatory organs of a specimen from Santa Elena, Ecuador (LACM 1934-81.15). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Figure 16. View largeDownload slide Penial morphology of Dolabrifera nicaraguana Pilsbry, 1896. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). B, male copulatory organs of a specimen from Bahía San Josecito, Costa Rica (CPIC 00551). C, male copulatory organs of a specimen from Santa Elena, Ecuador (LACM 1934-81.15). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Range Eastern Pacific Ocean, from Bahía Las Cruces, Baja California Sur, Mexico (Bertsch, 1970) to Tumbes, Peru (Nakamura, 2007; Uribe et al., 2013). Remarks Anatomical and sequence data reveal that all specimens here studied from the Eastern Pacific belong to the same species. All of them have a large and muscular penial canal, with a series of tubercles, as well as a very small, vesicular-like penial sheath containing a small penial papilla; the shells tend to be narrower than in Dolabrifera species from other regions and the lateral radular teeth have the apex bifurcated into two blunt denticles, one slightly larger than the other. The ABGD analysis reveals that Eastern Pacific specimens group together in the same species, this includes specimens ranging from Mexico to Peru, which constitutes the known range of Dolabrifera in this region. Moreover, specimens from the Eastern Pacific are genetically and morphologically distinct from other Dolabrifera specimens here examined, confirming the Eastern Pacific animals represent a different species. A review of the literature reveals that there is only one name available for Eastern Pacific Dolabrifera, D. nicaraguana Pilsbry, 1896, described from San Juan del Sur, Nicaragua. The original description included illustrations of a preserved specimen and a shell (Pilsbry, 1896: pl. 63, figs 12–16; Supporting Information, Fig. S2O–Q). The shell of the syntype is straight, very narrow and elongate (Fig. 15A, B), which is different from other shells from the Eastern Pacific here examined (Fig. 15C–H, K, P), but similar to others (Fig. 15I, J), underscoring the variability in shell morphology within this species. Here, we examined specimens from San Juan del Sur, Nicaragua and adjacent localities in Costa Rica, all of which share similar shell, penial and radular morphology with other specimens from the Eastern Pacific. Thus, we confidently assign the name D. nicaraguana to the Eastern Pacific species of Dolabrifera. Dolabrifera virens Verrill, 1901 (Figs 10G–H, 17–19) Dolabrifera virens Verrill, 1901: 24–25, pl. 2, fig. 4, 5, pl. 4, fig. 11. Type locality: Hungry Bay, Bermuda. Type material Dolabrifera virens Verrill, 1901, two syntypes at YPM IZ 048817 (wet) and YPM IZ 029268 (dry). Material examined Urchin Cove, St Ann’s Bay, Jamaica (18°27.20′N, 77°13.55′W), 0.3–0.6 m depth, 25 May 2006, two specimens 10–15 mm preserved length, leg. A. DuPont (LACM 173265). Lauderdale-by-the-Sea, Florida, USA, 8 m depth, 18 June 2014, one specimen 21 mm preserved length, leg. A. Dimitris (CPIC 01134). Vista Alegre, Curaçao (12°0.50′N, 68°53.30′W), intertidal, 23 April 1939, nine specimens 14–40 mm preserved length (LACM 1939-215.1). Description External morphology Body up to 80 mm long, oval, flattened, broader posteriorly, narrowing slightly towards head. Dorsum covered with densely packed tubercles of different sizes, each with elongate papilla on apex, some ramified at tips; larger tubercles surrounded by smaller ones, each bearing papilla, giving animal appearance of being densely covered with papillae (Fig. 10G–H). Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude; edge of parapodia tuberculate. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, red, brown or green, sometimes white, with spots and/or irregular patches of different colours. Mantle margin semi-translucent, with same general colour as rest of body and few small white dots. Ventral side typically same colour as rest of the body with numerous white spots. Digestive system Radular formulae: 32 × 61.1.60 (LACM 173265A); 27 × 50.1.57 (LACM 173265B); radulae nearly symmetrical, with about same number of teeth on each row. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 17A, D); teeth wider at base, both upper and lower ends of teeth convex. Lateral teeth elongate, with long bases (Fig. 17B, E); each cusp with two to three basal, blunt, inner denticles, apex bifurcated into two blunt denticles equal in size. Outer teeth very elongate, with secondary denticle, apex bifurcate (Fig. 17C, F). Jaws with simple, irregular denticles (Fig. 17J). Post-radular armature with numerous spines with elongate bases and low cusps (Fig. 17K). Gizzard plates variable in shape, typically short, wide, with some striations more visible near one end (Fig. 17G–I). Figure 17. