The origin of king crabs: hermit crab ancestry under the magnifying glass

The origin of king crabs: hermit crab ancestry under the magnifying glass Abstract The origin of king crabs from a hermit crab ancestor has caused controversy for more than a century. While the phylogenetic position of Lithodidae within the hermit crab family Paguridae has been strengthened in recent years, several key questions regarding the evolution of lithodid crabs have remained unanswered. In particular, it has been debated which hermit crabs constitute the closest extant relatives to lithodid crabs within Paguridae. Also, the relationships of the two lithodid subfamilies, Lithodinae and Hapalogastrinae, are unresolved. Answers to these questions are crucial to the understanding of the origin of king crabs, in particular which factors were the driving forces behind leaving a protective housing, transforming to a crab-like morphology and finally developing a large body size. To address these questions, we constructed the most comprehensive molecular phylogeny of Paguridae and Lithodidae to date. Our analyses revealed a species-rich clade of hermit crabs as closest relatives to lithodid crabs within Paguridae. Hermit crabs included in this clade have a predominantly shallow-water distribution in the North Pacific, agreeing with a proposed origin of lithodid crabs in this region. We suggest that the advances resulting from abandoning a shell-inhabiting lifestyle, rather than constraints of such shelters, played a central role in carcinization in this taxon. Phylogenetic relationships within Lithodidae revealed its two subfamilies to be non-monophyletic. Small-sized, shallow-water taxa are basal in the phylogenetic tree, while an increase in size and subsequent deep-sea distribution occurred later in the evolution of the group. INTRODUCTION King crabs (Lithodidae Samouelle, 1819) are anomuran crustaceans with a crab-like body, superficially resembling that of brachyuran (true) crabs. The evolutionary origin of the taxon has been discussed since the end of the 19th century when it was hypothesized that king crabs are secondarily calcified hermit crabs that left the protective gastropod housing and transformed to a crab-like form (Boas, 1880a, b; Bouvier, 1895). The process causing morphological modifications towards a crab-like body shape, with a reduced pleon, folded under a broadened and calcified cephalothorax, is termed carcinization (Borradaile, 1916; Scholtz, 2014). The hypothesis that lithodid crabs are derived from a hermit crab ancestor regained significant attention when investigated for the first time using molecular phylogenetic methods (Cunningham, Blackstone & Buss, 1992). Not only were king crabs placed within the Paguridae Latreille, 1802, but the molecules even suggested a nested position of the taxon within the hermit crab genus Pagurus Fabricius, 1775. All subsequent studies based on molecular data (Zaklan, 2002; Morrison et al., 2002; Tsang et al., 2008, 2011; Ahyong, Schnabel & Maas, 2009; Bracken et al., 2009; Chu et al., 2009; Schnabel, Ahyong & Maas, 2011; Bracken-Grissom et al., 2013) as well as various morphological studies (Richter & Scholtz, 1994; Keiler & Richter, 2011; Reimann, Richter & Scholtz, 2011; Keiler, Richter & Wirkner, 2015) further supported the placement of lithodid crabs within the asymmetrical hermit crab family Paguridae. Despite the growing evidence of a hermit crab ancestry of king crabs, McLaughlin et al. strongly opposed this evolutionary scenario (McLaughlin & Lemaitre, 1997; McLaughlin, Lemaitre & Tudge, 2004; McLaughlin, Lemaitre & Sorhannus, 2007; Lemaitre & McLaughlin, 2009). Reversal of complex characters related to dextral shell housings, like the morphology of the fourth pereopods, and a maladaptive scenario of a crab exposing the soft pleon made, in their view, this evolutionary pathway infeasible. Rather, the authors proposed the opposite evolutionary scenario, leading from king crabs to hermit crabs. The placement of lithodids within the Anomura has thus been heartily debated until recent years when steadily increasing evidence settled the position of the group within the Paguridae (Tsang et al., 2008, 2011; Ahyong et al., 2009; Chu et al., 2009; Schnabel et al., 2011; Bracken-Grissom et al., 2013). The overall body shape of lithodid crabs is highly altered from its hermit crab ancestor. However, pagurid hermit crab asymmetries, influenced by inhabiting dextral gastropod shells (Palmer, 2004), such as pleon and claw asymmetries (McLaughlin & Lemaitre, 1997, 2000; McLaughlin et al., 2004; Duguid, 2010), are still present in lithodids (Tsang et al., 2011). Further traces of their pagurid origin can, for example, be found in the mouthparts (Boas, 1924; Jaszkowiak et al., 2015), setation (Keiler & Richter, 2011), internal organ organization (Anker & Paulay, 2013; Keiler et al., 2015) or vascular system (Keiler, Richter & Wirkner, 2013; Keiler et al., 2015). Poor knowledge of phylogenetic relationships within the Paguridae (Matzen da Silva et al., 2011a) left the exact placement of lithodids within this hermit crab family uncertain. Various candidates have been highlighted as the possible closest extant relatives to lithodids within the Paguridae, yet conclusions from genetic studies so far have been restricted by limited taxon sampling. In early studies, king crabs were thought to be derived from an ancestor closely related to the genera Nematopagurus A. Milne-Edwards & Bouvier, 1892 and Pylopagurus A. Milne-Edwards & Bouvier, 1893 based on the presence of paired pleopods, found in females of these species and those of lithodids (Boas, 1924). Reimann et al. (2011), based on a cladistic analysis, also found a sister relationship of these genera to lithodids within the remaining Paguridae. An exclusively North Pacific genus, Discorsopagurus McLaughlin, 1974, has drawn attention as the possibly closest relative in recent studies (Morrison et al., 2002; Ahyong et al., 2009; Schnabel et al., 2011; Bracken-Grissom et al., 2013). Discorsopagurus inhabits non-coiled housings and has an almost symmetrical pleon (Komai, 2003), as also found in male lithodids. Other studies found a sister relationship with one or few other exclusively North Pacific genera (Labidochirus Benedict, 1892; Elassochirus Benedict, 1892; and Pagurodofleinia Asakura, 2005), as well as part of the genus Pagurus (Cunningham et al., 1992; Tsang et al., 2008, 2011; Chu et al., 2009). In particular, a possibly nested position of Lithodidae within Pagurus has been highlighted (Cunningham et al., 1992). Pagurus is a species-rich genus, and various informal morpho-groups have been established (Forest & de Saint Laurent, 1968; McLaughlin, 1974; Ingle, 1985; Lemaitre & Cruz-Castaño, 2004). Some of these informal groupings have been confirmed using molecular markers (Matzen da Silva et al., 2011a; Olguín & Mantelatto, 2013), and a highly polyphyletic pattern of the genus has been indicated (Cunningham et al., 1992; Reimann et al., 2011; Bracken-Grissom et al., 2013). Lithodidae are only found in temperate regions and deep-sea habitats. Few genera have a global distribution via the deep sea, while the largest number of lithodid genera is restricted to the North Pacific, where they display a high morphological diversity (Stevens & Lovrich, 2014). From the distribution pattern, combined with physiological and phylogenetic data, it has been concluded that lithodid crabs originated in the shallow North Pacific (Makarov, 1938; Zaklan, 2002; Hall & Thatje, 2009b). While the shallow-water genera of the North Pacific are monotypic or only contain few species, the king crab genera Lithodes Latreille, 1806; Neolithodes A. Milne-Edwards & Bouvier, 1894; and Paralomis White, 1856 are species rich, and new species have been frequently discovered in the last decades (Williams, Smith & Baco, 2000; Macpherson, 2001, 2003, 2004; Takeda & Nagai, 2004; Ahyong & Dawson, 2006; Spiridonov et al., 2006; Takeda & Bussarawit, 2007; Macpherson & Chan, 2008; Hall & Thatje, 2009a; Guzmán, 2009; Ahyong, 2010a, b; Ahyong & Chan, 2010; Muñoz & García-Isarch, 2013). The deep-sea lineages diversified on a global scale and constitute the majority of today’s species diversity of Lithodidae (Hall & Thatje, 2009b). Lithodidae exhibit a wide range of morphological diversity, ranging from small-sized species, such as Hapalogaster Brandt, 1850; Dermaturus Brandt, 1850; or Cryptolithodes Brandt, 1848, to the large box and king crabs. Balss (1924) pointed out the similarities between different body shapes of lithodids and brachyuran crabs as prime examples for convergent evolution. The gross morphology of the lithodid genus Cryptolithodes, for example, superficially resembles that of the brachyuran genus Aethra Latreille in Cuvier, 1816. The large king crabs on the other hand, in particular the genus Lithodes, resemble the brachyuran spider crabs of the genus Maja Lamarck, 1801. Knowledge of internal relationships within the Lithodidae is limited, and the association of the two lithodid subfamilies, Hapalogastrinae Brandt, 1850 and Lithodinae Samouelle, 1819, is uncertain (Hall & Thatje, 2009b; Bracken-Grissom et al., 2013). Bracken-Grissom et al. (2013) recovered different relationships between taxa of the two subfamilies, depending on their analyses. Using only molecular data, neither taxa were found to be monophyletic, while including morphological data in their analyses resolved both taxa as monophyletic. Hall and Thatje (2009a) resolved both subfamilies as monophyletic, but with a poorly supported placement of Cryptolithodes (Hapalogastrinae). Recently, Cryptolithodes was, however, placed outside Lithodinae and within Hapalogastrinae based on molecular data (Thatje & Hall, 2016). The two subfamilies are separated by the calcification of the third to fifth tergites of the pleon in Lithodinae or lack thereof in Hapalogastrinae (McLaughlin, 2014). Cryptolithodes, however, is the most heavily calcified lithodid crab, and its ambiguous phylogenetic placement makes the use of the degree of pleon calcification as an autapomorphy for the two subfamilies uncertain. Information on basal lithodid relationships is crucial for understanding the evolutionary pathway of lithodid crabs and the driving forces leading from a shell-inhabiting to a free-living lifestyle via the process of carcinization. In the present study, we construct an extensive molecular phylogeny of Paguridae and Lithodidae using nuclear ribosomal and mitochondrial genes to cover a suitable range of genetic variability (Toon et al., 2009). New and available sequence data are combined to construct the most comprehensive phylogeny of pagurid hermit crabs and lithodid crabs to date in the search for the closest living relatives of Lithodidae within the hermit crabs. MATERIAL AND METHODS Taxon sampling New sequences from 40 species of Paguridae and Lithodidae were obtained for this study. Specimens were obtained both in the field and from museum collections (Table 1). Collection of new material focussed on the temperate regions of the Northern Hemisphere, in particular the North Pacific. This region has been highlighted as the region of origin of Lithodidae (Hall & Thatje, 2009b), and the closest extant relatives to lithodids are likely found in this region. The data set was complemented by a large range of taxa of Paguridae and Lithodidae with sequences available in GenBank and the Barcode of Life Data Systems (BOLD), to cover a broad range of taxa and geographic regions (Table 1). Six representatives of the genera Paguristes and Areopaguristes (family Diogenidae Ortmann, 1892) were chosen as outgroup taxa, based on previous studies that indicated these genera as closest relatives to Paguridae and Lithodidae (Morrison et al., 2002; Bracken-Grissom et al., 2013). Table 1. Taxa included in this study with list of GenBank and BOLD accession numbers of the molecular marker. Accession numbers of new sequences are indicated with an asterisk Taxa  16S  18S  28S  COI  12S  Family Diogenidae   Areopaguristes hewatti  KF182535  KF182482  KF182644  –  –   Areopaguristes hummi  KF182542  KF182484  KF182641  –  –   Paguristes cadenati  KF182540  KF182493  KF182637  –  –   Paguristes puncticeps  KF182538  KF182487  KF182639  –  –   Paguristes triangulatus  KF182539  KF182489  KF182638  –  –   Paguristes turgidus  AF436056  AF436020  AF435997  DQ882097  –  Family Lithodidae   Subfamily Hapalogastrinae    Acantholithodes hispidus  –  –  –  DQ882026  –    Hapalogaster dentata  AF425327  –  AF425347  AF425306  AF425306    Hapalogaster grebnitzkii  KY426325*  –  –  –  KY426292*    Hapalogaster mertensii  AF425328  KF182451  KF182601  KY426267*  AF425307    Oedignathus inermis  AF425334  –  AF425313  AF425353  AF425313    Placetron wosnessenskii  KY426329*  –  KY454171*  DSALA006-06  –   Subfamily Lithodinae    Cryptolithodes sitchensis  AF425324  KF182453  KF182603  KC107820  AF425303    Cryptolithodes typicus  AF425325  AF436019  AF425345  AF425304  AF425304    Glyptolithodes cristatipes  AF425326  –  AF425346  AF425305  AF425305    Lithodes aequispinus  KY426332*  –  KY454183*  AF425308  AF425308    Lithodes confundens  HM020949  –  FJ462642  HM020901  –    Lithodes couesi  –  –  –  DQ882086  –    Lithodes ferox  HM020950  –  HM020856  KY426276*  KY426296*    Lithodes longispina  –  –  –  AB476815  –    Lithodes maja  KY426333*  KY454206*  AF425350  FJ581746  AF425309    Lithodes murrayi  HM020954  –  HM020857  HM020899  –    Lithodes nintokuae  AB769476  –  –  AB769476  AB769476    Lithodes paulayi  –  –  –  GU289677  –    Lithodes santolla  KF182572  AF439385  KF182602  KY426275*  AF425310    Lithodes turkayi  EU493268  –  –  KC196529  –    Lithodes turritus  KJ132573  –  –  –  –    Lopholithodes foraminatus  KY426330*  –  KY454182*  DQ882088  KY426295*    Lopholithodes mandtii  AF425333  KY454205*  AF425352/KY454174*  KY426271*  AF425312    Neolithodes asperrimus  HM020940  –  HM020847  HM020891  –    Neolithodes diomedeae  –  –  –  KC196528  –    Neolithodes duhameli  HM020946  –  HM020849  HM020892  –    Neolithodes grimladii  –  –  –  JQ305973  –    Neolithodes nr. brodiei  HM020942  –  FJ462640/HM020888  HM020894  –    Paralithodes brevipes  AF425337  –  AF425356  NC021458  AF425316    Paralithodes camtschaticus  AF425338  JN192147  AB193823  JF738154  AF425317    Paralithodes platypus  KY426328*  JN192152  AB193821  KY426274*  KY426297*    Paralomis aculeata  HM020958  –  HM020862  HM020904  –    Paralomis africana  EU493275  –  HM020864  HM020907  –    Paralomis anamerae  HM020959  –  HM020865  HM020906  –    Paralomis birsteini  KY426326*  –  HM020867  EU493260  KY426294*    Paralomis cristata  EU493267  –  –  HM020911  –    Paralomis cristulata  EU493271  –  HM020870  HM020908  –    Paralomis dofleini  HM020962  –  HM020871  HM020912  –    Paralomis elongata  –  –  HM020872/HM020887/ HM20884  HM020914  –    Paralomis erinacea  HM020966  –  HM020873  HM020915  –    Paralomis formosa  HM020971  –  FJ462641/HM020886  HM020918  –    Paralomis granulosa  AF425339  –  AF425358/HM020877  AF425318  AF425318    Paralomis hirtella  KY426327*  –  –  KY426272*  KY426293*    Paralomis multispina  –  –  –  AB211296  –    Paralomis pacifica  –  –  –  AB476750  –    Paralomis spinosissima  HM020982  –  HM020879  HM020927  –    Paralomis zealandica  HM020980  –  –  HM020935  –    Phyllolithodes papillosus  AF425340  KY454204*  AF425359/KY454175*  KY426273*  AF425319    Rhinolithodes wosnessenskii  KY426331*  –  AF425360  AF425320  AF425320  Family Paguridae   Agaricochirus alexandri  –  KF182447  KF182593  –  –   Anapagurus breviaculeatus  KY426316*  –  KY454162*  KY426262*  KY426286*   Anapagurus chiroacanthus  KY426315*  KY454187*  KY454161*  KY426263*  KY426285*   Anapagurus hydmanni  –  KJ182993  –  KJ183012  –   Anapagurus laevis  KY426317*  KY454186*  KY454163*  BNSC284-11  KY426287*   Cestopagurus timidus  KY426314*  KY454192*  KY454159*  KY426261*  KY426288*   Discorsopagurus schmitti  AF436055  AF436017  KY454176*  KY426283*  KY426298*   Elassochirus cavimanus  KY426342*  –  –  KY426281*  KY426302*   Elassochirus gilli  KY426343*  –  –  KY426282*  KY426300*   Elassochirus tenuimanus  KY426341*  KY454198*  KY454184*  KY426279*  KY426301*   Labidochirus splendescens  AF425332  –  AF425351  –  AF425311   Manucomplanus ungulatus  KF182575  KF182457  KF182612  –  –   Nematopagurus gardineri  –  –  –  MDECA670-10  –   Nematopagurus longicornis  KY426318*  KY454188*  KY454169*  KY426264*  KY426289*   Nematopagurus meiringae  –  –  –  MDECA669-10  –   Paguridium minimum  KY426319*  KY454191*  KY454168*  –  KY426308*   Pagurus acadianus  –  –  –  FJ581812  –   Pagurus alatus  KY426323*  –  JN107619  KY426270*  KY426309*   Pagurus aleuticus  KY426340*  –  KY454180*  KY426280*  KY426299*   Pagurus arcuatus  –  –  –  FJ581817  –   Pagurus armatus  –  –  –  AF483159  –   Pagurus beringanus  KY426337*  KY454201*  KY454173*  KY426277*  KY426307*   Pagurus bernhardus  KY426339*  KY454197*  JN107623/KY454185*  JN107580  AF425314   Pagurus brachiomastus  –  –  –  JN5990075  –   Pagurus brevidactylus  KF182563  KF182495  KF182610  –  –   Pagurus bullisi  KF182568  KF182454  KF182595  –  –   Pagurus caurinus  KY426336*  KY454200*  KY454181*  KY426278*  KY426306*   Pagurus chevreuxi  KY426312*  -  KY454160*  –  –   Pagurus comptus  FJ869145  KY454202*  KY454170*  KY426265*  KY426290*   Pagurus criniticornis  DQ369947  –  –  –  –   Pagurus cuanensis  KY426322*  KY454190*  JN107625  JN107584  KY426310*   Pagurus edwardsii  FJ869146  –  –  CFAD141-11  –   Pagurus excavatus  JN107610  –  JN107628  JN107587  –   Pagurus exilis  FJ869147  –  –  -  –   Pagurus forbesii  KF962984  –  –  KF962980  –   Pagurus forceps  FJ869150  –  –  –  –   Pagurus gladius  JX238503  –  –  –  –   Pagurus granosimanus  KY426338*  KY454196*  KY454178*  GU442314  KY426305*   Pagurus hirsutiusculus  KY426334*  KY454193*  KY454177*  GU442400  AF425315   Pagurus kennerlyi  KY426345*  KY454195*  KY454172*  KY426284*  KY426304*   Pagurus leptonyx  DQ369946  –  –  –  –   Pagurus longicarpus  AF150756  AF436018  AF425343/AY739185  AF150756  AF150756   Pagurus maclaughlinae  KF182566  KF182460  KF182611  –  –   Pagurus mbizi  KY426320*  –  KY454167*  –  KY426311*   Pagurus minutus  –  –  –  JX502978  –   Pagurus nr. carolinensis  KF182565  KF182465  KF182609  –  –   Pagurus ochotensis  KY426335*  KY454199*  KY454179*  JN590062  –   Pagurus pectinatus  –  –  –  JN5990060  –   