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera virens Verrill, 1901. A–C, radular teeth of a specimen from Jamaica (LACM 173265A). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173265B). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Jamaica (LACM 173265A). J, jaw elements of a specimen from Jamaica (LACM 173265B). K, post-radular armature of a specimen from Jamaica (LACM 173265A). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265B). N, O, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265A). Figure 17. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera virens Verrill, 1901. A–C, radular teeth of a specimen from Jamaica (LACM 173265A). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Jamaica (LACM 173265B). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Jamaica (LACM 173265A). J, jaw elements of a specimen from Jamaica (LACM 173265B). K, post-radular armature of a specimen from Jamaica (LACM 173265A). L, M, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265B). N, O, dorsal and ventral views of the shell apex of a specimen from Jamaica (LACM 173265A). Shell Shell flat, well calcified, elongate, width/length ratio greater than 1:2 (Fig. 18); nucleus inconspicuous, short, formed by single lobe. On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, widening gradually towards mid-length. Widest portion of shell mid-length or slightly backward. Sculpture of conspicuous growth lines. Figure 18. View largeDownload slide Shells of Dolabrifera virens Verrill, 1901. A, B, dorsal and ventral view of a specimen from Jamaica (LACM 173265A), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134), scale bar = 1 mm. E, F, dorsal and ventral view of a syntype from Bermuda (YPM IZ 029268), scale bar = 1 mm. Figure 18. View largeDownload slide Shells of Dolabrifera virens Verrill, 1901. A, B, dorsal and ventral view of a specimen from Jamaica (LACM 173265A), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134), scale bar = 1 mm. E, F, dorsal and ventral view of a syntype from Bermuda (YPM IZ 029268), scale bar = 1 mm. Male reproductive system Penial canal long, muscular, lacking tubercles, with two proximal retractor muscles, one on each side of opening into body wall (Fig. 19B, C). Penial canal ends in long retractor muscle connecting to ventral side of body wall internally. No distinguishable penial sheath. Penial papilla very long, occupies almost entire penial canal (Fig. 19A). Figure 19. View largeDownload slide Penial morphology of Dolabrifera virens Verrill, 1901. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Jamaica (LACM 173265A). B, male copulatory organs of a specimen from Jamaica (LACM 173265A). C, male copulatory organs of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Figure 19. View largeDownload slide Penial morphology of Dolabrifera virens Verrill, 1901. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Jamaica (LACM 173265A). B, male copulatory organs of a specimen from Jamaica (LACM 173265A). C, male copulatory organs of a specimen from Lauderdale-by-the-Sea, Florida (CPIC 01134). Abbreviations: pc, penial canal; pp, penial papilla; rm, retractor muscle. Range Western and South Atlantic from Jamaica to St. Helena. Confirmed records based on molecular data include Panama, Jamaica and St. Helena. Confirmed records based on morphology include Florida, Bermuda and Curaçao. Remarks The phylogenetic analyses recovered a clade including specimens from Bocas del Toro, Panama, Jamaica and St. Helena in the South Atlantic, distinct from the clade containing other Western Atlantic specimens, to which the name D. ascifera was assigned herein. The species delimitation analysis confirmed this clade is a distinct species. These animals have a male reproductive anatomy similar to that of D. ascifera, but they differ in other regards. For example, the rachidian radular teeth lack sharp conical extensions on the apical ends, the lateral radular teeth have a bifurcated apex with two blunt denticles equal in size, the shells are narrower and proportionally longer, narrowing gradually towards the nucleus; the nucleus is inconspicuous, formed by a single lobe. Because these animals are genetically