Pagurus perlatus  JQ805783  –  –  –  –   Pagurus pollicaris  FJ869152  KF182458  KF182589  AF483163  –   Pagurus prideaux  KY426321*  KY454189*  JN107629  JQ306249  –   Pagurus provenzanoi  FJ869154  –  –  –  –   Pagurus proximus  –  –  –  KC347562  –   Pagurus pseudosculptimanus  KF962986  –  KY454165*  KY426268*  –   Pagurus pubescens  KY426344*  KY454194*  JN107633  JQ305956  KY426303*   Pagurus pubescentulus  KY426324*  –  KY454166*  KY426269*  –   Pagurus samuelis  –  –  –  GU443022  –   Pagurus similis  –  –  –  HM180751  –   Pagurus stimpsoni  KF182564  KF182466  KF182613  –  –   Pagurus venturensis  –  –  –  GU442190  –   Pagurus villosus  FJ869155  –  –  CFAD136-11  –   Phimochirus holthuisi  KF182578  KF182455  KF182594  –  –   Phimochirus randalli  KF182577  KF182450  KF182591  –  –   Pylopaguridium markhami  KF182570  KF182478  KF182597  –  –   Pylopagurus discoidalis  KF182569  KF182496    –  –   Spiropagurus elegans  KY426313*  KY454203*  KY454164*  KY426266*  KY426291*   Spiropagurus profundorum  –  –  –  MDECA610-10  –   Tomopagurus merimaculosus  KF182567  KF182497  KF182590  –  –  Taxa  16S  18S  28S  COI  12S  Family Diogenidae   Areopaguristes hewatti  KF182535  KF182482  KF182644  –  –   Areopaguristes hummi  KF182542  KF182484  KF182641  –  –   Paguristes cadenati  KF182540  KF182493  KF182637  –  –   Paguristes puncticeps  KF182538  KF182487  KF182639  –  –   Paguristes triangulatus  KF182539  KF182489  KF182638  –  –   Paguristes turgidus  AF436056  AF436020  AF435997  DQ882097  –  Family Lithodidae   Subfamily Hapalogastrinae    Acantholithodes hispidus  –  –  –  DQ882026  –    Hapalogaster dentata  AF425327  –  AF425347  AF425306  AF425306    Hapalogaster grebnitzkii  KY426325*  –  –  –  KY426292*    Hapalogaster mertensii  AF425328  KF182451  KF182601  KY426267*  AF425307    Oedignathus inermis  AF425334  –  AF425313  AF425353  AF425313    Placetron wosnessenskii  KY426329*  –  KY454171*  DSALA006-06  –   Subfamily Lithodinae    Cryptolithodes sitchensis  AF425324  KF182453  KF182603  KC107820  AF425303    Cryptolithodes typicus  AF425325  AF436019  AF425345  AF425304  AF425304    Glyptolithodes cristatipes  AF425326  –  AF425346  AF425305  AF425305    Lithodes aequispinus  KY426332*  –  KY454183*  AF425308  AF425308    Lithodes confundens  HM020949  –  FJ462642  HM020901  –    Lithodes couesi  –  –  –  DQ882086  –    Lithodes ferox  HM020950  –  HM020856  KY426276*  KY426296*    Lithodes longispina  –  –  –  AB476815  –    Lithodes maja  KY426333*  KY454206*  AF425350  FJ581746  AF425309    Lithodes murrayi  HM020954  –  HM020857  HM020899  –    Lithodes nintokuae  AB769476  –  –  AB769476  AB769476    Lithodes paulayi  –  –  –  GU289677  –    Lithodes santolla  KF182572  AF439385  KF182602  KY426275*  AF425310    Lithodes turkayi  EU493268  –  –  KC196529  –    Lithodes turritus  KJ132573  –  –  –  –    Lopholithodes foraminatus  KY426330*  –  KY454182*  DQ882088  KY426295*    Lopholithodes mandtii  AF425333  KY454205*  AF425352/KY454174*  KY426271*  AF425312    Neolithodes asperrimus  HM020940  –  HM020847  HM020891  –    Neolithodes diomedeae  –  –  –  KC196528  –    Neolithodes duhameli  HM020946  –  HM020849  HM020892  –    Neolithodes grimladii  –  –  –  JQ305973  –    Neolithodes nr. brodiei  HM020942  –  FJ462640/HM020888  HM020894  –    Paralithodes brevipes  AF425337  –  AF425356  NC021458  AF425316    Paralithodes camtschaticus  AF425338  JN192147  AB193823  JF738154  AF425317    Paralithodes platypus  KY426328*  JN192152  AB193821  KY426274*  KY426297*    Paralomis aculeata  HM020958  –  HM020862  HM020904  –    Paralomis africana  EU493275  –  HM020864  HM020907  –    Paralomis anamerae  HM020959  –  HM020865  HM020906  –    Paralomis birsteini  KY426326*  –  HM020867  EU493260  KY426294*    Paralomis cristata  EU493267  –  –  HM020911  –    Paralomis cristulata  EU493271  –  HM020870  HM020908  –    Paralomis dofleini  HM020962  –  HM020871  HM020912  –    Paralomis elongata  –  –  HM020872/HM020887/ HM20884  HM020914  –    Paralomis erinacea  HM020966  –  HM020873  HM020915  –    Paralomis formosa  HM020971  –  FJ462641/HM020886  HM020918  –    Paralomis granulosa  AF425339  –  AF425358/HM020877  AF425318  AF425318    Paralomis hirtella  KY426327*  –  –  KY426272*  KY426293*    Paralomis multispina  –  –  –  AB211296  –    Paralomis pacifica  –  –  –  AB476750  –    Paralomis spinosissima  HM020982  –  HM020879  HM020927  –    Paralomis zealandica  HM020980  –  –  HM020935  –    Phyllolithodes papillosus  AF425340  KY454204*  AF425359/KY454175*  KY426273*  AF425319    Rhinolithodes wosnessenskii  KY426331*  –  AF425360  AF425320  AF425320  Family Paguridae   Agaricochirus alexandri  –  KF182447  KF182593  –  –   Anapagurus breviaculeatus  KY426316*  –  KY454162*  KY426262*  KY426286*   Anapagurus chiroacanthus  KY426315*  KY454187*  KY454161*  KY426263*  KY426285*   Anapagurus hydmanni  –  KJ182993  –  KJ183012  –   Anapagurus laevis  KY426317*  KY454186*  KY454163*  BNSC284-11  KY426287*   Cestopagurus timidus  KY426314*  KY454192*  KY454159*  KY426261*  KY426288*   Discorsopagurus schmitti  AF436055  AF436017  KY454176*  KY426283*  KY426298*   Elassochirus cavimanus  KY426342*  –  –  KY426281*  KY426302*   Elassochirus gilli  KY426343*  –  –  KY426282*  KY426300*   Elassochirus tenuimanus  KY426341*  KY454198*  KY454184*  KY426279*  KY426301*   Labidochirus splendescens  AF425332  –  AF425351  –  AF425311   Manucomplanus ungulatus  KF182575  KF182457  KF182612  –  –   Nematopagurus gardineri  –  –  –  MDECA670-10  –   Nematopagurus longicornis  KY426318*  KY454188*  KY454169*  KY426264*  KY426289*   Nematopagurus meiringae  –  –  –  MDECA669-10  –   Paguridium minimum  KY426319*  KY454191*  KY454168*  –  KY426308*   Pagurus acadianus  –  –  –  FJ581812  –   Pagurus alatus  KY426323*  –  JN107619  KY426270*  KY426309*   Pagurus aleuticus  KY426340*  –  KY454180*  KY426280*  KY426299*   Pagurus arcuatus  –  –  –  FJ581817  –   Pagurus armatus  –  –  –  AF483159  –   Pagurus beringanus  KY426337*  KY454201*  KY454173*  KY426277*  KY426307*   Pagurus bernhardus  KY426339*  KY454197*  JN107623/KY454185*  JN107580  AF425314   Pagurus brachiomastus  –  –  –  JN5990075  –   Pagurus brevidactylus  KF182563  KF182495  KF182610  –  –   Pagurus bullisi  KF182568  KF182454  KF182595  –  –   Pagurus caurinus  KY426336*  KY454200*  KY454181*  KY426278*  KY426306*   Pagurus chevreuxi  KY426312*  -  KY454160*  –  –   Pagurus comptus  FJ869145  KY454202*  KY454170*  KY426265*  KY426290*   Pagurus criniticornis  DQ369947  –  –  –  –   Pagurus cuanensis  KY426322*  KY454190*  JN107625  JN107584  KY426310*   Pagurus edwardsii  FJ869146  –  –  CFAD141-11  –   Pagurus excavatus  JN107610  –  JN107628  JN107587  –   Pagurus exilis  FJ869147  –  –  -  –   Pagurus forbesii  KF962984  –  –  KF962980  –   Pagurus forceps  FJ869150  –  –  –  –   Pagurus gladius  JX238503  –  –  –  –   Pagurus granosimanus  KY426338*  KY454196*  KY454178*  GU442314  KY426305*   Pagurus hirsutiusculus  KY426334*  KY454193*  KY454177*  GU442400  AF425315   Pagurus kennerlyi  KY426345*  KY454195*  KY454172*  KY426284*  KY426304*   Pagurus leptonyx  DQ369946  –  –  –  –   Pagurus longicarpus  AF150756  AF436018  AF425343/AY739185  AF150756  AF150756   Pagurus maclaughlinae  KF182566  KF182460  KF182611  –  –   Pagurus mbizi  KY426320*  –  KY454167*  –  KY426311*   Pagurus minutus  –  –  –  JX502978  –   Pagurus nr. carolinensis  KF182565  KF182465  KF182609  –  –   Pagurus ochotensis  KY426335*  KY454199*  KY454179*  JN590062  –   Pagurus pectinatus  –  –  –  JN5990060  –   Pagurus perlatus  JQ805783  –  –  –  –   Pagurus pollicaris  FJ869152  KF182458  KF182589  AF483163  –   Pagurus prideaux  KY426321*  KY454189*  JN107629  JQ306249  –   Pagurus provenzanoi  FJ869154  –  –  –  –   Pagurus proximus  –  –  –  KC347562  –   Pagurus pseudosculptimanus  KF962986  –  KY454165*  KY426268*  –   Pagurus pubescens  KY426344*  KY454194*  JN107633  JQ305956  KY426303*   Pagurus pubescentulus  KY426324*  –  KY454166*  KY426269*  –   Pagurus samuelis  –  –  –  GU443022  –   Pagurus similis  –  –  –  HM180751  –   Pagurus stimpsoni  KF182564  KF182466  KF182613  –  –   Pagurus venturensis  –  –  –  GU442190  –   Pagurus villosus  FJ869155  –  –  CFAD136-11  –   Phimochirus holthuisi  KF182578  KF182455  KF182594  –  –   Phimochirus randalli  KF182577  KF182450  KF182591  –  –   Pylopaguridium markhami  KF182570  KF182478  KF182597  –  –   Pylopagurus discoidalis  KF182569  KF182496    –  –   Spiropagurus elegans  KY426313*  KY454203*  KY454164*  KY426266*  KY426291*   Spiropagurus profundorum  –  –  –  MDECA610-10  –   Tomopagurus merimaculosus  KF182567  KF182497  KF182590  –  –  View Large Molecular work Extraction Specimens collected for this study were preserved in 96% ethanol prior to DNA extraction. Molecular work was conducted in the Biodiversity Laboratories, University of Bergen, Norway. Total genomic DNA was extracted from muscle tissue using a Gene Mole automatic nucleic acid extractor from Mole Genetics AS, Norway, or using the Qiagen DNeasy Blood and Tissue kit (QIAGEN Inc., Valencia, CA, USA), following the manufacturer’s standard protocols. PCR and sequencing Sequences of five genes were amplified by PCR: three mitochondrial markers [ribosomal 12S and 16S rRNA subunits, and cytochrome c oxidase 1 (COI)] and two nuclear markers (18S and 28S rRNA subunits). PCR reactions were carried out on a Bio-Rad C1000 Thermal Cycler using Takara polymerase in 25-µL reactions. Primers from previous studies as well as newly designed primers were used (Table 2). The 28S gene was amplified using two primer pairs, resulting in two overlapping fragments. For some species, the 12S and COI genes, situated next to each other in the mitochondrial genome, were amplified in a single PCR run, using the primers 12S-A-Paguridae and COI-B-Paguridae. All PCR products were checked for successful amplification on 1% agarose gels stained with GelRed. PCR products were purified and sequenced in both directions at Macrogen Inc. using the same primers as for amplification. For the 18S fragment two additional primers (18S-A− and 18S-B+) were used for sequencing the entire PCR product. Contigs were assembled using Lasergene SeqMan Pro 8.1. To exclude a possible presence of pseudogenes or gene duplicates, the individual sequence chromatogram files were checked for the presence of double peaks, and the COI alignment was translated into amino acids and checked for premature stop codons and frame shifts using BioEdit 7.2.3. Sequences are deposited in GenBank (Table 1). Table 2. Primers used for PCR amplification and sequencing Marker  Primer  Primer sequence (5′–3′)  Reference  COI  HCO2198  TAA ACT TCA GGG TGA CCA AAA AAT CA  Folmer et al. (1994)   LCO1490  GGT CAA CAA ATC ATA AAG ATA TTG G  Folmer et al. (1994)   COI-A-Paguridae  TCT TAT ATT TCC ACT ATA AAG CC  This study  COI-B-Paguridae  ATT CTT GAC TTA CAA TRT GTG A  This study  16S  LR-N-13398  CGC CTG TTT AAC AAA AAC AT  Simon et al. (1994)   LR-J-12887  CCG GTC TGA ACT CAG ATC ACG T  Simon et al. (1994)   16S-A-Paguridae  AAG ATA GAA ACC AAC CTG GCT C  This study  16S-B-Paguridae  TGC CTG TTT AAC AAA AAC ATG TC  This study  12S  12S-A-Paguridae  ATT ATA ATA GGG TAT CTA ATC CTA G  This study  12S-B-Paguridae  AAT GTT CCA ATR TCT TTA TGG  This study  18S  18S-329  TAA TGA TCC TTC CGC AGG TT  Spears et al. (1992)   18S-328  CCT GGT TGA TCC TGC CAG  Spears et al. (1992)   18S-A− (sequencing)  CAG CMG CC GCG GTA ATW C  Spears et al. (1992)   18S-B+ (sequencing)  ATT CCC CGT TAC CCG  Spears et al. (1992)   28S  28S-OI  GCG GAG GAA AAG AAA CTA AC  Zaklan (2001)   28S-R443  CCT CAC GGT ACT TGT TCG CTA TCG G  Ahyong et al. (2009)   28S-Paguridae-F1  CGT AGA GTC GGG TTG CTT GA  This study  28S-Paguridae-R1  CTT TCG GGT CCC AAC ATG TC  This study  Marker  Primer  Primer sequence (5′–3′)  Reference  COI  HCO2198  TAA ACT TCA GGG TGA CCA AAA AAT CA  Folmer et al. (1994)   LCO1490  GGT CAA CAA ATC ATA AAG ATA TTG G  Folmer et al. (1994)   COI-A-Paguridae  TCT TAT ATT TCC ACT ATA AAG CC  This study  COI-B-Paguridae  ATT CTT GAC TTA CAA TRT GTG A  This study  16S  LR-N-13398  CGC CTG TTT AAC AAA AAC AT  Simon et al. (1994)   LR-J-12887  CCG GTC TGA ACT CAG ATC ACG T  Simon et al. (1994)   16S-A-Paguridae  AAG ATA GAA ACC AAC CTG GCT C  This study  16S-B-Paguridae  TGC CTG TTT AAC AAA AAC ATG TC  This study  12S  12S-A-Paguridae  ATT ATA ATA GGG TAT CTA ATC CTA G  This study  12S-B-Paguridae  AAT GTT CCA ATR TCT TTA TGG  This study  18S  18S-329  TAA TGA TCC TTC CGC AGG TT  Spears et al. (1992)   18S-328  CCT GGT TGA TCC TGC CAG  Spears et al. (1992)   18S-A− (sequencing)  CAG CMG CC GCG GTA ATW C  Spears et al. (1992)   18S-B+ (sequencing)  ATT CCC CGT TAC CCG  Spears et al. (1992)   28S  28S-OI  GCG GAG GAA AAG AAA CTA AC  Zaklan (2001)   28S-R443  CCT CAC GGT ACT TGT TCG CTA TCG G  Ahyong et al. (2009)   28S-Paguridae-F1  CGT AGA GTC GGG TTG CTT GA  This study  28S-Paguridae-R1  CTT TCG GGT CCC AAC ATG TC  This study  View Large Sequence alignments The data set included 49 Lithodidae and 69 Paguridae species, as well as six Diogenidae species as outgroup taxa. Sequences for COI, 16S, 12S and 18S were aligned in eBioX 1.5 using the MUSCLE algorithm (Edgar, 2004). 28S sequences were aligned in the online version of MAFFT 7 (Katoh & Standley, 2013) using the E-INS-i strategy. Individual alignments were checked by eye. The COI alignment was checked by translation into amino acids using Seaview 4.4 with the genetic code set to ‘Invertebrate mt’. Sequences downloaded from GenBank were cut to the corresponding sequence region of our own PCRs. BLAST searches of sequences that appeared suspicious in the alignments revealed few published sequences to be contaminations, which therefore were removed from the data set. Those sequences are 28S for Pylopagurus discoidalis (A. Milne-Edwards, 1880) (KF182614); COI for Nematopagurus squamichelis Alcock, 1905 (KJ150706); and COI for Pylopaguropsis magnimanus (Henderson, 1896) (KM043479). A 16S sequence attributed to Cestopagurus timidus (Roux, 1830) (FR849637) available on GenBank is misidentified and appears to belong to Pagurus prideaux Leach, 1815 based on comparison with our data from these species. The 18S sequence of Pylopaguridium markhami McLaughlin & Lemaitre, 2001 (KF182478) was trimmed, since the end of the sequence consists of a repetition of a previous section of the same sequence, possibly generated during processing of the sequence data. The 18S sequence of Oedignathus inermis (Stimpson, 1860) (Z14062) was excluded due to few obvious minor sequencing errors in highly conservative regions. The 18S (KF182453) and 28S (KF182603) sequences of Cryptolithodes sp. were assigned to Cryptolithodes sitchensis Brandt, 1853, based on the 16S gene of Cryptolithodes sp. from the same study (KF182574) (Bracken-Grissom et al., 2013), which is identical to sequences of C. sitchensis from other studies. GenBank sequences attributed to Neolithodes brodiei Dawson & Yaldwyn, 1970, sampled from Vanuatu (Snow, 2010) appear to come from an undescribed species (Ahyong, 2010b) and are here referred to as ‘Neolithodes nr. brodiei’. The alignments of the non-protein coding genes were subsequently run in Gblocks 0.91b (Castresana, 2000) to exclude ambiguous aligned regions, using the Gblocks server. Gblocks criteria used for this were for a less stringent selection, allowing for gaps within blocks, smaller final blocks and less strict flanking positions. The 28S alignment was not complete for all taxa over the entire length. Gblocks treats missing data like gaps and would, therefore, also remove highly conserved regions in this alignment. Ambiguously aligned regions in 28S were removed by hand, using the same parameters as Gblocks, but considering only gaps. The single gene alignments were concatenated to a single file using MacClade 4.06. PartitionFinder 1.1.1 (Lanfear et al., 2012) was used to determine the best partitioning scheme and best-fit nucleotide substitution models for the concatenated data set under the Bayesian information criterion. The ‘greedy’ algorithm was used with branch lengths of alternative partitions ‘linked’. The analysis suggested a partitioning of the data set by each marker as well as each codon position for the protein-coding COI. PartitionFinder suggested as the best-fit substitution models SYM + I + G for the first codon position of COI and 18S, F81 for the second codon position of COI, GTR + G for the third codon position of COI, GTR + I + G for 16S and 28S, and HKY + I + G for 12S. Phylogenetic analyses The concatenated data set was analysed using maximum likelihood (ML) and Bayesian inference (BI) approaches. The ML analysis were conducted using RAxML 8.2.4 (Stamatakis, 2014), on the CIPRES science gateway (Miller, Pfeiffer & Schwartz, 2010). A unique GTR model of sequence evolution was specified for each partition following the scheme given by PartitionFinder with corrections for a discrete gamma distribution for site-rate heterogeneity (GTRGAMMA). The GTRCAT model was used for the bootstrapping phase. Thousand rapid bootstrap iterations were conducted to search for the best-scoring ML tree in one single program run. BI was conducted in MrBayes 3.2.2 (Ronquist et al., 2012), on the Lifeportal, University of Oslo. The concatenated data set was partitioned following the scheme given by PartitionFinder. Each partition was run under the best-fit model of evolution, and all model parameter values were ‘unlinked’ among partitions. Two independent runs using four Metropolis-coupled Markov chain Monte Carlo analyses were performed. The chains were run for 20 million generations and sampled every 500 generations. The first 10000 trees were discarded as burn-in, and a 50% majority-rule consensus tree was obtained from the remaining saved trees. The average standard deviation of split frequencies was checked for convergence towards zero, and MrBayes parameter files were examined in Tracer 1.6 (Rambaut et al., 2014) to assess if runs had reached a stationary phase and converged on model parameters. A second data set was produced, based on the phylogenetic tree obtained from the initial analyses, comprising only representatives of Lithodidae and pagurid hermit crabs that had been identified as sister clade to Lithodidae, as well as one outgroup taxon (Pagurus comptus White, 1847). Ambiguously aligned positions of the non-protein-coding gene alignments were removed as described for the initial data set. Since the gene alignments with this limited number of taxa contained fewer ambiguous positions, the resulting alignments were longer and contained more phylogenetic information. The single gene alignments were concatenated, and PartitionFinder was used as described above. The analyses suggested the same partition scheme as in the previous data set, with the same best-fit substitution models, except F81 + I for the second codon position of COI, HKY + I + G for 16S and K80 + I for 18S. The data set was analysed using ML and BI as described for the initial data set. The resulting phylogenetic trees were visualized using Dendroscope 3.2 (Huson & Scornavacca, 2012). RESULTS The phylogenetic trees obtained from ML and BI analyses were largely corresponding. Support values, however, were lower in the trees resulting from the ML analyses than in the trees obtained by BI (Figs 1, 2). We included the mitochondrial 12S and 16S rRNAs and the mitochondrial cytochrome c oxidase 1 (COI), as well as parts of the nuclear 18S and 28S rRNAs. All these genes have previously been proven to be useful in systematic studies of crustaceans (Schubart, Neigel & Felder, 2000). Different rates of evolution among the genes make a concatenated set of these markers a valuable phylogenetic tool for resolving a range of taxonomic levels (Toon et al., 2009). Additional sequences from a large range of pagurid and lithodid species were downloaded from GenBank and BOLD, allowing us to compile the most complete data set to date, in the search for the closest extant relatives of lithodid crabs. The resulting data set was fragmentary for many taxa, since for most species only one or few genes were available in GenBank and BOLD. Deep phylogenetic nodes were resolved by taxa with a larger coverage in the matrix, while species with low coverage, for example only COI, clearly affiliated with taxa that had a larger coverage, usually congeneric species. Figure 1. View largeDownload slide Bayesian 50% majority-rule tree of Paguridae, Lithodidae and Diogenidae (outgroup) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Colour fields indicating Lithodidae (blue), Paguridae (yellow) and Diogenidae (green). The red dot indicates the node corresponding to the LCA shared by Lithodidae and pagurid hermit crabs. Pagurus species assigned to informal species groups are highlighted with a grey overlay. Figure 1. View largeDownload slide Bayesian 50% majority-rule tree of Paguridae, Lithodidae and Diogenidae (outgroup) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Colour fields indicating Lithodidae (blue), Paguridae (yellow) and Diogenidae (green). The red dot indicates the node corresponding to the LCA shared by Lithodidae and pagurid hermit crabs. Pagurus species assigned to informal species groups are highlighted with a grey overlay. Figure 2. View largeDownload slide Bayesian 50% majority-rule tree of Lithodidae (blue) and the hermit crab clade, which constitutes the closest relatives within the Paguridae, the ‘pagurid–lithodid sister clade’ (PLS clade) (yellow) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Species of the subfamily Hapalogastrinae are highlighted by dark blue overlay. Pagurus s.s. species of the ‘bernhardus’ group are highlighted by dark yellow overlay. Red stars indicating clades with deep-sea radiations outside the North Pacific. Illustrated specimens are not to scale. Figure 2. View largeDownload slide Bayesian 50% majority-rule tree of Lithodidae (blue) and the hermit crab clade, which constitutes the closest relatives within the Paguridae, the ‘pagurid–lithodid sister clade’ (PLS clade) (yellow) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Species of the subfamily Hapalogastrinae are highlighted by dark blue overlay. Pagurus s.s. species of the ‘bernhardus’ group are highlighted by dark yellow overlay. Red stars indicating clades with deep-sea radiations outside the North Pacific. Illustrated specimens are not to scale. Our results show a monophyletic Lithodidae, deeply nested within a paraphyletic hermit crab family Paguridae (Fig. 1). Paguridae with the contained Lithodidae was found clearly distinct from the diogenid outgroup taxa (Fig. 1). A clade of pagurid hermit crabs was clearly resolved as the sister taxon to Lithodidae within the Paguridae, which we refer to as ‘pagurid–lithodid sister clade’ (PLS clade) (Fig. 1). This clade shared the last common ancestor (LCA) with lithodid crabs within the hermit crabs. All species included in this clade are shallow-water hermit crabs, mainly distributed in the North Pacific. It consists of some genera exclusively found in the North Pacific: Discorsopagurus, Elassochirus and Labidochirus, as well as various species of Pagurus. Included representatives of Pagurus in the PLS clade belong to several of the established informal Pagurus morpho-groups; the ‘bernhardus’, ‘trigonocheirus’, ‘capillatus’ and part of the ‘comptus’ group (Forest & de Saint Laurent, 1968; McLaughlin, 1974; Lemaitre & Cruz-Castaño, 2004), as well as species that have not been assigned to any of these informal groupings. Pagurus species included in this PLS clade are also exclusive to the North Pacific, except for four species from the North Atlantic, which have close related species in the Pacific. The usage of Pagurus as a catch-all genus for species with a general pagurid hermit crab morphology is well illustrated in the phylogenetic tree (Fig. 1), as representatives of the genus are dispersed throughout the family. The analyses largely confirm previously established informal morphological Pagurus groups (Fig. 1). Only representatives of the ‘comptus’ group show a clear separation between species from South America (‘comptus’ group I) and the North Pacific (‘comptus’ group II). Two Pagurus clades, corresponding to the ‘provenzanoi’ group and subdivision I by Ingle (1985), also contain species that are assigned to other genera, Manucomplanus McLaughlin, 1981 and Paguridium Forest, 1961, respectively. Except for Pagurus, all other hermit crab genera included in the phylogenetic analyses with multiple representatives were resolved as monophyletic. While Lithodidae was resolved as monophyletic overall, the two subfamilies Lithodinae and Hapalogastrinae appear not to be monophyletic (Fig. 2). Basal to the remaining lithodid taxa are the two hapalogastrine genera Oedignathus Benedict, 1895 and Hapalogaster (Fig. 2). The lithodine genus Cryptolithodes nests among the hapalogastrine and likely forms the sister taxon to all remaining lithodids, including all Lithodinae and the two monotypic hapalogastrine genera Placetron Schalfeew, 1892 and Acantholithodes Holmes, 1895. The internal phylogeny of the Lithodidae is not fully resolved in our analyses, especially with respect to the genus Paralithodes Brandt, 1848. A sister relationship between the monotypic genera Rhinolithodes Brandt, 1848 and Phyllolithodes Brandt, 1848 is highly supported. The genus Paralomis forms a highly supported clade, also including the monotypic genus Glyptolithodes Faxon, 1895. Sister taxon to Paralomis/Glyptolithodes is the genus Lopholithodes Brandt, 1848, which consists of only two species that were both included in the analyses. Another highly supported clade consists of Lithodes and Neolithodes. DISCUSSION Internal relationships within Lithodidae A monophyletic origin of lithodid crabs has been confirmed in several studies, both using molecular and morphological data (e.g. McLaughlin et al., 2007; Reimann et al., 2011; Bracken-Grissom et al., 2013). Recent molecular studies, however, have left the status of the two lithodid subfamilies, Hapalogastrinae and Lithodinae, ambiguous (Hall & Thatje, 2009b; Bracken-Grissom et al., 2013; Thatje & Hall, 2016). The two taxa are separated based on the presence of a calcified or uncalcified pleon (McLaughlin, 2014). Hapalogastrinae are mostly small-sized crabs with a soft, uncalcified pleon, and have been suggested to represent a morphological intermediate form between pagurid hermit crabs and the large-sized king crabs, most closely resembling the lithodid stem species (Bouvier, 1895; Boas, 1924; Richter & Scholtz, 1994; Keiler et al., 2015). Recently, detailed morphological examinations of Hapalogaster mertensii Brandt, 1850 showed that this small lithodid crab possesses anatomical features of both Paguridae and Lithodidae. Most strikingly, Hapalogaster exhibits an uncalcified sac-like pleon and also features characters of both groups in the vascular system (Keiler et al., 2015). Morphological studies have supported the separation of lithodid crabs into the two distinct subfamilies. Hapalogastrinae were, however, only represented by the single genus Hapalogaster in these studies (Richter & Scholtz, 1994; Keiler & Richter, 2011; Keiler et al., 2013). Few previous molecular studies included representatives of Hapalogastrinae. In two studies, only one representative of each of Hapalogastrinae and Lithodinae was included (Ahyong et al., 2009; Schnabel et al., 2011). Two species of Hapalogastrinae were included in other studies (Hall & Thatje, 2009b; Bracken-Grissom et al., 2013), together with a number of lithodine species. Hall & Thatje (2009a) found two monophyletic subfamilies, but with only low support. Bracken-Grissom et al. (2013) found different internal relationships within the Lithodidae, depending on whether only molecular data were used, or if the data set was combined with morphological data. In the combined data set, Hapalogaster and Oedignathus were resolved as basal within the Lithodidae. In analyses using molecular data alone on the other hand, Oedignathus was placed among the lithodid crabs and distant from Hapalogaster. This placement of Oedignathus, however, appears to be due to a number of apparent sequencing errors in the included 18S gene from this species (GenBank: Z14062) (Kim, Min & Kim, 1992), which we found to be highly conserved in Lithodidae. For this reason, this sequence was excluded from our analyses. Instead of a clear separation into Hapalogastrinae and Lithodinae, various small-sized, shallow-water taxa appear basal in the phylogenetic tree of Lithodidae from our analysis (Fig. 2). The Hapalogastrinae genera Hapalogaster and Oedignathus are resolved as basal to all other lithodids. Two other genera of Hapalogastrinae (Placetron and Acantholithodes) had not previously been included in any phylogenetic study. These were found nested within Lithodinae and appear more derived than the small-sized lithodine genus Cryptolithodes, rendering both subfamilies non-monophyletic. The basal position of some genera of Hapalogastrinae, as indicated by previous studies (Richter & Scholtz, 1994; Keiler & Richter, 2011; Keiler et al., 2013), is in agreement with our results. A soft pleon, as a remnant of the hermit crab origin, must be considered as the plesiomorphic state of Lithodidae. The phylogenetic position of the small-sized lithodine genus Cryptolithodes, however, which is throughout heavily calcified, indicates that calcification of the pleon evolved at least twice within Lithodidae. The closest extant relatives to Lithodidae Our molecular analyses clearly show that a distinct, species-rich clade of hermit crabs forms the direct sister group to Lithodidae within the Paguridae (Fig. 1). Such a morphologically diverse and species-rich hermit crab sister clade to the lithodids is not as surprising as it might appear at first glance, given the species richness and morphological diversity of lithodid crabs themselves. Previous molecular phylogenetic studies have suggested different pagurid taxa as the closest relatives to the king crabs, depending on the representation of pagurid species in the analyses. With internal relationships of the Paguridae largely unknown, especially regarding the polyphyletic genus Pagurus, the topology of previous phylogenetic studies depended on which Pagurus species were included in the data sets. The first molecular study on king crabs (Cunningham et al., 1992) found the closest sister taxa to the lithodids to be a clade containing two species of the genus Pagurus [Pagurus bernhardus (Linnaeus, 1758) and Pagurus acadianus Benedict, 1901], together with the genera Labidochirus and Elassochirus. Two other Pagurus species included in the analyses were found to be more distantly related. Cunningham et al. highlighted the nested position of lithodids within the genus Pagurus. Richter & Scholtz (1994) subsequently noted that strong similarities of the first antennae between P. bernhardus and lithodid crabs support the results of Cunningham et al. (1992). Our analyses are in accordance with the finding of this first molecular study, as species resolved as closest relatives are also found in our PLS clade. Boas (1880b) initially assumed lithodids to be derived from the former Eupagurus Brandt, 1851, which contained some species of the genus Pagurus. Bouvier (1895) assumed lithodids as derived from an ancestor in which females have paired first pleopods, as present in the genus Pylopagurus. However, this taxon has since undergone major taxonomic revisions (McLaughlin, 1981; Lemaitre & McLaughlin, 2003), and it is thus unclear to which species Bouvier actually referred (Reimann et al., 2011). Boas (1924) later suggested Pylopagurus and Nematopagurus as candidates for the closest relatives to lithodids. Also in a cladistic analysis based on foregut morphology, Nematopagurus and Pylopagurus s.s. were resolved as sister group to lithodids within other Paguridae (Reimann et al., 2011), suggesting the same position to lithodids as assumed by Boas (1924). Our data, however, clearly show that neither Nematopagurus, Pylopagurus, nor any of the other genera of the ‘Pylopagurus–Tomopagurus’ group after Lemaitre & McLaughlin (2003) included in our analyses are particularly closely related to the Lithodidae (Fig. 1). The occurrence of first pleopods in different Paguridae appears to be plesiomorphic (Richter & Scholtz, 1994) and might not have been present in the LCA of hermit crabs and lithodids. Another genus of hermit crabs, Discorsopagurus, came into focus as possibly the closest relative to lithodids after being included in a molecular phylogeny by Morrison et al. (2002). In addition to Discorsopagurus schmitti (Stevens, 1925), Pagurus longicarpus Say, 1817 and the lithodid Cryptolithodes typicus Brandt, 1848 were included in this study. Discorsopagurus and Cryptolithodes showed a sister relationship, while P. longicarpus was found one node lower in the tree. This result might have initiated a focus on Discorsopagurus as a possible closest relative to lithodids, as it appeared more closely related than Pagurus. However, the reason that Discorsopagurus was resolved as a closer relative than a representative of Pagurus is merely due to the fact that Discorsopagurus is included in the PLS clade, while P. longicarpus is not found in this group (Fig. 1). Later studies confirmed a close relationship of Discorsopagurus to Lithodidae (Ahyong et al., 2009; Schnabel et al., 2011). Bracken-Grissom et al. (2013) included a larger number of Paguridae in their analyses and also here species of our PLS clade [Labidochirus splendescens (Owen, 1839), D. schmitti and P. bernhardus] were resolved with an equal sister relationship to lithodids based on molecular data alone. The authors, however, focused their discussion on the apparently closer relationship of Discorsopagurus, which was found when morphological data were added to the analyses. Based on their phylogenetic results, they suggested a Discorsopagurus-like hermit crab as the precursor to lithodids, which appeared plausible considering the North Pacific distribution of the genus (the region where lithodids are assumed to have originated; Hall & Thatje, 2009b). Discorsopagurus inhabits non-coiled housings, like polychaete tubes, and possesses an almost symmetrical pleon, with asymmetry restricted to the pleopods (Gherardi, 1996; Komai, 2003). Our data show that while Discorsopagurus is included in the PLS clade and thus shares the LCA to lithodid crabs within the Paguridae, it is not more closely related than other members of this clade with more typical pagurid morphologies. This indicates that the secondary pleon symmetry in Discorsopagurus and male lithodid crabs evolved independently and was unlikely present in the LCA. Interestingly, no extant hermit crabs with tendencies towards a crab-like body shape have been suggested as precursor to lithodid crabs. Two species that show signs of carcinization from a typical pagurid morphology (Blackstone, 1989; Anker & Paulay, 2013) are found inside the PLS clade: L. splendescens and Pagurus hirsutiusculus (Dana, 1851) (Blackstone, 1985; Cunningham et al., 1992). Labidochirus, a hermit crab with a fully calcified carapace, has been referred to as resembling a ‘missing link’ between hermit crabs and lithodid crabs (Jensen, 1995; Seeb et al., 2002). The large number of conventional pagurid hermit crabs in the PLS clade, however, points to a LCA with a rather typical hermit crab morphology. Polyphyly of the genus pagurus The finding that Lithodidae are not only nested within the pagurid hermit crabs, but even inside the genus Pagurus (Cunningham et al., 1992), caused much attention and disputes (McLaughlin et al., 2004; Lemaitre & McLaughlin, 2009). The genus Pagurus was originally established by Fabricius (1775) as a heterogeneous group of non-crab-like species of Linnaeus’ genus Cancer Linnaeus, 1758. A large range of hermit crabs was initially included in Pagurus and later assigned to new genera (McLaughlin, 1974). Our phylogenetic analyses show that today Pagurus is still highly polyphyletic. The species assigned to Pagurus do not possess unique morphological features, but rather display a ‘standard’ pagurid body plan (McLaughlin, 2003). The genus has been grouped into several informal morphological species groups (Forest & de Saint Laurent, 1968; McLaughlin, 1974; Lemaitre, McLaughlin & García-Gómez, 1982; Ingle, 1985; Lemaitre & Cruz-Castaño, 2004). In our phylogenetic tree, species of the genus Pagurus are divided into numerous distinct genetic lineages, often confirming previously recognized morpho-groups, for example the ‘exilis’, ‘bernhardus’, ‘capillatus’ and ‘provenzanoi’ groups, and subdivision I by Ingle (1985) (Fig. 1). Only representatives of the informal ‘comptus’ group are found in two very distinct clades within the