and morphologically distinct from D. ascifera, it is necessary to determine whether an available name exists for this taxon. Dolabrifera virens Verrill, 1901 was described from Hungry Bay, Bermuda, based on several specimens, two of which remain at the YPM collections. One of the syntypes was photographed alive (Verrill, 1901: text fig. 1, pl. 4, fig. 11) and is covered by numerous tubercles with elongate, branched papillae (Supporting Information, Fig. S2J). This is similar to the animals here described, which contrary to D. ascifera appear to be covered by densely arranged, ramified papillae. The shells of several specimens were illustrated in the original description (Verrill, 1901: pl. 2, figs 4, 5), and photographs of one of the syntypes are reproduced here (Fig. 18E, F). The syntype shell (YPM IZ 029268) is very narrow and elongate, width/length ratio greater than 1:2, very similar to other shells dissected from specimens clustering into the unnamed Western Atlantic clade (Fig. 18). These shells are consistently longer and narrower than all the shells of D. ascifera here examined (Fig. 12). Because specimens in this unnamed Western Atlantic clade match the original description of D. virens, this name is assigned to it. Dolabrifera virens and D. ascifera are externally undistinguishable and sympatric in large portions of the Western Atlantic, making the identification of these species problematic and requiring anatomical examination and/or DNA sequence data. Dolabrifera edmundsi sp. nov. (Figs 10E–F, 20–22) Type material Holotype at ZSM Mol 20160193, Porto Moniz, Madeira, September 2014, 18 mm preserved length. Material examined Porto Moniz, Madeira, September 2014, one specimen 17 mm preserved length, leg. P. Wirtz (ZSM Mol 20160194), one specimen 19 mm preserved length, leg. P. Wirtz (ZSM Mol 20160195), one specimen 20 mm preserved length, leg. P. Wirtz (ZSM Mol 20160196), one specimen 20 mm preserved length, leg. P. Wirtz (ZSM Mol 20160197). Ilhéu Bombom, Ilha do Príncipe, São Tomé and Príncipe, 20 January 2009, one specimen 11 mm preserved length (CASIZ 179395). South of Terrebonne Bay, LA, USA (28°5.57′N, 91°1.34′W), 56–58 m depth, 4 June 2005, four specimens 12–14 mm preserved length, leg. E. Garcia (LACM 179615). Marsh Island, LA, USA (27°98.34′N, 92°22.42′W), 68–86 m depth, 22 June 2005, two specimens 11–16 mm preserved length, leg. E. Garcia (LACM 179619). Description External morphology Body up to 80 mm long, oval to elongate, flattened, broader posteriorly, narrowing gradually towards head. Dorsum covered with small, rounded, scattered tubercles, each with retractile, highly ramified, papilla on apex (Fig. 10E, F); tubercles typically surrounded by large areas of smooth tissue. Rhinophores enrolled, elongated. Oral tentacles wide, short, enrolled, with narrower bases. Parapodia fused together except for short region in posterior midline, forming two small flaps. Flaps partially overlapping in middle (right parapodium partially covers left one), allowing enough space anteriorly for inhalant opening and posteriorly for exhalant siphon to protrude. External sperm groove clearly visible on right side of animal, between mantle cavity and base of right oral tentacle. External coloration Background colour variable, typically greenish-grey or brown, sometimes red, with spots and/or irregular patches of different colours. Mantle margin with same general colour as rest of body. Digestive system Radular formula: 40 × 70.1.71 (ZSM Mol 20160195); 38 × 71.1.72 (ZSM Mol 20160196); radulae symmetrical, with just about same number teeth in both half-rows. Rachidian teeth with robust central cusp, two large denticles on each side (Fig. 20A, D), all similar in size; cusp with or without denticles on both sides; rachidian teeth wider at base, both upper and lower ends of teeth convex. Some rachidian teeth with small extensions on apical ends. Lateral teeth elongate, with long bases (Fig. 20B, E); each cusp with basal, narrow, inner denticle, apex bifurcated into two blunt denticles equal in length. Outer teeth very elongate, with or without secondary denticle, apex bifurcated (Fig. 20C, F). Jaws with simple denticles (Fig. 20J). Post-radular armature with numerous spines with elongate bases and triangular cusps (Fig. 20K). Gizzard plates variable in shape, typically irregular, with numerous striations (Fig. 20G–I). Figure 20. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera edmundsi sp. nov. A–C, radular teeth of a specimen from Madeira (ZSM Mol 20160195). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Maderia (ZSM Mol 20160196). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Madeira (ZSM Mol 20160195). J, jaw elements of a specimen from Madeira (ZSM Mol 20160196). K, post-radular armature of a specimen from Madeira (ZSM Mol 20160196). L, M, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160195). N, O, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160196). Figure 20. View largeDownload slide SEM micrographs of the internal anatomy of Dolabrifera edmundsi sp. nov. A–C, radular teeth of a specimen from Madeira (ZSM Mol 20160195). A, rachidian and innermost lateral teeth; B, mid-lateral teeth; C, outer lateral teeth. D–F, radular teeth of a specimen from Maderia (ZSM Mol 20160196). D, rachidian and innermost lateral teeth; E, mid-lateral teeth; F, outer lateral teeth. G–I, gizzard plates of a specimen from Madeira (ZSM Mol 20160195). J, jaw elements of a specimen from Madeira (ZSM Mol 20160196). K, post-radular armature of a specimen from Madeira (ZSM Mol 20160196). L, M, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160195). N, O, dorsal and ventral views of the shell apex of a specimen from Madeira (ZSM Mol 20160196). Shell Shell flat, well calcified, oval to elongate (Fig. 21); nucleus conspicuous, formed by single lobe (Fig. 20L–O). On apertural view with nucleus on upper end, left side of shell either concave or straight. Right side of shell convex, narrower near nucleus, widening slightly towards mid-length. Widest portion of shell mid-length. Sculpture of conspicuous growth lines. Figure 21. View largeDownload slide Shells of Dolabrifera edmundsi sp. nov. A, B, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160195), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160196), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160197), scale bar = 1 mm. Figure 21. View largeDownload slide Shells of Dolabrifera edmundsi sp. nov. A, B, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160195), scale bar = 1 mm. C, D, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160196), scale bar = 1 mm. E, F, dorsal and ventral view of a specimen from Madeira (ZSM Mol 20160197), scale bar = 1 mm. Male reproductive system Penial canal large, muscular, with two proximal retractor muscles, on each side of opening into body wall (Fig. 22B–D). Penial canal widens gradually into oval penial sheath, containing penial papilla. Distal end of penial sheath connects to thick and strong retractor muscle attaching to ventral side of body wall internally. Penial papilla elongate, triangular, with longitudinal groove (Fig. 22A). Figure 22. View largeDownload slide Penial morphology of Dolabrifera edmundsi sp. nov. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Madeira (ZSM Mol 20160196). B, male copulatory organs of a specimen from Madeira (ZSM Mol 20160195). C, male copulatory organs of a specimen from Madeira (ZSM Mol 20160196). D, male copulatory organs of a specimen from Louisiana, USA (LACM 176306). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Figure 22. View largeDownload slide Penial morphology of Dolabrifera edmundsi sp. nov. A, SEM micrograph of the penial papilla (dissected out the penial sheath) of a specimen from Madeira (ZSM Mol 20160196). B, male copulatory organs of a specimen from Madeira (ZSM Mol 20160195). C, male copulatory organs of a specimen from Madeira (ZSM Mol 20160196). D, male copulatory organs of a specimen from Louisiana, USA (LACM 176306). Abbreviations: pc, penial canal; pp, penial papilla; ps, penial sheath; rm, retractor muscle; tb, tubercle. Range Amphiatlantic. Primarily found in the eastern Atlantic, confirmed records include the Canary Islands, Madeira, Cape Verde, and São Tomé and Príncipe. Also present in St. Helena (South Atlantic) and the Gulf of Mexico (Western Atlantic). Etymology Named in memory of our colleague Dr Malcolm Edmunds to recognize his seminal contribution to the study of sea slugs, including the discovery of many tropical Atlantic species and his insights on their biogeographical patterns. Remarks Specimens from the Eastern Atlantic and some specimens from the South Atlantic (St. Helena) and deep waters in the Gulf of Mexico are genetically distinct from other species of Dolabrifera. These animals have tubercles with long, ramified papillae. Their shells are elongate, wider near mid-length and much narrower posteriorly and anteriorly, and the male reproductive organs contain a well-developed