phylogenetic tree. However, due to the deviation from the group diagnostic characters in North Pacific representatives of this group, the ‘comptus’ group has been highlighted as likely polyphyletic (McLaughlin, 1974). McLaughlin (1974) suggests that Pagurus ‘s.s.’ will eventually be restricted to a few species typified by P. bernhardus, which was selected as type species for the genus by Latreille (1810). Our phylogenetic analyses support this prediction, as a group of only few morphologically very similar species form a clade with P. bernhardus, without rendering the genus polyphyletic (Fig. 2). The species in this Pagurus ‘s.s.’ group are the Northeastern Atlantic P. bernhardus; the Northwestern Atlantic P. acadianus; and the North Pacific Pagurus aleuticus (Benedict, 1892), Pagurus armatus (Dana, 1851) and Pagurus ochotensis Brandt, 1851, which have been grouped together in the ‘bernhardus’ group (McLaughlin, 1974). Included in this ‘bernhardus’ group are also three other species from the North Pacific (Komai, 1998; McLaughlin & Asakura, 2003; Lemaitre & Watabe, 2005), which were not included in our analyses. Of all the ~180 species currently assigned to Pagurus (Türkay, 2016), likely only these eight species can be included in the genus without rendering it polyphyletic. Taxonomic hierarchy of the Lithodid crabs The deeply nested position of lithodid crabs within the hermit crab family Paguridae makes a phylogenetic classification difficult to apply. To further complicate issues, McLaughlin et al. (2007) proposed the taxonomic elevation of lithodid crabs to superfamily level, Lithodoidea, since the authors did not agree with the concept of a pagurid ancestry of the taxon. The two subfamilies of lithodid crabs were accordingly elevated from subfamily to family rankings: Lithodidae and Hapalogastridae. However, the phylogenetic position of lithodids outside the Paguroidea (McLaughlin et al., 2007) contradicted all molecular and many morphological studies. The placement of a superfamily, Lithodoidea, within the family Paguridae subsequently has been highlighted as problematic, since it obscures evolutionary relationships (Ahyong et al., 2009; Keiler et al., 2013, 2015; Anker & Paulay, 2013). Further highlighting the problem caused by the elevation of the taxon is that Lithodidae sensuMcLaughlin et al. (2007) is exclusive of hapalogastrids, while these were previously recognized as part of this family via the subfamily Hapalogastrinae. The concept of a ‘Lithodoidea’ has subsequently only been used by a few authors (Ahyong et al., 2009; De Grave et al., 2009; Schnabel et al., 2011; Tsang et al., 2011; Bracken-Grissom et al., 2013). Some authors adopted the two distinct family rankings (Lithodidae and Hapalogastridae) while rejecting the superfamily Lithodoidea to combine the two taxa (Keiler et al., 2013, 2015). Others used the family Lithodidae in its former sense, containing the two subfamilies Lithodinae and Hapalogastrinae (Guzmán, 2009; Hall & Thatje, 2009b; Macpherson & Wehrtmann, 2010; Anker & Paulay, 2013), a system which we also use in this study. However, even the nested position of a family (Lithodidae) within another family (Paguridae) masks the true relationships of the groups. The fact that lithodids, in addition, appear nested within the polyphyletic genus Pagurus highlights this problem. Both Hapalogastrinae and Lithodinae appear non-monophyletic in our analyses. We, therefore, suggest a rather opposite taxonomic ranking to the one proposed by McLaughlin et al. (2007), by combining all lithodid crabs in a single taxon, and recognize its position within Paguridae by using the rank of a subfamily, Lithodinae. Hermit crabs within Paguridae, subsequently categorized under Pagurinae, are, however, still paraphyletic under this ranking. Pagurid hermit crabs are in need of an extensive taxonomic revision, which will need in-depth morphological and molecular investigations. Geographic origin of the Lithodidae Our finding that the closest hermit crab relatives to lithodid crabs predominantly consist of North Pacific species adds further support to a Northern Pacific origin of lithodids, as it suggests that the split between the PLS lineage and lithodids also occurred here. The four species of the PLS clade with a North Atlantic distribution have closely allied species in the Pacific: P. bernhardus from the North East Atlantic and P. acadianus from the North West Atlantic are sister species, with closely related species in the North Pacific, forming the ‘bernhardus’ group (Fig. 2). Pagurus pubescens Krøyer, 1838, found on both sides of the North Atlantic, has closely allied species in the North Pacific, forming the ‘trigonocheirus’ group. Pagurus arcuatus Squires, 1964, from the North West Atlantic, has closely allied species in the North Pacific, forming the ‘capillatus’ group (McLaughlin, 1974). The terminal nodes in the phylogenetic tree leading to these Atlantic species show that their predecessors, one for the representatives of each group, must independently have entered the Atlantic via the Bering Strait. The evolution of the deep-sea lineages followed a diversification of the taxon in the shallow North Pacific before changes in larval biology enabled certain taxa to extend their distribution into the deep sea (Hall & Thatje, 2009b; Thatje & Hall, 2016). Our data confirm the distribution of basal taxa of Lithodidae in the North Pacific and show two clear independent events of deep-sea radiation (Fig. 2). One event for Paralomis, including the monotypic Glyptolithodes which is found to be nested within the otherwise monophyletic genus Paralomis (Hall & Thatje, 2010), and one for Lithodes and Neolithodes (Fig. 2), of which Neolithodes reaches abyssal depths (Hall & Thatje, 2009b). Confining temperature boundaries have allowed only a few species from the boreal regions to re-emerge from the deep-sea into shallow-water habitats (Hall & Thatje, 2009b). Age of the origin of the Lithodidae The origin of Lithodidae has been estimated from 15 to 13 Mya (mid to lower Miocene) based on molecular clock analyses of the mitochondrial 16S rRNA (Cunningham et al., 1992). Following this study, the first lithodid crab known from the fossil record was described. This fossil species, Paralomis debodeorum Feldmann, 1998, has been dated to the mid to late Miocene of New Zealand and documents the presence of king crabs to at least 10 Mya in the South Pacific (Feldmann, 1998). In this context, the estimated lithodid origin obtained by Cunningham et al. (1992) appears quite recent. A slightly older origin, between 29 and 18 Mya, has been estimated using multiple genes, with fossil calibrations from the entire Anomura (Bracken-Grissom et al., 2013). Molecular clock analyses might, however, be influenced by a low sequence diversity within the Lithodidae (Snow, 2010; Matzen da Silva et al., 2011b). On the other hand, the deposit from which P. debodeorum has been discovered is difficult to date precisely (Feldmann, 1998; Feldmann, Schweitzer & McLauchlan, 2006), leaving a relatively recent radiation, reflected in low genetic variation, as a possibility. A low sequence divergence within Lithodidae was also found in our own sequence data, especially for the nuclear rRNA subunits. The entire ~1800 bp long 18S fragment only showed minimal variation, and even sequences obtained from different lithodid genera were found to be identical. The taxonomic assignment of fossil hermit crabs is problematic, and ‘lump genera’ such as Palaeopagurus Van Straelen, 1924 or Pagurus have been used for most species (Jagt et al., 2006). The highly polyphyletic pattern of extant species of Pagurus, as shown in our phylogeny (Fig. 1), further highlights this problem. Accurate divergence timing using fossil calibrations for the Paguridae is, therefore, problematic. Detailed investigation of phylogenetic relationships within various Paguridae taxa, and the species-rich deep-sea lithodid genera, in conjunction with biogeography might eventually provide a more reliable divergence estimate through the timing of geological events. Evolutionary scenarios for the Lithodidae Taxa within Lithodidae that are found basal in our phylogenetic analyses inhabit shallow-water, rocky habitats, which is in agreement with previous studies that suggested a shallow-water origin of lithodids (Makarov, 1938; Zaklan, 2001; Hall & Thatje, 2009b). Our finding that the closest hermit crab relatives also inhabit shallow habitats further supports this theory. This habitat must have played a key factor in the process of changing from a shell-utilizing to a free-living lifestyle. Crab-like forms appear to have evolved multiple times in shallow-water habitats (Morrison et al., 2002; Tsang et al., 2011), and these independent transitions offer strong evidence for the adaptive advantages of the crab-like form in relation to habitat type (Tsang et al., 2011). Most anomurans with a crab-like morphology are found living in hard bottom habitats, under boulders and stones, where a short, compact pleon is advantageous in exploring crevices (Tsang et al., 2011). Carcinization in hermit crabs is more complex than in other decapods, since this process, besides the broadening of the cephalothorax and reduction and underfolding of the pleon, also implies reorganization and calcification of the cephalothorax and pleon as these animals abandon the use of domiciles (Anker & Paulay, 2013). Besides the lithodids, tendencies towards carcinization, involving reduction or armouring of the pleon, tendency to lose domiciles and calcification of the cephalothorax, occurred independently in several groups of hermit crabs (Anker & Paulay, 2013). The abandonment or reduced use of a portable domicile must be seen as the most important step towards this morphological transformation. Different alternative pathways of leaving a protective housing are possible, and multiple scenarios for the cause of the predecessor of lithodid crabs leaving a protective shell exist. The various degrees of carcinization present in different hermit crabs give insights into the possibilities for morphological transition, but none of these taxa represent direct evolutionary intermediate forms between hermit crabs and lithodid crabs. Cunningham et al. (1992) explained carcinization in king crabs via a heterochronic shift in developmental timing, the extension of the ancestral hermit crab ontogeny to produce a carcinized adult, termed peramorphosis. In this scenario, ancestral hermit crab allometries were modified to accommodate an extended ontogeny and larger body size. In particular, Cunningham et al. (1992) highlighted the terrestrial hermit crab Birgus latro (Linnaeus, 1767), which, having a normal hermit crab habitus as a juvenile, outgrows its protective shell during ontogeny and develops a crab-like, calcified body (Greenaway, 2003). In Lithodidae, a crab-like morphology is, however, already apparent at metamorphosis (Morrison et al., 2002; McLaughlin et al., 2004), and Morrison et al. (2002) suggested a somewhat different mechanism of heterochronic shift, in the form of displacement heterochrony (Alberch et al., 1979). The size of the largest available gastropod shell limits the size of hermit crabs (Cunningham et al., 1992), and a lack of suitable shells has been discussed as a factor in the carcinization of lithodids (Richter & Scholtz, 1994). An absence of sufficiently large shells, due to an increase in size in the ancestral lineage of Lithodidae, leading to limited resources of suitable housings was rejected, since many lithodid species, in particular Hapalogastrinae, are not very large. As a more likely alternative, the lack of suitable shells in certain habitats was suggested as a possible starting point for lithodid evolution (Richter & Scholtz, 1994). Our results also point to a small-sized LCA, making the limitation of large shells as causation behind the evolution of lithodids unlikely. Some species of hermit crabs with tendencies towards carcinization are restricted by the availability of large gastropod shells, for example due to a deep-sea habitat, such as Porcellanopagurus Filhol, 1885; Solitariopagurus Türkay, 1986; and Patagurus Anker & Paulay, 2013 (McLaughlin & Lemaitre, 1997; Anker & Paulay, 2013). In some of these taxa, the pleon is reduced in size and only covered by a shell, which is too small for the animal to retract into (McLaughlin & Lemaitre, 1997; Anker & Paulay, 2013). From the early Miocene, large gastropod species were, however, never rare in the shallow North Pacific (Vermeij, 2012), and also the parallel diversification of hermit crabs in the same region, as indicated from our phylogeny, further points to a scenario without a general lack of gastropod shells. Furthermore, the large increase in body size of some lithodid taxa clearly occurred after the acquisition of the crab-like form, as basal taxa within the Lithodidae are of only moderate size. Advantages of abandoning of a domicile While the disadvantages of abandoning a protective housing and subsequently exposing the soft pleon have been highlighted as a maladaptive evolutionary scenario (McLaughlin & Lemaitre, 1997; McLaughlin et al., 2004), becoming independent from a housing also brings clear advantages. Competition for housings, and the need to find and change suitable housings during ontogeny are probably the most obvious ones. Inhabiting a gastropod shell, however, also requires a heavy object to be carried, greatly reducing mobility. Leaving the constraint of being bound to a foreign shell results in an increase in agility and speed, potentially making new prey sources available and enabling escape from predators (Blackstone, 1989; Anker & Paulay, 2013). The advantages of higher mobility also include the possibility of inhabiting new microhabitats precluded by carrying a bulky and heavy shell, like crevices or rock overhangs. As basal lithodids are found in such habitats today (Jensen, 1995), the enhanced mobility resulting from abandoning a protective housing is likely a key factor behind the evolutionary pathway of Lithodidae. An example of enhanced mobility by reducing the weight of a protective housing is found in the intertidal hermit crab P. hirsutiusculus, which uses only small shells in which the animal cannot fully retract. Pagurus hirsutiusculus shows tendencies towards carcinization, such as a broadened carapace and stronger armature of the pleon (McLaughlin, 1974). This species is very agile and often abandons its housing in escape reactions (Blackstone, 1989). A higher level of activity permits the animal to rely on speed of escape, rather than a housing for protection, which could favour shell loss and carcinization (Blackstone, 1989). This example illustrates how slight changes in ecology and shell-use might lead to carcinization in hermit crabs, without a restriction of housings. A number of hermit crab species have obligate commensal relationships with certain species of actinarians (Williams & McDermott, 2004). In the most advanced of these symbiotic relationships, the sea anemone builds the entire housing for the crab or greatly enlarges an originally present small gastropod shell. This lightweight housing protects its inhabitant not by a heavily calcified structure, but by a soft housing with protrudable thread-like acontias, which are loaded with poisonous nematocysts for defence. An increase in mobility, via reducing the weight of the shelter, is also an advantage of these relationships. The pagurid L. splendescens, which is found in the PLS clade, has such a symbiotic relationship. The lightweight housing, together with long walking legs for rapid locomotion, gives the animal a much higher mobility than seen in conventional hermit crabs. Labidochirus splendescens also shows tendency towards carcinization, like a broadened, fully calcified carapace, and an only moderate-sized pleon (McLaughlin & Lemaitre, 1997; Anker & Paulay, 2013). CONCLUSIONS The Lithodidae are deeply nested within the hermit crab family Paguridae and show a clear sister relationship with a clade of predominantly North Pacific shallow-water hermit crabs. Lithodid crabs are even found nested within a highly polyphyletic hermit crab genus Pagurus, confirming the results of the very first molecular study on the taxon by Cunningham et al. (1992). The stem species of Lithodidae inhabited shallow waters of the North Pacific with no general shortage of suitable gastropod shells. A crab-like morphology likely evolved gradually due to the adoption of smaller housings for the benefit of higher mobility. The basal position of small-sized taxa clearly indicates that an increase in body size was not the trigger for developing a crab-like habitus in the Lithodidae. The abandonment of a domicile, however, enabled the development of gigantism in lithodid crabs, since available gastropod shells for housing no longer set a size limitation. Enhanced armour in the form of spines and calcification, and an increase in size, enabled king crabs to leave the initial protective environment and expand into non-sheltered habitats. In the deep sea, the taxon could finally diversify on a global scale. Knowledge about phylogenetic relationships within the diverse deep-sea genera is still fragmentary. However, the species richness of these genera might eventually enable a detailed reconstruction of the dispersal routes within the deep-sea lineages of king crabs. ACKNOWLEDGEMENTS Funding for this study was provided by a grant from the Meltzer Foundation, University of Bergen. The Shannon Point Marine Center (Western Washington University, Anacortes, WA, USA) helped in sampling of specimens and provided laboratory space. Specimens for DNA extractions were kindly loaned from the University Museum of Bergen, Norway; the Royal BC Museum, Victoria, BC, Canada; and the NIWA Invertebrate Collection, Wellington, New Zealand. We thank the reviewers and the editors for their comments, which improved the manuscript. REFERENCES Ahyong ST. 2010a. The marine fauna of New Zealand: king crabs of New Zealand, Australia, and the Ross Sea (Crustacea: Decapoda: Lithodidae). NIWA Biodiversity Memoir  123: 194. Ahyong ST. 2010b. Neolithodes flindersi, a new species of king crab from southeastern Australia (Crustacea: Decapoda: Lithodidae). Zootaxa  2362: 55– 62. Ahyong ST, Chan TY. 2010. 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The origin of king crabs: hermit crab ancestry under the magnifying glass

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Abstract

Abstract The origin of king crabs from a hermit crab ancestor has caused controversy for more than a century. While the phylogenetic position of Lithodidae within the hermit crab family Paguridae has been strengthened in recent years, several key questions regarding the evolution of lithodid crabs have remained unanswered. In particular, it has been debated which hermit crabs constitute the closest extant relatives to lithodid crabs within Paguridae. Also, the relationships of the two lithodid subfamilies, Lithodinae and Hapalogastrinae, are unresolved. Answers to these questions are crucial to the understanding of the origin of king crabs, in particular which factors were the driving forces behind leaving a protective housing, transforming to a crab-like morphology and finally developing a large body size. To address these questions, we constructed the most comprehensive molecular phylogeny of Paguridae and Lithodidae to date. Our analyses revealed a species-rich clade of hermit crabs as closest relatives to lithodid crabs within Paguridae. Hermit crabs included in this clade have a predominantly shallow-water distribution in the North Pacific, agreeing with a proposed origin of lithodid crabs in this region. We suggest that the advances resulting from abandoning a shell-inhabiting lifestyle, rather than constraints of such shelters, played a central role in carcinization in this taxon. Phylogenetic relationships within Lithodidae revealed its two subfamilies to be non-monophyletic. Small-sized, shallow-water taxa are basal in the phylogenetic tree, while an increase in size and subsequent deep-sea distribution occurred later in the evolution of the group. INTRODUCTION King crabs (Lithodidae Samouelle, 1819) are anomuran crustaceans with a crab-like body, superficially resembling that of brachyuran (true) crabs. The evolutionary origin of the taxon has been discussed since the end of the 19th century when it was hypothesized that king crabs are secondarily calcified hermit crabs that left the protective gastropod housing and transformed to a crab-like form (Boas, 1880a, b; Bouvier, 1895). The process causing morphological modifications towards a crab-like body shape, with a reduced pleon, folded under a broadened and calcified cephalothorax, is termed carcinization (Borradaile, 1916; Scholtz, 2014). The hypothesis that lithodid crabs are derived from a hermit crab ancestor regained significant attention when investigated for the first time using molecular phylogenetic methods (Cunningham, Blackstone & Buss, 1992). Not only were king crabs placed within the Paguridae Latreille, 1802, but the molecules even suggested a nested position of the taxon within the hermit crab genus Pagurus Fabricius, 1775. All subsequent studies based on molecular data (Zaklan, 2002; Morrison et al., 2002; Tsang et al., 2008, 2011; Ahyong, Schnabel & Maas, 2009; Bracken et al., 2009; Chu et al., 2009; Schnabel, Ahyong & Maas, 2011; Bracken-Grissom et al., 2013) as well as various morphological studies (Richter & Scholtz, 1994; Keiler & Richter, 2011; Reimann, Richter & Scholtz, 2011; Keiler, Richter & Wirkner, 2015) further supported the placement of lithodid crabs within the asymmetrical hermit crab family Paguridae. Despite the growing evidence of a hermit crab ancestry of king crabs, McLaughlin et al. strongly opposed this evolutionary scenario (McLaughlin & Lemaitre, 1997; McLaughlin, Lemaitre & Tudge, 2004; McLaughlin, Lemaitre & Sorhannus, 2007; Lemaitre & McLaughlin, 2009). Reversal of complex characters related to dextral shell housings, like the morphology of the fourth pereopods, and a maladaptive scenario of a crab exposing the soft pleon made, in their view, this evolutionary pathway infeasible. Rather, the authors proposed the opposite evolutionary scenario, leading from king crabs to hermit crabs. The placement of lithodids within the Anomura has thus been heartily debated until recent years when steadily increasing evidence settled the position of the group within the Paguridae (Tsang et al., 2008, 2011; Ahyong et al., 2009; Chu et al., 2009; Schnabel et al., 2011; Bracken-Grissom et al., 2013). The overall body shape of lithodid crabs is highly altered from its hermit crab ancestor. However, pagurid hermit crab asymmetries, influenced by inhabiting dextral gastropod shells (Palmer, 2004), such as pleon and claw asymmetries (McLaughlin & Lemaitre, 1997, 2000; McLaughlin et al., 2004; Duguid, 2010), are still present in lithodids (Tsang et al., 2011). Further traces of their pagurid origin can, for example, be found in the mouthparts (Boas, 1924; Jaszkowiak et al., 2015), setation (Keiler & Richter, 2011), internal organ organization (Anker & Paulay, 2013; Keiler et al., 2015) or vascular system (Keiler, Richter & Wirkner, 2013; Keiler et al., 2015). Poor knowledge of phylogenetic relationships within the Paguridae (Matzen da Silva et al., 2011a) left the exact placement of lithodids within this hermit crab family uncertain. Various candidates have been highlighted as the possible closest extant relatives to lithodids within the Paguridae, yet conclusions from genetic studies so far have been restricted by limited taxon sampling. In early studies, king crabs were thought to be derived from an ancestor closely related to the genera Nematopagurus A. Milne-Edwards & Bouvier, 1892 and Pylopagurus A. Milne-Edwards & Bouvier, 1893 based on the presence of paired pleopods, found in females of these species and those of lithodids (Boas, 1924). Reimann et al. (2011), based on a cladistic analysis, also found a sister relationship of these genera to lithodids within the remaining Paguridae. An exclusively North Pacific genus, Discorsopagurus McLaughlin, 1974, has drawn attention as the possibly closest relative in recent studies (Morrison et al., 2002; Ahyong et al., 2009; Schnabel et al., 2011; Bracken-Grissom et al., 2013). Discorsopagurus inhabits non-coiled housings and has an almost symmetrical pleon (Komai, 2003), as also found in male lithodids. Other studies found a sister relationship with one or few other exclusively North Pacific genera (Labidochirus Benedict, 1892; Elassochirus Benedict, 1892; and Pagurodofleinia Asakura, 2005), as well as part of the genus Pagurus (Cunningham et al., 1992; Tsang et al., 2008, 2011; Chu et al., 2009). In particular, a possibly nested position of Lithodidae within Pagurus has been highlighted (Cunningham et al., 1992). Pagurus is a species-rich genus, and various informal morpho-groups have been established (Forest & de Saint Laurent, 1968; McLaughlin, 1974; Ingle, 1985; Lemaitre & Cruz-Castaño, 2004). Some of these informal groupings have been confirmed using molecular markers (Matzen da Silva et al., 2011a; Olguín & Mantelatto, 2013), and a highly polyphyletic pattern of the genus has been indicated (Cunningham et al., 1992; Reimann et al., 2011; Bracken-Grissom et al., 2013). Lithodidae are only found in temperate regions and deep-sea habitats. Few genera have a global distribution via the deep sea, while the largest number of lithodid genera is restricted to the North Pacific, where they display a high morphological diversity (Stevens & Lovrich, 2014). From the distribution pattern, combined with physiological and phylogenetic data, it has been concluded that lithodid crabs originated in the shallow North Pacific (Makarov, 1938; Zaklan, 2002; Hall & Thatje, 2009b). While the shallow-water genera of the North Pacific are monotypic or only contain few species, the king crab genera Lithodes Latreille, 1806; Neolithodes A. Milne-Edwards & Bouvier, 1894; and Paralomis White, 1856 are species rich, and new species have been frequently discovered in the last decades (Williams, Smith & Baco, 2000; Macpherson, 2001, 2003, 2004; Takeda & Nagai, 2004; Ahyong & Dawson, 2006; Spiridonov et al., 2006; Takeda & Bussarawit, 2007; Macpherson & Chan, 2008; Hall & Thatje, 2009a; Guzmán, 2009; Ahyong, 2010a, b; Ahyong & Chan, 2010; Muñoz & García-Isarch, 2013). The deep-sea lineages diversified on a global scale and constitute the majority of today’s species diversity of Lithodidae (Hall & Thatje, 2009b). Lithodidae exhibit a wide range of morphological diversity, ranging from small-sized species, such as Hapalogaster Brandt, 1850; Dermaturus Brandt, 1850; or Cryptolithodes Brandt, 1848, to the large box and king crabs. Balss (1924) pointed out the similarities between different body shapes of lithodids and brachyuran crabs as prime examples for convergent evolution. The gross morphology of the lithodid genus Cryptolithodes, for example, superficially resembles that of the brachyuran genus Aethra Latreille in Cuvier, 1816. The large king crabs on the other hand, in particular the genus Lithodes, resemble the brachyuran spider crabs of the genus Maja Lamarck, 1801. Knowledge of internal relationships within the Lithodidae is limited, and the association of the two lithodid subfamilies, Hapalogastrinae Brandt, 1850 and Lithodinae Samouelle, 1819, is uncertain (Hall & Thatje, 2009b; Bracken-Grissom et al., 2013). Bracken-Grissom et al. (2013) recovered different relationships between taxa of the two subfamilies, depending on their analyses. Using only molecular data, neither taxa were found to be monophyletic, while including morphological data in their analyses resolved both taxa as monophyletic. Hall and Thatje (2009a) resolved both subfamilies as monophyletic, but with a poorly supported placement of Cryptolithodes (Hapalogastrinae). Recently, Cryptolithodes was, however, placed outside Lithodinae and within Hapalogastrinae based on molecular data (Thatje & Hall, 2016). The two subfamilies are separated by the calcification of the third to fifth tergites of the pleon in Lithodinae or lack thereof in Hapalogastrinae (McLaughlin, 2014). Cryptolithodes, however, is the most heavily calcified lithodid crab, and its ambiguous phylogenetic placement makes the use of the degree of pleon calcification as an autapomorphy for the two subfamilies uncertain. Information on basal lithodid relationships is crucial for understanding the evolutionary pathway of lithodid crabs and the driving forces leading from a shell-inhabiting to a free-living lifestyle via the process of carcinization. In the present study, we construct an extensive molecular phylogeny of Paguridae and Lithodidae using nuclear ribosomal and mitochondrial genes to cover a suitable range of genetic variability (Toon et al., 2009). New and available sequence data are combined to construct the most comprehensive phylogeny of pagurid hermit crabs and lithodid crabs to date in the search for the closest living relatives of Lithodidae within the hermit crabs. MATERIAL AND METHODS Taxon sampling New sequences from 40 species of Paguridae and Lithodidae were obtained for this study. Specimens were obtained both in the field and from museum collections (Table 1). Collection of new material focussed on the temperate regions of the Northern Hemisphere, in particular the North Pacific. This region has been highlighted as the region of origin of Lithodidae (Hall & Thatje, 2009b), and the closest extant relatives to lithodids are likely found in this region. The data set was complemented by a large range of taxa of Paguridae and Lithodidae with sequences available in GenBank and the Barcode of Life Data Systems (BOLD), to cover a broad range of taxa and geographic regions (Table 1). Six representatives of the genera Paguristes and Areopaguristes (family Diogenidae Ortmann, 1892) were chosen as outgroup taxa, based on previous studies that indicated these genera as closest relatives to Paguridae and Lithodidae (Morrison et al., 2002; Bracken-Grissom et al., 2013). Table 1. Taxa included in this study with list of GenBank and BOLD accession numbers of the molecular marker. Accession numbers of new sequences are indicated with an asterisk Taxa  16S  18S  28S  COI  12S  Family Diogenidae   Areopaguristes hewatti  KF182535  KF182482  KF182644  –  –   Areopaguristes hummi  KF182542  KF182484  KF182641  –  –   Paguristes cadenati  KF182540  KF182493  KF182637  –  –   Paguristes puncticeps  KF182538  KF182487  KF182639  –  –   Paguristes triangulatus  KF182539  KF182489  KF182638  –  –   Paguristes turgidus  AF436056  AF436020  AF435997  DQ882097  –  Family Lithodidae   Subfamily Hapalogastrinae    Acantholithodes hispidus  –  –  –  DQ882026  –    Hapalogaster dentata  AF425327  –  AF425347  AF425306  AF425306    Hapalogaster grebnitzkii  KY426325*  –  –  –  KY426292*    Hapalogaster mertensii  AF425328  KF182451  KF182601  KY426267*  AF425307    Oedignathus inermis  AF425334  –  AF425313  AF425353  AF425313    Placetron wosnessenskii  KY426329*  –  KY454171*  DSALA006-06  –   Subfamily Lithodinae    Cryptolithodes sitchensis  AF425324  KF182453  KF182603  KC107820  AF425303    Cryptolithodes typicus  AF425325  AF436019  AF425345  AF425304  AF425304    Glyptolithodes cristatipes  AF425326  –  AF425346  AF425305  AF425305    Lithodes aequispinus  KY426332*  –  KY454183*  AF425308  AF425308    Lithodes confundens  HM020949  –  FJ462642  HM020901  –    Lithodes couesi  –  –  –  DQ882086  –    Lithodes ferox  HM020950  –  HM020856  KY426276*  KY426296*    Lithodes longispina  –  –  –  AB476815  –    Lithodes maja  KY426333*  KY454206*  AF425350  FJ581746  AF425309    Lithodes murrayi  HM020954  –  HM020857  HM020899  –    Lithodes nintokuae  AB769476  –  –  AB769476  AB769476    Lithodes paulayi  –  –  –  GU289677  –    Lithodes santolla  KF182572  AF439385  KF182602  KY426275*  AF425310    Lithodes turkayi  EU493268  –  –  KC196529  –    Lithodes turritus  KJ132573  –  –  –  –    Lopholithodes foraminatus  KY426330*  –  KY454182*  DQ882088  KY426295*    Lopholithodes mandtii  AF425333  KY454205*  AF425352/KY454174*  KY426271*  AF425312    Neolithodes asperrimus  HM020940  –  HM020847  HM020891  –    Neolithodes diomedeae  –  –  –  KC196528  –    Neolithodes duhameli  HM020946  –  HM020849  HM020892  –    Neolithodes grimladii  –  –  –  JQ305973  –    Neolithodes nr. brodiei  HM020942  –  FJ462640/HM020888  HM020894  –    Paralithodes brevipes  AF425337  –  AF425356  NC021458  AF425316    Paralithodes camtschaticus  AF425338  JN192147  AB193823  JF738154  AF425317    Paralithodes platypus  KY426328*  JN192152  AB193821  KY426274*  KY426297*    Paralomis aculeata  HM020958  –  HM020862  HM020904  –    Paralomis africana  EU493275  –  HM020864  HM020907  –    Paralomis anamerae  HM020959  –  HM020865  HM020906  –    Paralomis birsteini  KY426326*  –  HM020867  EU493260  KY426294*    Paralomis cristata  EU493267  –  –  HM020911  –    Paralomis cristulata  EU493271  –  HM020870  HM020908  –    Paralomis dofleini  HM020962  –  HM020871  HM020912  –    Paralomis elongata  –  –  HM020872/HM020887/ HM20884  HM020914  –    Paralomis erinacea  HM020966  –  HM020873  HM020915  –    Paralomis formosa  HM020971  –  FJ462641/HM020886  HM020918  –    Paralomis granulosa  AF425339  –  AF425358/HM020877  AF425318  AF425318    Paralomis hirtella  KY426327*  –  –  KY426272*  KY426293*    Paralomis multispina  –  –  –  AB211296  –    Paralomis pacifica  –  –  –  AB476750  –    Paralomis spinosissima  HM020982  –  HM020879  HM020927  –    Paralomis zealandica  HM020980  –  –  HM020935  –    Phyllolithodes papillosus  AF425340  KY454204*  AF425359/KY454175*  KY426273*  AF425319    Rhinolithodes wosnessenskii  KY426331*  –  AF425360  AF425320  AF425320  Family Paguridae   Agaricochirus alexandri  –  KF182447  KF182593  –  –   Anapagurus breviaculeatus  KY426316*  –  KY454162*  KY426262*  KY426286*   Anapagurus chiroacanthus  KY426315*  KY454187*  KY454161*  KY426263*  KY426285*   Anapagurus hydmanni  –  KJ182993  –  KJ183012  –   Anapagurus laevis  KY426317*  KY454186*  KY454163*  BNSC284-11  KY426287*   Cestopagurus timidus  KY426314*  KY454192*  KY454159*  KY426261*  KY426288*   Discorsopagurus schmitti  AF436055  AF436017  KY454176*  KY426283*  KY426298*   Elassochirus cavimanus  KY426342*  –  –  KY426281*  KY426302*   Elassochirus gilli  KY426343*  –  –  KY426282*  KY426300*   Elassochirus tenuimanus  KY426341*  KY454198*  KY454184*  KY426279*  KY426301*   Labidochirus splendescens  AF425332  –  AF425351  –  AF425311   Manucomplanus ungulatus  KF182575  KF182457  KF182612  –  –   Nematopagurus gardineri  –  –  –  MDECA670-10  –   Nematopagurus longicornis  KY426318*  KY454188*  KY454169*  KY426264*  KY426289*   Nematopagurus meiringae  –  –  –  MDECA669-10  –   Paguridium minimum  KY426319*  KY454191*  KY454168*  –  KY426308*   Pagurus acadianus  –  –  –  FJ581812  –   Pagurus alatus  KY426323*  –  JN107619  KY426270*  KY426309*   Pagurus aleuticus  KY426340*  –  KY454180*  KY426280*  KY426299*   Pagurus arcuatus  –  –  –  FJ581817  –   Pagurus armatus  –  –  –  AF483159  –   Pagurus beringanus  KY426337*  KY454201*  KY454173*  KY426277*  KY426307*   Pagurus bernhardus  KY426339*  KY454197*  JN107623/KY454185*  JN107580  AF425314   Pagurus brachiomastus  –  –  –  JN5990075  –   Pagurus brevidactylus  KF182563  KF182495  KF182610  –  –   Pagurus bullisi  KF182568  KF182454  KF182595  –  –   Pagurus caurinus  KY426336*  KY454200*  KY454181*  KY426278*  KY426306*   Pagurus chevreuxi  KY426312*  -  KY454160*  –  –   Pagurus comptus  FJ869145  KY454202*  KY454170*  KY426265*  KY426290*   Pagurus criniticornis  DQ369947  –  –  –  –   Pagurus cuanensis  KY426322*  KY454190*  JN107625  JN107584  KY426310*   Pagurus edwardsii  FJ869146  –  –  CFAD141-11  –   Pagurus excavatus  JN107610  –  JN107628  JN107587  –   Pagurus exilis  FJ869147  –  –  -  –   Pagurus forbesii  KF962984  –  –  KF962980  –   Pagurus forceps  FJ869150  –  –  –  –   Pagurus gladius  JX238503  –  –  –  –   Pagurus granosimanus  KY426338*  KY454196*  KY454178*  GU442314  KY426305*   Pagurus hirsutiusculus  KY426334*  KY454193*  KY454177*  GU442400  AF425315   Pagurus kennerlyi  KY426345*  KY454195*  KY454172*  KY426284*  KY426304*   Pagurus leptonyx  DQ369946  –  –  –  –   Pagurus longicarpus  AF150756  AF436018  AF425343/AY739185  AF150756  AF150756   Pagurus maclaughlinae  KF182566  KF182460  KF182611  –  –   Pagurus mbizi  KY426320*  –  KY454167*  –  KY426311*   Pagurus minutus  –  –  –  JX502978  –   Pagurus nr. carolinensis  KF182565  KF182465  KF182609  –  –   Pagurus ochotensis  KY426335*  KY454199*  KY454179*  JN590062  –   Pagurus pectinatus  –  –  –  JN5990060  –   Pagurus perlatus  JQ805783  –  –  –  –   Pagurus pollicaris  