penial papillae. These characteristics are very different from the other two Atlantic species here recognized. Dolabrifera virens is the only Atlantic species with ramified papillae, but they are shorter. A review of the literature reveals that there is no name available for this taxon, and therefore, it is here described as a new species. DISCUSSION Species identification and characterization The genus Dolabrifera, which was often considered to include the single species D. dolabrifera (see Engel & Hummelinck, 1936; Eales, 1944), now includes five different tropical species, in addition to the temperate species D. brazieri, which was already considered distinct by some authors (Klussman-Kolb, 2004; Burn, 2006). These results are hardly surprising, considering that other species of sea hares and heterobranch sea slugs with broad tropical ranges studied to date have been found to constitute species complexes (e.g. Ornelas-Gatdula et al., 2012; Alexander & Valdés, 2013; Krug et al., 2013; Carmona et al., 2014a, b; Goodheart et al., 2015; Kienberger et al., 2016). However, the case of Dolabrifera is unique in several respects. For example, in most other cases, the species complexes are composed of primarily allopatric taxa, often found in different ocean basins (Ornelas-Gatdula et al., 2012; Alexander & Valdés, 2013; Carmona et al., 2014b) or with multiple sympatric species in the Indo-Pacific tropics (Krug et al., 2013; Carmona et al., 2014a). On the contrary, in Dolabrifera, there is a single tropical Indo-Pacific species ranging from the western Indian Ocean to the Hawaiian Islands and displaying virtually no geographical structure; at the same time, there are three partially sympatric species in the Atlantic Ocean, two of them widely distributed across the ocean basin. Another difference is that in most other species complexes identified to date, newly characterized species have been found to be pseudocryptic, meaning that they can be distinguished based on external morphological traits, but those differences only became evident once molecular data were available (Sáez & Lozano, 2005). In the case of Dolabrifera, we have been unable to identify reliable external traits for species identification; this is due to the high degree of intraspecific variation in both colour and papillae morphology in all species and the lack of unique external characteristics in individual species. Reliable differences found among species are in the morphology of the male reproductive organs and the shells (Table 2), but these differences are not always consistent. For instance, shell morphology is variable in some species (i.e. D. dolabrifera, D. ascifera, D. nicaraguana) and there is a certain degree of overlap among species, for example some shells of D. dolabrifera (Fig. 5M, N) are similar to shells of Dolabrifera edmundsi (Fig. 16). Regarding the penial morphology, there is also some overlap, for example between D. ascifera and D. virens and between D. edmundsi and D. brazieri. The lack of reliable morphological traits for species identification and characterization is particularly problematic in the Atlantic Ocean where three species co-occur. Based on the information collected in this study, species of Dolabrifera appear to be completely cryptic externally and only molecular data, and to some extent internal morphological data, can be used to reliably identify specimens. Table 2. Summary of the principal morphological characteristics that differentiate species of Dolabrifera Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  View Large Table 2. Summary of the principal morphological characteristics that differentiate species of Dolabrifera Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  Species  Shell  Penial canal  Penial sheath  Penial papilla  D. dolabrifera  Narrow, elongate, variable in shape  Large and muscular, lacking tubercles  Oval  Elongate, curved cusp, with longitudinal groove  D. brazieri  Narrow, elongate, variable in shape, nucleus connected to rest of the shell by narrow area  Large and muscular, lacking tubercles  Elongate  Elongate, curved cusp, with longitudinal groove  D. ascifera  Narrow, typically widening abruptly near mid-length  Large and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. nicaraguana  Narrow to very narrow, elongate, variable in shape  Large and muscular, with tubercles  Small, vesicle-like  Small, triangular, with longitudinal groove  D. virens  Narrow, elongate, variable in shape  Long and muscular, lacking tubercles  Undifferentiated  Very long, occupying almost entire penial canal  D. edmundsi sp. nov.  