FJ869152  KF182458  KF182589  AF483163  –   Pagurus prideaux  KY426321*  KY454189*  JN107629  JQ306249  –   Pagurus provenzanoi  FJ869154  –  –  –  –   Pagurus proximus  –  –  –  KC347562  –   Pagurus pseudosculptimanus  KF962986  –  KY454165*  KY426268*  –   Pagurus pubescens  KY426344*  KY454194*  JN107633  JQ305956  KY426303*   Pagurus pubescentulus  KY426324*  –  KY454166*  KY426269*  –   Pagurus samuelis  –  –  –  GU443022  –   Pagurus similis  –  –  –  HM180751  –   Pagurus stimpsoni  KF182564  KF182466  KF182613  –  –   Pagurus venturensis  –  –  –  GU442190  –   Pagurus villosus  FJ869155  –  –  CFAD136-11  –   Phimochirus holthuisi  KF182578  KF182455  KF182594  –  –   Phimochirus randalli  KF182577  KF182450  KF182591  –  –   Pylopaguridium markhami  KF182570  KF182478  KF182597  –  –   Pylopagurus discoidalis  KF182569  KF182496    –  –   Spiropagurus elegans  KY426313*  KY454203*  KY454164*  KY426266*  KY426291*   Spiropagurus profundorum  –  –  –  MDECA610-10  –   Tomopagurus merimaculosus  KF182567  KF182497  KF182590  –  –  Taxa  16S  18S  28S  COI  12S  Family Diogenidae   Areopaguristes hewatti  KF182535  KF182482  KF182644  –  –   Areopaguristes hummi  KF182542  KF182484  KF182641  –  –   Paguristes cadenati  KF182540  KF182493  KF182637  –  –   Paguristes puncticeps  KF182538  KF182487  KF182639  –  –   Paguristes triangulatus  KF182539  KF182489  KF182638  –  –   Paguristes turgidus  AF436056  AF436020  AF435997  DQ882097  –  Family Lithodidae   Subfamily Hapalogastrinae    Acantholithodes hispidus  –  –  –  DQ882026  –    Hapalogaster dentata  AF425327  –  AF425347  AF425306  AF425306    Hapalogaster grebnitzkii  KY426325*  –  –  –  KY426292*    Hapalogaster mertensii  AF425328  KF182451  KF182601  KY426267*  AF425307    Oedignathus inermis  AF425334  –  AF425313  AF425353  AF425313    Placetron wosnessenskii  KY426329*  –  KY454171*  DSALA006-06  –   Subfamily Lithodinae    Cryptolithodes sitchensis  AF425324  KF182453  KF182603  KC107820  AF425303    Cryptolithodes typicus  AF425325  AF436019  AF425345  AF425304  AF425304    Glyptolithodes cristatipes  AF425326  –  AF425346  AF425305  AF425305    Lithodes aequispinus  KY426332*  –  KY454183*  AF425308  AF425308    Lithodes confundens  HM020949  –  FJ462642  HM020901  –    Lithodes couesi  –  –  –  DQ882086  –    Lithodes ferox  HM020950  –  HM020856  KY426276*  KY426296*    Lithodes longispina  –  –  –  AB476815  –    Lithodes maja  KY426333*  KY454206*  AF425350  FJ581746  AF425309    Lithodes murrayi  HM020954  –  HM020857  HM020899  –    Lithodes nintokuae  AB769476  –  –  AB769476  AB769476    Lithodes paulayi  –  –  –  GU289677  –    Lithodes santolla  KF182572  AF439385  KF182602  KY426275*  AF425310    Lithodes turkayi  EU493268  –  –  KC196529  –    Lithodes turritus  KJ132573  –  –  –  –    Lopholithodes foraminatus  KY426330*  –  KY454182*  DQ882088  KY426295*    Lopholithodes mandtii  AF425333  KY454205*  AF425352/KY454174*  KY426271*  AF425312    Neolithodes asperrimus  HM020940  –  HM020847  HM020891  –    Neolithodes diomedeae  –  –  –  KC196528  –    Neolithodes duhameli  HM020946  –  HM020849  HM020892  –    Neolithodes grimladii  –  –  –  JQ305973  –    Neolithodes nr. brodiei  HM020942  –  FJ462640/HM020888  HM020894  –    Paralithodes brevipes  AF425337  –  AF425356  NC021458  AF425316    Paralithodes camtschaticus  AF425338  JN192147  AB193823  JF738154  AF425317    Paralithodes platypus  KY426328*  JN192152  AB193821  KY426274*  KY426297*    Paralomis aculeata  HM020958  –  HM020862  HM020904  –    Paralomis africana  EU493275  –  HM020864  HM020907  –    Paralomis anamerae  HM020959  –  HM020865  HM020906  –    Paralomis birsteini  KY426326*  –  HM020867  EU493260  KY426294*    Paralomis cristata  EU493267  –  –  HM020911  –    Paralomis cristulata  EU493271  –  HM020870  HM020908  –    Paralomis dofleini  HM020962  –  HM020871  HM020912  –    Paralomis elongata  –  –  HM020872/HM020887/ HM20884  HM020914  –    Paralomis erinacea  HM020966  –  HM020873  HM020915  –    Paralomis formosa  HM020971  –  FJ462641/HM020886  HM020918  –    Paralomis granulosa  AF425339  –  AF425358/HM020877  AF425318  AF425318    Paralomis hirtella  KY426327*  –  –  KY426272*  KY426293*    Paralomis multispina  –  –  –  AB211296  –    Paralomis pacifica  –  –  –  AB476750  –    Paralomis spinosissima  HM020982  –  HM020879  HM020927  –    Paralomis zealandica  HM020980  –  –  HM020935  –    Phyllolithodes papillosus  AF425340  KY454204*  AF425359/KY454175*  KY426273*  AF425319    Rhinolithodes wosnessenskii  KY426331*  –  AF425360  AF425320  AF425320  Family Paguridae   Agaricochirus alexandri  –  KF182447  KF182593  –  –   Anapagurus breviaculeatus  KY426316*  –  KY454162*  KY426262*  KY426286*   Anapagurus chiroacanthus  KY426315*  KY454187*  KY454161*  KY426263*  KY426285*   Anapagurus hydmanni  –  KJ182993  –  KJ183012  –   Anapagurus laevis  KY426317*  KY454186*  KY454163*  BNSC284-11  KY426287*   Cestopagurus timidus  KY426314*  KY454192*  KY454159*  KY426261*  KY426288*   Discorsopagurus schmitti  AF436055  AF436017  KY454176*  KY426283*  KY426298*   Elassochirus cavimanus  KY426342*  –  –  KY426281*  KY426302*   Elassochirus gilli  KY426343*  –  –  KY426282*  KY426300*   Elassochirus tenuimanus  KY426341*  KY454198*  KY454184*  KY426279*  KY426301*   Labidochirus splendescens  AF425332  –  AF425351  –  AF425311   Manucomplanus ungulatus  KF182575  KF182457  KF182612  –  –   Nematopagurus gardineri  –  –  –  MDECA670-10  –   Nematopagurus longicornis  KY426318*  KY454188*  KY454169*  KY426264*  KY426289*   Nematopagurus meiringae  –  –  –  MDECA669-10  –   Paguridium minimum  KY426319*  KY454191*  KY454168*  –  KY426308*   Pagurus acadianus  –  –  –  FJ581812  –   Pagurus alatus  KY426323*  –  JN107619  KY426270*  KY426309*   Pagurus aleuticus  KY426340*  –  KY454180*  KY426280*  KY426299*   Pagurus arcuatus  –  –  –  FJ581817  –   Pagurus armatus  –  –  –  AF483159  –   Pagurus beringanus  KY426337*  KY454201*  KY454173*  KY426277*  KY426307*   Pagurus bernhardus  KY426339*  KY454197*  JN107623/KY454185*  JN107580  AF425314   Pagurus brachiomastus  –  –  –  JN5990075  –   Pagurus brevidactylus  KF182563  KF182495  KF182610  –  –   Pagurus bullisi  KF182568  KF182454  KF182595  –  –   Pagurus caurinus  KY426336*  KY454200*  KY454181*  KY426278*  KY426306*   Pagurus chevreuxi  KY426312*  -  KY454160*  –  –   Pagurus comptus  FJ869145  KY454202*  KY454170*  KY426265*  KY426290*   Pagurus criniticornis  DQ369947  –  –  –  –   Pagurus cuanensis  KY426322*  KY454190*  JN107625  JN107584  KY426310*   Pagurus edwardsii  FJ869146  –  –  CFAD141-11  –   Pagurus excavatus  JN107610  –  JN107628  JN107587  –   Pagurus exilis  FJ869147  –  –  -  –   Pagurus forbesii  KF962984  –  –  KF962980  –   Pagurus forceps  FJ869150  –  –  –  –   Pagurus gladius  JX238503  –  –  –  –   Pagurus granosimanus  KY426338*  KY454196*  KY454178*  GU442314  KY426305*   Pagurus hirsutiusculus  KY426334*  KY454193*  KY454177*  GU442400  AF425315   Pagurus kennerlyi  KY426345*  KY454195*  KY454172*  KY426284*  KY426304*   Pagurus leptonyx  DQ369946  –  –  –  –   Pagurus longicarpus  AF150756  AF436018  AF425343/AY739185  AF150756  AF150756   Pagurus maclaughlinae  KF182566  KF182460  KF182611  –  –   Pagurus mbizi  KY426320*  –  KY454167*  –  KY426311*   Pagurus minutus  –  –  –  JX502978  –   Pagurus nr. carolinensis  KF182565  KF182465  KF182609  –  –   Pagurus ochotensis  KY426335*  KY454199*  KY454179*  JN590062  –   Pagurus pectinatus  –  –  –  JN5990060  –   Pagurus perlatus  JQ805783  –  –  –  –   Pagurus pollicaris  FJ869152  KF182458  KF182589  AF483163  –   Pagurus prideaux  KY426321*  KY454189*  JN107629  JQ306249  –   Pagurus provenzanoi  FJ869154  –  –  –  –   Pagurus proximus  –  –  –  KC347562  –   Pagurus pseudosculptimanus  KF962986  –  KY454165*  KY426268*  –   Pagurus pubescens  KY426344*  KY454194*  JN107633  JQ305956  KY426303*   Pagurus pubescentulus  KY426324*  –  KY454166*  KY426269*  –   Pagurus samuelis  –  –  –  GU443022  –   Pagurus similis  –  –  –  HM180751  –   Pagurus stimpsoni  KF182564  KF182466  KF182613  –  –   Pagurus venturensis  –  –  –  GU442190  –   Pagurus villosus  FJ869155  –  –  CFAD136-11  –   Phimochirus holthuisi  KF182578  KF182455  KF182594  –  –   Phimochirus randalli  KF182577  KF182450  KF182591  –  –   Pylopaguridium markhami  KF182570  KF182478  KF182597  –  –   Pylopagurus discoidalis  KF182569  KF182496    –  –   Spiropagurus elegans  KY426313*  KY454203*  KY454164*  KY426266*  KY426291*   Spiropagurus profundorum  –  –  –  MDECA610-10  –   Tomopagurus merimaculosus  KF182567  KF182497  KF182590  –  –  View Large Molecular work Extraction Specimens collected for this study were preserved in 96% ethanol prior to DNA extraction. Molecular work was conducted in the Biodiversity Laboratories, University of Bergen, Norway. Total genomic DNA was extracted from muscle tissue using a Gene Mole automatic nucleic acid extractor from Mole Genetics AS, Norway, or using the Qiagen DNeasy Blood and Tissue kit (QIAGEN Inc., Valencia, CA, USA), following the manufacturer’s standard protocols. PCR and sequencing Sequences of five genes were amplified by PCR: three mitochondrial markers [ribosomal 12S and 16S rRNA subunits, and cytochrome c oxidase 1 (COI)] and two nuclear markers (18S and 28S rRNA subunits). PCR reactions were carried out on a Bio-Rad C1000 Thermal Cycler using Takara polymerase in 25-µL reactions. Primers from previous studies as well as newly designed primers were used (Table 2). The 28S gene was amplified using two primer pairs, resulting in two overlapping fragments. For some species, the 12S and COI genes, situated next to each other in the mitochondrial genome, were amplified in a single PCR run, using the primers 12S-A-Paguridae and COI-B-Paguridae. All PCR products were checked for successful amplification on 1% agarose gels stained with GelRed. PCR products were purified and sequenced in both directions at Macrogen Inc. using the same primers as for amplification. For the 18S fragment two additional primers (18S-A− and 18S-B+) were used for sequencing the entire PCR product. Contigs were assembled using Lasergene SeqMan Pro 8.1. To exclude a possible presence of pseudogenes or gene duplicates, the individual sequence chromatogram files were checked for the presence of double peaks, and the COI alignment was translated into amino acids and checked for premature stop codons and frame shifts using BioEdit 7.2.3. Sequences are deposited in GenBank (Table 1). Table 2. Primers used for PCR amplification and sequencing Marker  Primer  Primer sequence (5′–3′)  Reference  COI  HCO2198  TAA ACT TCA GGG TGA CCA AAA AAT CA  Folmer et al. (1994)   LCO1490  GGT CAA CAA ATC ATA AAG ATA TTG G  Folmer et al. (1994)   COI-A-Paguridae  TCT TAT ATT TCC ACT ATA AAG CC  This study  COI-B-Paguridae  ATT CTT GAC TTA CAA TRT GTG A  This study  16S  LR-N-13398  CGC CTG TTT AAC AAA AAC AT  Simon et al. (1994)   LR-J-12887  CCG GTC TGA ACT CAG ATC ACG T  Simon et al. (1994)   16S-A-Paguridae  AAG ATA GAA ACC AAC CTG GCT C  This study  16S-B-Paguridae  TGC CTG TTT AAC AAA AAC ATG TC  This study  12S  12S-A-Paguridae  ATT ATA ATA GGG TAT CTA ATC CTA G  This study  12S-B-Paguridae  AAT GTT CCA ATR TCT TTA TGG  This study  18S  18S-329  TAA TGA TCC TTC CGC AGG TT  Spears et al. (1992)   18S-328  CCT GGT TGA TCC TGC CAG  Spears et al. (1992)   18S-A− (sequencing)  CAG CMG CC GCG GTA ATW C  Spears et al. (1992)   18S-B+ (sequencing)  ATT CCC CGT TAC CCG  Spears et al. (1992)   28S  28S-OI  GCG GAG GAA AAG AAA CTA AC  Zaklan (2001)   28S-R443  CCT CAC GGT ACT TGT TCG CTA TCG G  Ahyong et al. (2009)   28S-Paguridae-F1  CGT AGA GTC GGG TTG CTT GA  This study  28S-Paguridae-R1  CTT TCG GGT CCC AAC ATG TC  This study  Marker  Primer  Primer sequence (5′–3′)  Reference  COI  HCO2198  TAA ACT TCA GGG TGA CCA AAA AAT CA  Folmer et al. (1994)   LCO1490  GGT CAA CAA ATC ATA AAG ATA TTG G  Folmer et al. (1994)   COI-A-Paguridae  TCT TAT ATT TCC ACT ATA AAG CC  This study  COI-B-Paguridae  ATT CTT GAC TTA CAA TRT GTG A  This study  16S  LR-N-13398  CGC CTG TTT AAC AAA AAC AT  Simon et al. (1994)   LR-J-12887  CCG GTC TGA ACT CAG ATC ACG T  Simon et al. (1994)   16S-A-Paguridae  AAG ATA GAA ACC AAC CTG GCT C  This study  16S-B-Paguridae  TGC CTG TTT AAC AAA AAC ATG TC  This study  12S  12S-A-Paguridae  ATT ATA ATA GGG TAT CTA ATC CTA G  This study  12S-B-Paguridae  AAT GTT CCA ATR TCT TTA TGG  This study  18S  18S-329  TAA TGA TCC TTC CGC AGG TT  Spears et al. (1992)   18S-328  CCT GGT TGA TCC TGC CAG  Spears et al. (1992)   18S-A− (sequencing)  CAG CMG CC GCG GTA ATW C  Spears et al. (1992)   18S-B+ (sequencing)  ATT CCC CGT TAC CCG  Spears et al. (1992)   28S  28S-OI  GCG GAG GAA AAG AAA CTA AC  Zaklan (2001)   28S-R443  CCT CAC GGT ACT TGT TCG CTA TCG G  Ahyong et al. (2009)   28S-Paguridae-F1  CGT AGA GTC GGG TTG CTT GA  This study  28S-Paguridae-R1  CTT TCG GGT CCC AAC ATG TC  This study  View Large Sequence alignments The data set included 49 Lithodidae and 69 Paguridae species, as well as six Diogenidae species as outgroup taxa. Sequences for COI, 16S, 12S and 18S were aligned in eBioX 1.5 using the MUSCLE algorithm (Edgar, 2004). 28S sequences were aligned in the online version of MAFFT 7 (Katoh & Standley, 2013) using the E-INS-i strategy. Individual alignments were checked by eye. The COI alignment was checked by translation into amino acids using Seaview 4.4 with the genetic code set to ‘Invertebrate mt’. Sequences downloaded from GenBank were cut to the corresponding sequence region of our own PCRs. BLAST searches of sequences that appeared suspicious in the alignments revealed few published sequences to be contaminations, which therefore were removed from the data set. Those sequences are 28S for Pylopagurus discoidalis (A. Milne-Edwards, 1880) (KF182614); COI for Nematopagurus squamichelis Alcock, 1905 (KJ150706); and COI for Pylopaguropsis magnimanus (Henderson, 1896) (KM043479). A 16S sequence attributed to Cestopagurus timidus (Roux, 1830) (FR849637) available on GenBank is misidentified and appears to belong to Pagurus prideaux Leach, 1815 based on comparison with our data from these species. The 18S sequence of Pylopaguridium markhami McLaughlin & Lemaitre, 2001 (KF182478) was trimmed, since the end of the sequence consists of a repetition of a previous section of the same sequence, possibly generated during processing of the sequence data. The 18S sequence of Oedignathus inermis (Stimpson, 1860) (Z14062) was excluded due to few obvious minor sequencing errors in highly conservative regions. The 18S (KF182453) and 28S (KF182603) sequences of Cryptolithodes sp. were assigned to Cryptolithodes sitchensis Brandt, 1853, based on the 16S gene of Cryptolithodes sp. from the same study (KF182574) (Bracken-Grissom et al., 2013), which is identical to sequences of C. sitchensis from other studies. GenBank sequences attributed to Neolithodes brodiei Dawson & Yaldwyn, 1970, sampled from Vanuatu (Snow, 2010) appear to come from an undescribed species (Ahyong, 2010b) and are here referred to as ‘Neolithodes nr. brodiei’. The alignments of the non-protein coding genes were subsequently run in Gblocks 0.91b (Castresana, 2000) to exclude ambiguous aligned regions, using the Gblocks server. Gblocks criteria used for this were for a less stringent selection, allowing for gaps within blocks, smaller final blocks and less strict flanking positions. The 28S alignment was not complete for all taxa over the entire length. Gblocks treats missing data like gaps and would, therefore, also remove highly conserved regions in this alignment. Ambiguously aligned regions in 28S were removed by hand, using the same parameters as Gblocks, but considering only gaps. The single gene alignments were concatenated to a single file using MacClade 4.06. PartitionFinder 1.1.1 (Lanfear et al., 2012) was used to determine the best partitioning scheme and best-fit nucleotide substitution models for the concatenated data set under the Bayesian information criterion. The ‘greedy’ algorithm was used with branch lengths of alternative partitions ‘linked’. The analysis suggested a partitioning of the data set by each marker as well as each codon position for the protein-coding COI. PartitionFinder suggested as the best-fit substitution models SYM + I + G for the first codon position of COI and 18S, F81 for the second codon position of COI, GTR + G for the third codon position of COI, GTR + I + G for 16S and 28S, and HKY + I + G for 12S. Phylogenetic analyses The concatenated data set was analysed using maximum likelihood (ML) and Bayesian inference (BI) approaches. The ML analysis were conducted using RAxML 8.2.4 (Stamatakis, 2014), on the CIPRES science gateway (Miller, Pfeiffer & Schwartz, 2010). A unique GTR model of sequence evolution was specified for each partition following the scheme given by PartitionFinder with corrections for a discrete gamma distribution for site-rate heterogeneity (GTRGAMMA). The GTRCAT model was used for the bootstrapping phase. Thousand rapid bootstrap iterations were conducted to search for the best-scoring ML tree in one single program run. BI was conducted in MrBayes 3.2.2 (Ronquist et al., 2012), on the Lifeportal, University of Oslo. The concatenated data set was partitioned following the scheme given by PartitionFinder. Each partition was run under the best-fit model of evolution, and all model parameter values were ‘unlinked’ among partitions. Two independent runs using four Metropolis-coupled Markov chain Monte Carlo analyses were performed. The chains were run for 20 million generations and sampled every 500 generations. The first 10000 trees were discarded as burn-in, and a 50% majority-rule consensus tree was obtained from the remaining saved trees. The average standard deviation of split frequencies was checked for convergence towards zero, and MrBayes parameter files were examined in Tracer 1.6 (Rambaut et al., 2014) to assess if runs had reached a stationary phase and converged on model parameters. A second data set was produced, based on the phylogenetic tree obtained from the initial analyses, comprising only representatives of Lithodidae and pagurid hermit crabs that had been identified as sister clade to Lithodidae, as well as one outgroup taxon (Pagurus comptus White, 1847). Ambiguously aligned positions of the non-protein-coding gene alignments were removed as described for the initial data set. Since the gene alignments with this limited number of taxa contained fewer ambiguous positions, the resulting alignments were longer and contained more phylogenetic information. The single gene alignments were concatenated, and PartitionFinder was used as described above. The analyses suggested the same partition scheme as in the previous data set, with the same best-fit substitution models, except F81 + I for the second codon position of COI, HKY + I + G for 16S and K80 + I for 18S. The data set was analysed using ML and BI as described for the initial data set. The resulting phylogenetic trees were visualized using Dendroscope 3.2 (Huson & Scornavacca, 2012). RESULTS The phylogenetic trees obtained from ML and BI analyses were largely corresponding. Support values, however, were lower in the trees resulting from the ML analyses than in the trees obtained by BI (Figs 1, 2). We included the mitochondrial 12S and 16S rRNAs and the mitochondrial cytochrome c oxidase 1 (COI), as well as parts of the nuclear 18S and 28S rRNAs. All these genes have previously been proven to be useful in systematic studies of crustaceans (Schubart, Neigel & Felder, 2000). Different rates of evolution among the genes make a concatenated set of these markers a valuable phylogenetic tool for resolving a range of taxonomic levels (Toon et al., 2009). Additional sequences from a large range of pagurid and lithodid species were downloaded from GenBank and BOLD, allowing us to compile the most complete data set to date, in the search for the closest extant relatives of lithodid crabs. The resulting data set was fragmentary for many taxa, since for most species only one or few genes were available in GenBank and BOLD. Deep phylogenetic nodes were resolved by taxa with a larger coverage in the matrix, while species with low coverage, for example only COI, clearly affiliated with taxa that had a larger coverage, usually congeneric species. Figure 1. View largeDownload slide Bayesian 50% majority-rule tree of Paguridae, Lithodidae and Diogenidae (outgroup) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Colour fields indicating Lithodidae (blue), Paguridae (yellow) and Diogenidae (green). The red dot indicates the node corresponding to the LCA shared by Lithodidae and pagurid hermit crabs. Pagurus species assigned to informal species groups are highlighted with a grey overlay. Figure 1. View largeDownload slide Bayesian 50% majority-rule tree of Paguridae, Lithodidae and Diogenidae (outgroup) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Colour fields indicating Lithodidae (blue), Paguridae (yellow) and Diogenidae (green). The red dot indicates the node corresponding to the LCA shared by Lithodidae and pagurid hermit crabs. Pagurus species assigned to informal species groups are highlighted with a grey overlay. Figure 2. View largeDownload slide Bayesian 50% majority-rule tree of Lithodidae (blue) and the hermit crab clade, which constitutes the closest relatives within the Paguridae, the ‘pagurid–lithodid sister clade’ (PLS clade) (yellow) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Species of the subfamily Hapalogastrinae are highlighted by dark blue overlay. Pagurus s.s. species of the ‘bernhardus’ group are highlighted by dark yellow overlay. Red stars indicating clades with deep-sea radiations outside the North Pacific. Illustrated specimens are not to scale. Figure 2. View largeDownload slide Bayesian 50% majority-rule tree of Lithodidae (blue) and the hermit crab clade, which constitutes the closest relatives within the Paguridae, the ‘pagurid–lithodid sister clade’ (PLS clade) (yellow) for the five-gene concatenated data set. Bayesian posterior probabilities (PP) and maximum likelihood bootstrap values over 50% are given on the nodes, respectively. Species of the subfamily Hapalogastrinae are highlighted by dark blue overlay. Pagurus s.s. species of the ‘bernhardus’ group are highlighted by dark yellow overlay. Red stars indicating clades with deep-sea radiations outside the North Pacific. Illustrated specimens are not to scale. Our results show a monophyletic Lithodidae, deeply nested within a paraphyletic hermit crab family Paguridae (Fig. 1). Paguridae with the contained Lithodidae was found clearly distinct from the diogenid outgroup taxa (Fig. 1). A clade of pagurid hermit crabs was clearly resolved as the sister taxon to Lithodidae within the Paguridae, which we refer to as ‘pagurid–lithodid sister clade’ (PLS clade) (Fig. 1). This clade shared the last common ancestor (LCA) with lithodid crabs within the hermit crabs. All species included in this clade are shallow-water hermit crabs, mainly distributed in the North Pacific. It consists of some genera exclusively found in the North Pacific: Discorsopagurus, Elassochirus and Labidochirus, as well as various species of Pagurus. Included representatives of Pagurus in the PLS clade belong to several of the established informal Pagurus morpho-groups; the ‘bernhardus’, ‘trigonocheirus’, ‘capillatus’ and part of the ‘comptus’ group (Forest & de Saint Laurent, 1968; McLaughlin, 1974; Lemaitre & Cruz-Castaño, 2004), as well as species that have not been assigned to any of these informal groupings. Pagurus species included in this PLS clade are also exclusive to the North Pacific, except for four species from the North Atlantic, which have close related species in the Pacific. The usage of Pagurus as a catch-all genus for species with a general pagurid hermit crab morphology is well illustrated in the phylogenetic tree (Fig. 1), as representatives of the genus are dispersed throughout the family. The analyses largely confirm previously established informal morphological Pagurus groups (Fig. 1). Only representatives of the ‘comptus’ group show a clear separation between species from South America (‘comptus’ group I) and the North Pacific (‘comptus’ group II). Two Pagurus clades, corresponding to the ‘provenzanoi’ group and subdivision I by Ingle (1985), also contain species that are assigned to other genera, Manucomplanus McLaughlin, 1981 and Paguridium Forest, 1961, respectively. Except for Pagurus, all other hermit crab genera included in the phylogenetic analyses with multiple representatives were resolved as monophyletic. While Lithodidae was resolved as monophyletic overall, the two subfamilies Lithodinae and Hapalogastrinae appear not to be monophyletic (Fig. 2). Basal to the remaining lithodid taxa are the two hapalogastrine genera Oedignathus Benedict, 1895 and Hapalogaster (Fig. 2). The lithodine genus Cryptolithodes nests among the hapalogastrine and likely forms the sister taxon to all remaining lithodids, including all Lithodinae and the two monotypic hapalogastrine genera Placetron Schalfeew, 1892 and Acantholithodes Holmes, 1895. The internal phylogeny of the Lithodidae is not fully resolved in our analyses, especially with respect to the genus Paralithodes Brandt, 1848. A sister relationship between the monotypic genera Rhinolithodes Brandt, 1848 and Phyllolithodes Brandt, 1848 is highly supported. The genus Paralomis forms a highly supported clade, also including the monotypic genus Glyptolithodes Faxon, 1895. Sister taxon to Paralomis/Glyptolithodes is the genus Lopholithodes Brandt, 1848, which consists of only two species that were both included in the analyses. Another highly supported clade consists of Lithodes and Neolithodes. DISCUSSION Internal relationships within Lithodidae A monophyletic origin of lithodid crabs has been confirmed in several studies, both using molecular and morphological data (e.g. McLaughlin et al., 2007; Reimann et al., 2011; Bracken-Grissom et al., 2013). Recent molecular studies, however, have left the status of the two lithodid subfamilies, Hapalogastrinae and Lithodinae, ambiguous (Hall & Thatje, 2009b; Bracken-Grissom et al., 2013; Thatje & Hall, 2016). The two taxa are separated based on the presence of a calcified or uncalcified pleon (McLaughlin, 2014). Hapalogastrinae are mostly small-sized crabs with a soft, uncalcified pleon, and have been suggested to represent a morphological intermediate form between pagurid hermit crabs and the large-sized king crabs, most closely resembling the lithodid stem species (Bouvier, 1895; Boas, 1924; Richter & Scholtz, 1994; Keiler et al., 2015). Recently, detailed morphological examinations of Hapalogaster mertensii Brandt, 1850 showed that this small lithodid crab possesses anatomical features of both Paguridae and Lithodidae. Most strikingly, Hapalogaster exhibits an uncalcified sac-like pleon and also features characters of both groups in the vascular system (Keiler et al., 2015). Morphological studies have supported the separation of lithodid crabs into the two distinct subfamilies. Hapalogastrinae were, however, only represented by the single genus Hapalogaster in these studies (Richter & Scholtz, 1994; Keiler & Richter, 2011; Keiler et al., 2013). Few previous molecular studies included representatives of Hapalogastrinae. In two studies, only one representative of each of Hapalogastrinae and Lithodinae was included (Ahyong et al., 2009; Schnabel et al., 2011). Two species of Hapalogastrinae were included in other studies (Hall & Thatje, 2009b; Bracken-Grissom et al., 2013), together with a number of lithodine species. Hall & Thatje (2009a) found two monophyletic subfamilies, but with only low support. Bracken-Grissom et al. (2013) found different internal relationships within the Lithodidae, depending on whether only molecular data were used, or if the data set was combined with morphological data. In the combined data set, Hapalogaster and Oedignathus were resolved as basal within the Lithodidae. In analyses using molecular data alone on the other hand, Oedignathus was placed among the lithodid crabs and distant from Hapalogaster. This placement of Oedignathus, however, appears to be due to a number of apparent sequencing errors in the included 18S gene from this species (GenBank: Z14062) (Kim, Min & Kim, 1992), which we found to be highly conserved in Lithodidae. For this reason, this sequence was excluded from our analyses. Instead of a clear separation into Hapalogastrinae and Lithodinae, various small-sized, shallow-water taxa appear basal in the phylogenetic tree of Lithodidae from our analysis (Fig. 2). The Hapalogastrinae genera Hapalogaster and Oedignathus are resolved as basal to all other lithodids. Two other genera of Hapalogastrinae (Placetron and Acantholithodes) had not previously been included in any phylogenetic study. These were found nested within Lithodinae and appear more derived than the small-sized lithodine genus Cryptolithodes, rendering both subfamilies non-monophyletic. The basal position of some genera of Hapalogastrinae, as indicated by previous studies (Richter & Scholtz, 1994; Keiler & Richter, 2011; Keiler et al., 2013), is in agreement with our results. A soft pleon, as a remnant of the hermit crab origin, must be considered as the plesiomorphic state of Lithodidae. The phylogenetic position of the small-sized lithodine genus Cryptolithodes, however, which is throughout heavily calcified, indicates that calcification of the pleon evolved at least twice within Lithodidae. The closest extant relatives to Lithodidae Our molecular analyses clearly show that a distinct, species-rich clade of hermit crabs forms the direct sister group to Lithodidae within the Paguridae (Fig. 1). Such a morphologically diverse and species-rich hermit crab sister clade to the lithodids is not as surprising as it might appear at first glance, given the species richness and morphological diversity of lithodid crabs themselves. Previous molecular phylogenetic studies have suggested different pagurid taxa as the closest relatives to the king crabs, depending on the representation of pagurid species in the analyses. With internal relationships of the Paguridae largely unknown, especially regarding the polyphyletic genus Pagurus, the topology of previous phylogenetic studies depended on which Pagurus species were included in the data sets. The first molecular study on king crabs (Cunningham et al., 1992) found the closest sister taxa to the lithodids to be a clade containing two species of the genus Pagurus [Pagurus bernhardus (Linnaeus, 1758) and Pagurus acadianus Benedict, 1901], together with the genera Labidochirus and Elassochirus. Two other Pagurus species included in the analyses were found to be more distantly related. Cunningham et al. highlighted the nested position of lithodids within the genus Pagurus. Richter & Scholtz (1994) subsequently noted that strong similarities of the first antennae between P. bernhardus and lithodid crabs support the results of Cunningham et al. (1992). Our analyses are in accordance with the finding of this first molecular study, as species resolved as closest relatives are also found in our PLS clade. Boas (1880b) initially assumed lithodids to be derived from the former Eupagurus Brandt, 1851, which contained some species of the genus Pagurus. Bouvier (1895) assumed lithodids as derived from an ancestor in which females have paired first pleopods, as present in the genus Pylopagurus. However, this taxon has since undergone major taxonomic revisions (McLaughlin, 1981; Lemaitre & McLaughlin, 2003), and it is thus unclear to which species Bouvier actually referred (Reimann et al., 2011). Boas (1924) later suggested Pylopagurus and Nematopagurus as candidates for the closest relatives to lithodids. Also in a cladistic analysis based on foregut morphology, Nematopagurus and Pylopagurus s.s. were resolved as sister group to lithodids within other Paguridae (Reimann et al., 2011), suggesting the same position to lithodids as assumed by Boas (1924). Our data, however, clearly show that neither Nematopagurus, Pylopagurus, nor any of the other genera of the ‘Pylopagurus–Tomopagurus’ group after Lemaitre & McLaughlin (2003) included in our analyses are particularly closely related to the Lithodidae (Fig. 1). The occurrence of first pleopods in different Paguridae appears to be plesiomorphic (Richter & Scholtz, 1994) and might not have been present in the LCA of hermit crabs and lithodids. Another genus of hermit crabs, Discorsopagurus, came into focus as possibly the closest relative to lithodids after being included in a molecular phylogeny by Morrison et al. (2002). In addition to Discorsopagurus schmitti (Stevens, 1925), Pagurus longicarpus Say, 1817 and the lithodid Cryptolithodes typicus Brandt, 1848 were included in this study. Discorsopagurus and Cryptolithodes showed a sister relationship, while P. longicarpus was found one node lower in the tree. This result might have initiated a focus on Discorsopagurus as a possible closest relative to lithodids, as it appeared more closely related than Pagurus. However, the reason that Discorsopagurus was resolved as a closer relative than a representative of Pagurus is merely due to the fact that Discorsopagurus is included in the PLS clade, while P. longicarpus is not found in this group (Fig. 1). Later studies confirmed a close relationship of Discorsopagurus to Lithodidae (Ahyong et al., 2009; Schnabel et al., 2011). Bracken-Grissom et al. (2013) included a larger number of Paguridae in their analyses and also here species of our PLS clade [Labidochirus splendescens (Owen, 1839), D. schmitti and P. bernhardus] were resolved with an equal sister relationship to lithodids based on molecular data alone. The authors, however, focused their discussion on the apparently closer relationship of Discorsopagurus, which was found when morphological data were added to the analyses. Based on their phylogenetic results, they suggested a Discorsopagurus-like hermit crab as the precursor to lithodids, which appeared plausible considering the North Pacific distribution of the genus (the region where lithodids are assumed to have originated; Hall & Thatje, 2009b). Discorsopagurus inhabits non-coiled housings, like polychaete tubes, and possesses an almost symmetrical pleon, with asymmetry restricted to the pleopods (Gherardi, 1996; Komai, 2003). Our data show that while Discorsopagurus is included in the PLS clade and thus shares the LCA to lithodid crabs within the Paguridae, it is not more closely related than other members of this clade with more typical pagurid morphologies. This indicates that the secondary pleon symmetry in Discorsopagurus and male lithodid crabs evolved independently and was unlikely present in the LCA. Interestingly, no extant hermit crabs with tendencies towards a crab-like body shape have been suggested as precursor to lithodid crabs. Two species that show signs of carcinization from a typical pagurid morphology (Blackstone, 1989; Anker & Paulay, 2013) are found inside the PLS clade: L. splendescens and Pagurus hirsutiusculus (Dana, 1851) (Blackstone, 1985; Cunningham et al., 1992). Labidochirus, a hermit crab with a fully calcified carapace, has been referred to as resembling a ‘missing link’ between hermit crabs and lithodid crabs (Jensen, 1995; Seeb et al., 2002). The large number of conventional pagurid hermit crabs in the PLS clade, however, points to a LCA with a rather typical hermit crab morphology. Polyphyly of the genus pagurus The finding that Lithodidae are not only nested within the pagurid hermit crabs, but even inside the genus Pagurus (Cunningham et al., 1992), caused much attention and disputes (McLaughlin et al., 2004; Lemaitre & McLaughlin, 2009). The genus Pagurus was originally established by Fabricius (1775) as a heterogeneous group of non-crab-like species of Linnaeus’ genus Cancer Linnaeus, 1758. A large range of hermit crabs was initially included in Pagurus and later assigned to new genera (McLaughlin, 1974). Our phylogenetic analyses show that today Pagurus is still highly polyphyletic. The species assigned to Pagurus do not possess unique morphological features, but rather display a ‘standard’ pagurid body plan (McLaughlin, 2003). The genus has been grouped into several informal morphological species groups (Forest & de Saint Laurent, 1968; McLaughlin, 1974; Lemaitre, McLaughlin & García-Gómez, 1982; Ingle, 1985; Lemaitre & Cruz-Castaño, 2004). In our phylogenetic tree, species of the genus Pagurus are divided into numerous distinct genetic lineages, often confirming previously recognized morpho-groups, for example the ‘exilis’, ‘bernhardus’, ‘capillatus’ and ‘provenzanoi’ groups, and subdivision I by Ingle (1985) (Fig. 1). Only representatives of the informal ‘comptus’ group