Oval to elongate  Large and muscular, lacking tubercles  Oval  Elongate, triangular, with longitudinal groove  View Large Biogeography Dolabrifera displays an unusual biogeographical pattern, with the lowest species diversity in the Indo-Pacific tropics and the highest species diversity in the Atlantic region. A single species, D. dolabrifera, occurs from the Hawaiian Islands to the eastern Indian Ocean, the longest stretch of uninterrupted tropical ocean in the planet, which contains the highest species diversity for most groups of marine organisms (Briggs, 1999). In addition, the phylogenetic analyses recovered no geographical structure and very short branches in D. dolabrifera, suggesting this species is probably panmictic across its range. Further research using population genetics approaches is necessary to verify this hypothesis, but it is clear that D. dolabrifera must have a great dispersal potential. Contrary to expectations, the Atlantic Ocean, a much smaller body of water with comparatively lower levels of marine biodiversity, contains three species of Dolabrifera, and all three overlap in range in the Caribbean region. Although D. edmundsi is primarily found in the eastern Atlantic, several specimens collected in deep water from the Gulf of Mexico share the same penial morphology as other material here examined; one of them was successfully sequenced confirming that it belongs to D. edmundsi. The other two Atlantic species, D. virens and D. ascifera, are sympatric in the Caribbean region, and specimens collected in the same localities in Jamaica and Panama were confirmed to belong to these two species. Also, D. virens and D. edmundsi are sympatric in St. Helena. The patterns of cladogenesis and speciation that produced this unique pattern are unclear. Further research on other groups of heterobranch sea slugs and other marine invertebrates is necessary to elucidate the unusual biogeographical pattern and complex evolutionary history of species of Dolabrifera. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Individual Bayesian consensus trees of specimens of Dolabrifera + outgroup taxa for each gene analysed individually (16S, COI, H3), including posterior probabilities (above nodes) and bootstrap support values from the maximum likelihood analysis (below nodes). Figure S2. Illustrations of specimens and/or shells in the original descriptions of various species of Dolabrifera. A–C, Dolabrifera jacksoniensis by Pilsbry (1896); D–F, Aplysia ascifera by Rang (1828); G–J, Dolabrifera virens by Verrill (1901); K, L, Dolabrifera sowerbyi by Guilding inReeve (1868); M, N, Dolabrifera swiftii by Pilsbry (1896); O–Q, Dolabrifera nicaraguana by Pilsbry (1896); R, Dolabrifera pelsartensis by O’Donoghue (1924); S–U, Dolabrifera holboelli by Bergh (1872); V–W, Dolabrifera triangularis by Watson (1884). Figure S3. Illustrations of specimens and/or shells in the original descriptions of species of Dolabrifera from the tropical Indo-Pacific region (excluding temperate South Africa and Australia). A–D, Aplysia dolabrifera by Rang (1828); E–H, Aplysia oahouensis by Eydoux & Souleyet (1846–49); I–J, Dolabrifera cuvieri by H. Adams & A. Adams (1854); K, L, Dolabrifera maillardi by Deshayes (1863); M, N, Dolabrifera fusca by Pease (1868); O, P, Dolabrifera tahitensis by Pease (1868); Q, R, Dolabrifera vitraea by G. B. Sowerby II inReeve (1868); S, Dolabrifera pacifica by Pease in Reeve (1868); T–U, Dolabrifera marmorea by Pease in Reeve (1868). [Version of Record, published online 29 December 2017; http://zoobank.org/urn:lsid:zoobank.org:pub:1BDCBB96-B722-4095-9D6C-1E418A2D961E] ACKNOWLEDGEMENTS Several individuals provided access to museum specimens, photographs and data: Lindsey Groves (LACM), Janet Waterhouse (AM), Mandy Reid (AM), Wilma Blom (AM), Elizabeth Kools (CASIZ), Andreia Salvador (NHMUK), Juliana Bahia (ZSM), Kevin Webb (NHMUK), Edna Naranjo-García (CNMO), Paul Callomon (ANSP), Rachele Trevisi (ZSM) and Eric Lazo-Wasem (YPM). Judith Brown (Ascension Government) and Peter Wirtz made available important specimens from St. Helena and Madeira, respectively. Gary Cobb provided a photograph of a live animal of Dolabrifera brazieri. The SEM was conducted at the California State Polytechnic University SEM Laboratory supported by the US National Science Foundation (NSF) grant DMR-1429674. 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