are found in two very distinct clades within the phylogenetic tree. However, due to the deviation from the group diagnostic characters in North Pacific representatives of this group, the ‘comptus’ group has been highlighted as likely polyphyletic (McLaughlin, 1974). McLaughlin (1974) suggests that Pagurus ‘s.s.’ will eventually be restricted to a few species typified by P. bernhardus, which was selected as type species for the genus by Latreille (1810). Our phylogenetic analyses support this prediction, as a group of only few morphologically very similar species form a clade with P. bernhardus, without rendering the genus polyphyletic (Fig. 2). The species in this Pagurus ‘s.s.’ group are the Northeastern Atlantic P. bernhardus; the Northwestern Atlantic P. acadianus; and the North Pacific Pagurus aleuticus (Benedict, 1892), Pagurus armatus (Dana, 1851) and Pagurus ochotensis Brandt, 1851, which have been grouped together in the ‘bernhardus’ group (McLaughlin, 1974). Included in this ‘bernhardus’ group are also three other species from the North Pacific (Komai, 1998; McLaughlin & Asakura, 2003; Lemaitre & Watabe, 2005), which were not included in our analyses. Of all the ~180 species currently assigned to Pagurus (Türkay, 2016), likely only these eight species can be included in the genus without rendering it polyphyletic. Taxonomic hierarchy of the Lithodid crabs The deeply nested position of lithodid crabs within the hermit crab family Paguridae makes a phylogenetic classification difficult to apply. To further complicate issues, McLaughlin et al. (2007) proposed the taxonomic elevation of lithodid crabs to superfamily level, Lithodoidea, since the authors did not agree with the concept of a pagurid ancestry of the taxon. The two subfamilies of lithodid crabs were accordingly elevated from subfamily to family rankings: Lithodidae and Hapalogastridae. However, the phylogenetic position of lithodids outside the Paguroidea (McLaughlin et al., 2007) contradicted all molecular and many morphological studies. The placement of a superfamily, Lithodoidea, within the family Paguridae subsequently has been highlighted as problematic, since it obscures evolutionary relationships (Ahyong et al., 2009; Keiler et al., 2013, 2015; Anker & Paulay, 2013). Further highlighting the problem caused by the elevation of the taxon is that Lithodidae sensuMcLaughlin et al. (2007) is exclusive of hapalogastrids, while these were previously recognized as part of this family via the subfamily Hapalogastrinae. The concept of a ‘Lithodoidea’ has subsequently only been used by a few authors (Ahyong et al., 2009; De Grave et al., 2009; Schnabel et al., 2011; Tsang et al., 2011; Bracken-Grissom et al., 2013). Some authors adopted the two distinct family rankings (Lithodidae and Hapalogastridae) while rejecting the superfamily Lithodoidea to combine the two taxa (Keiler et al., 2013, 2015). Others used the family Lithodidae in its former sense, containing the two subfamilies Lithodinae and Hapalogastrinae (Guzmán, 2009; Hall & Thatje, 2009b; Macpherson & Wehrtmann, 2010; Anker & Paulay, 2013), a system which we also use in this study. However, even the nested position of a family (Lithodidae) within another family (Paguridae) masks the true relationships of the groups. The fact that lithodids, in addition, appear nested within the polyphyletic genus Pagurus highlights this problem. Both Hapalogastrinae and Lithodinae appear non-monophyletic in our analyses. We, therefore, suggest a rather opposite taxonomic ranking to the one proposed by McLaughlin et al. (2007), by combining all lithodid crabs in a single taxon, and recognize its position within Paguridae by using the rank of a subfamily, Lithodinae. Hermit crabs within Paguridae, subsequently categorized under Pagurinae, are, however, still paraphyletic under this ranking. Pagurid hermit crabs are in need of an extensive taxonomic revision, which will need in-depth morphological and molecular investigations. Geographic origin of the Lithodidae Our finding that the closest hermit crab relatives to lithodid crabs predominantly consist of North Pacific species adds further support to a Northern Pacific origin of lithodids, as it suggests that the split between the PLS lineage and lithodids also occurred here. The four species of the PLS clade with a North Atlantic distribution have closely allied species in the Pacific: P. bernhardus from the North East Atlantic and P. acadianus from the North West Atlantic are sister species, with closely related species in the North Pacific, forming the ‘bernhardus’ group (Fig. 2). Pagurus pubescens Krøyer, 1838, found on both sides of the North Atlantic, has closely allied species in the North Pacific, forming the ‘trigonocheirus’ group. Pagurus arcuatus Squires, 1964, from the North West Atlantic, has closely allied species in the North Pacific, forming the ‘capillatus’ group (McLaughlin, 1974). The terminal nodes in the phylogenetic tree leading to these Atlantic species show that their predecessors, one for the representatives of each group, must independently have entered the Atlantic via the Bering Strait. The evolution of the deep-sea lineages followed a diversification of the taxon in the shallow North Pacific before changes in larval biology enabled certain taxa to extend their distribution into the deep sea (Hall & Thatje, 2009b; Thatje & Hall, 2016). Our data confirm the distribution of basal taxa of Lithodidae in the North Pacific and show two clear independent events of deep-sea radiation (Fig. 2). One event for Paralomis, including the monotypic Glyptolithodes which is found to be nested within the otherwise monophyletic genus Paralomis (Hall & Thatje, 2010), and one for Lithodes and Neolithodes (Fig. 2), of which Neolithodes reaches abyssal depths (Hall & Thatje, 2009b). Confining temperature boundaries have allowed only a few species from the boreal regions to re-emerge from the deep-sea into shallow-water habitats (Hall & Thatje, 2009b). Age of the origin of the Lithodidae The origin of Lithodidae has been estimated from 15 to 13 Mya (mid to lower Miocene) based on molecular clock analyses of the mitochondrial 16S rRNA (Cunningham et al., 1992). Following this study, the first lithodid crab known from the fossil record was described. This fossil species, Paralomis debodeorum Feldmann, 1998, has been dated to the mid to late Miocene of New Zealand and documents the presence of king crabs to at least 10 Mya in the South Pacific (Feldmann, 1998). In this context, the estimated lithodid origin obtained by Cunningham et al. (1992) appears quite recent. A slightly older origin, between 29 and 18 Mya, has been estimated using multiple genes, with fossil calibrations from the entire Anomura (Bracken-Grissom et al., 2013). Molecular clock analyses might, however, be influenced by a low sequence diversity within the Lithodidae (Snow, 2010; Matzen da Silva et al., 2011b). On the other hand, the deposit from which P. debodeorum has been discovered is difficult to date precisely (Feldmann, 1998; Feldmann, Schweitzer & McLauchlan, 2006), leaving a relatively recent radiation, reflected in low genetic variation, as a possibility. A low sequence divergence within Lithodidae was also found in our own sequence data, especially for the nuclear rRNA subunits. The entire ~1800 bp long 18S fragment only showed minimal variation, and even sequences obtained from different lithodid genera were found to be identical. The taxonomic assignment of fossil hermit crabs is problematic, and ‘lump genera’ such as Palaeopagurus Van Straelen, 1924 or Pagurus have been used for most species (Jagt et al., 2006). The highly polyphyletic pattern of extant species of Pagurus, as shown in our phylogeny (Fig. 1), further highlights this problem. Accurate divergence timing using fossil calibrations for the Paguridae is, therefore, problematic. Detailed investigation of phylogenetic relationships within various Paguridae taxa, and the species-rich deep-sea lithodid genera, in conjunction with biogeography might eventually provide a more reliable divergence estimate through the timing of geological events. Evolutionary scenarios for the Lithodidae Taxa within Lithodidae that are found basal in our phylogenetic analyses inhabit shallow-water, rocky habitats, which is in agreement with previous studies that suggested a shallow-water origin of lithodids (Makarov, 1938; Zaklan, 2001; Hall & Thatje, 2009b). Our finding that the closest hermit crab relatives also inhabit shallow habitats further supports this theory. This habitat must have played a key factor in the process of changing from a shell-utilizing to a free-living lifestyle. Crab-like forms appear to have evolved multiple times in shallow-water habitats (Morrison et al., 2002; Tsang et al., 2011), and these independent transitions offer strong evidence for the adaptive advantages of the crab-like form in relation to habitat type (Tsang et al., 2011). Most anomurans with a crab-like morphology are found living in hard bottom habitats, under boulders and stones, where a short, compact pleon is advantageous in exploring crevices (Tsang et al., 2011). Carcinization in hermit crabs is more complex than in other decapods, since this process, besides the broadening of the cephalothorax and reduction and underfolding of the pleon, also implies reorganization and calcification of the cephalothorax and pleon as these animals abandon the use of domiciles (Anker & Paulay, 2013). Besides the lithodids, tendencies towards carcinization, involving reduction or armouring of the pleon, tendency to lose domiciles and calcification of the cephalothorax, occurred independently in several groups of hermit crabs (Anker & Paulay, 2013). The abandonment or reduced use of a portable domicile must be seen as the most important step towards this morphological transformation. Different alternative pathways of leaving a protective housing are possible, and multiple scenarios for the cause of the predecessor of lithodid crabs leaving a protective shell exist. The various degrees of carcinization present in different hermit crabs give insights into the possibilities for morphological transition, but none of these taxa represent direct evolutionary intermediate forms between hermit crabs and lithodid crabs. Cunningham et al. (1992) explained carcinization in king crabs via a heterochronic shift in developmental timing, the extension of the ancestral hermit crab ontogeny to produce a carcinized adult, termed peramorphosis. In this scenario, ancestral hermit crab allometries were modified to accommodate an extended ontogeny and larger body size. In particular, Cunningham et al. (1992) highlighted the terrestrial hermit crab Birgus latro (Linnaeus, 1767), which, having a normal hermit crab habitus as a juvenile, outgrows its protective shell during ontogeny and develops a crab-like, calcified body (Greenaway, 2003). In Lithodidae, a crab-like morphology is, however, already apparent at metamorphosis (Morrison et al., 2002; McLaughlin et al., 2004), and Morrison et al. (2002) suggested a somewhat different mechanism of heterochronic shift, in the form of displacement heterochrony (Alberch et al., 1979). The size of the largest available gastropod shell limits the size of hermit crabs (Cunningham et al., 1992), and a lack of suitable shells has been discussed as a factor in the carcinization of lithodids (Richter & Scholtz, 1994). An absence of sufficiently large shells, due to an increase in size in the ancestral lineage of Lithodidae, leading to limited resources of suitable housings was rejected, since many lithodid species, in particular Hapalogastrinae, are not very large. As a more likely alternative, the lack of suitable shells in certain habitats was suggested as a possible starting point for lithodid evolution (Richter & Scholtz, 1994). Our results also point to a small-sized LCA, making the limitation of large shells as causation behind the evolution of lithodids unlikely. Some species of hermit crabs with tendencies towards carcinization are restricted by the availability of large gastropod shells, for example due to a deep-sea habitat, such as Porcellanopagurus Filhol, 1885; Solitariopagurus Türkay, 1986; and Patagurus Anker & Paulay, 2013 (McLaughlin & Lemaitre, 1997; Anker & Paulay, 2013). In some of these taxa, the pleon is reduced in size and only covered by a shell, which is too small for the animal to retract into (McLaughlin & Lemaitre, 1997; Anker & Paulay, 2013). From the early Miocene, large gastropod species were, however, never rare in the shallow North Pacific (Vermeij, 2012), and also the parallel diversification of hermit crabs in the same region, as indicated from our phylogeny, further points to a scenario without a general lack of gastropod shells. Furthermore, the large increase in body size of some lithodid taxa clearly occurred after the acquisition of the crab-like form, as basal taxa within the Lithodidae are of only moderate size. Advantages of abandoning of a domicile While the disadvantages of abandoning a protective housing and subsequently exposing the soft pleon have been highlighted as a maladaptive evolutionary scenario (McLaughlin & Lemaitre, 1997; McLaughlin et al., 2004), becoming independent from a housing also brings clear advantages. Competition for housings, and the need to find and change suitable housings during ontogeny are probably the most obvious ones. Inhabiting a gastropod shell, however, also requires a heavy object to be carried, greatly reducing mobility. Leaving the constraint of being bound to a foreign shell results in an increase in agility and speed, potentially making new prey sources available and enabling escape from predators (Blackstone, 1989; Anker & Paulay, 2013). The advantages of higher mobility also include the possibility of inhabiting new microhabitats precluded by carrying a bulky and heavy shell, like crevices or rock overhangs. As basal lithodids are found in such habitats today (Jensen, 1995), the enhanced mobility resulting from abandoning a protective housing is likely a key factor behind the evolutionary pathway of Lithodidae. An example of enhanced mobility by reducing the weight of a protective housing is found in the intertidal hermit crab P. hirsutiusculus, which uses only small shells in which the animal cannot fully retract. Pagurus hirsutiusculus shows tendencies towards carcinization, such as a broadened carapace and stronger armature of the pleon (McLaughlin, 1974). This species is very agile and often abandons its housing in escape reactions (Blackstone, 1989). A higher level of activity permits the animal to rely on speed of escape, rather than a housing for protection, which could favour shell loss and carcinization (Blackstone, 1989). This example illustrates how slight changes in ecology and shell-use might lead to carcinization in hermit crabs, without a restriction of housings. A number of hermit crab species have obligate commensal relationships with certain species of actinarians (Williams & McDermott, 2004). In the most advanced of these symbiotic relationships, the sea anemone builds the entire housing for the crab or greatly enlarges an originally present small gastropod shell. This lightweight housing protects its inhabitant not by a heavily calcified structure, but by a soft housing with protrudable thread-like acontias, which are loaded with poisonous nematocysts for defence. An increase in mobility, via reducing the weight of the shelter, is also an advantage of these relationships. The pagurid L. splendescens, which is found in the PLS clade, has such a symbiotic relationship. The lightweight housing, together with long walking legs for rapid locomotion, gives the animal a much higher mobility than seen in conventional hermit crabs. Labidochirus splendescens also shows tendency towards carcinization, like a broadened, fully calcified carapace, and an only moderate-sized pleon (McLaughlin & Lemaitre, 1997; Anker & Paulay, 2013). CONCLUSIONS The Lithodidae are deeply nested within the hermit crab family Paguridae and show a clear sister relationship with a clade of predominantly North Pacific shallow-water hermit crabs. Lithodid crabs are even found nested within a highly polyphyletic hermit crab genus Pagurus, confirming the results of the very first molecular study on the taxon by Cunningham et al. (1992). The stem species of Lithodidae inhabited shallow waters of the North Pacific with no general shortage of suitable gastropod shells. A crab-like morphology likely evolved gradually due to the adoption of smaller housings for the benefit of higher mobility. The basal position of small-sized taxa clearly indicates that an increase in body size was not the trigger for developing a crab-like habitus in the Lithodidae. The abandonment of a domicile, however, enabled the development of gigantism in lithodid crabs, since available gastropod shells for housing no longer set a size limitation. Enhanced armour in the form of spines and calcification, and an increase in size, enabled king crabs to leave the initial protective environment and expand into non-sheltered habitats. In the deep sea, the taxon could finally diversify on a global scale. Knowledge about phylogenetic relationships within the diverse deep-sea genera is still fragmentary. However, the species richness of these genera might eventually enable a detailed reconstruction of the dispersal routes within the deep-sea lineages of king crabs. ACKNOWLEDGEMENTS Funding for this study was provided by a grant from the Meltzer Foundation, University of Bergen. The Shannon Point Marine Center (Western Washington University, Anacortes, WA, USA) helped in sampling of specimens and provided laboratory space. Specimens for DNA extractions were kindly loaned from the University Museum of Bergen, Norway; the Royal BC Museum, Victoria, BC, Canada; and the NIWA Invertebrate Collection, Wellington, New Zealand. We thank the reviewers and the editors for their comments, which improved the manuscript. REFERENCES Ahyong ST. 2010a. The marine fauna of New Zealand: king crabs of New Zealand, Australia, and the Ross Sea (Crustacea: Decapoda: Lithodidae). NIWA Biodiversity Memoir  123: 194. Ahyong ST. 2010b. Neolithodes flindersi, a new species of king crab from southeastern Australia (Crustacea: Decapoda: Lithodidae). Zootaxa  2362: 55– 62. Ahyong ST, Chan TY. 2010. 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