Abstract The taxonomic status of lineages within the Australian allodapine bees has been unstable over the last six decades, with multiple changes in generic and subgeneric assignments. This is unhelpful given the continuing attention to these bees for understanding social evolution and biogeography. The Australian genus Exoneurella (Michener, 1963 Hymenoptera: Apidae) has received substantial attention because it contains a highly eusocial species, Exoneurella tridentata (Houston, 1976), as well as casteless species. Here, we describe a new Exoneurella species Exoneurella micheneri sp. nov. With the addition of this species, and re-examination of the four other Exoneurella species, we redefine the genus and use both genetic and morphological data to explore its phylogenetic relationships to the other Australian allodapine genera. We show that E. tridentata is highly unusual, not just in terms of queen/worker morphology but also in terms of male genitalia. As with other allodapine genera, larval morphology is highly divergent between genera, while female morphology is more conserved. Bees in the tribe Allodapini (Apidae: Xylocopinae) have been widely used to explore social evolution, because of their range of social forms (Schwarz et al. 2007). These bees nest in the dead stems and branches of plants and are most abundant and diverse in sub-Saharan Africa and Australia (Michener 1965, Michener 1971). The Australian allodapine bees comprise two well supported clades: Braunsapis (which also occurs in the Oriental region, southern parts of the Arabian Peninsula, Africa and Madagascar), and two ‘exoneurine’ genera, Exoneura and Exoneurella. Exoneura is further divided into three subgenera, Exoneura s.s., Brevineura, and the socially parasitic Inquilina (Michener 2007). Fuller et al. (2005) showed via molecular phylogenetic reconstruction that the Australian species of Braunsapis resulted from a dispersal from southern Asia sometime in the late Miocene, while Chenoweth and Schwarz (2011) showed that the exoneurines comprise a monophyletic clade that resulted from a single dispersal event from Africa, probably via Antarctica, in the Oligocene. The exoneurines have received substantial attention because of their relevance to studies on social behavior (Schwarz et al. 2007; Stevens et al. 2007; Schwarz et al. 2011, Dew et al. 2012). Exoneurella in particular present a unique system for the study of social evolution. Exoneurella tridentataHouston, 1976 is eusocial with morphologically distinct reproductive cates (Hurst 2001). In contrast, Exoneurella setosaHouston, 1976 and Exoneurella eremophilaHouston, 1976 are casteless, with all females in social groups having equal reproductive opportunities (Dew et al. 2017a,b). For future research to continue, it is important to establish a formal taxonomic framework for the exoneurines based on a well-supported phylogeny using an integrative approach (e.g., Stevens et al. 2011, Schmidt et al. 2015, Packer and Ruz 2016, Stevens and D’Hease 2017). Exoneurella was first described as a monospecific genus by Michener (1963), based on re-examination of Exoneura lawsoniRayment, 1946. Generic status was justified by a number of traits including forewing venation, shape of the sixth tergum with lack of pubescence at its apex, and larval morphology (Michener 1963). Michener (1965) relegated Exoneurella to a subgenus of Exoneura which contained two other subgenera, Exoneura s. s. and Brevineura. This reduced the number of genera in the group, we now regard as the exoneurines to two—namely, Exoneura and parasitic genus Inquilina (Michener 1961). Subsequently, three other Exoneurella species, E. (E.) eremophila, E. (E.) setosa and E. (E.) tridentata, were described by Houston (1976) and diagnostic traits for the subgenus were expanded by broadening the description of the apex of the sixth tergum, and including metasomal pigmentation and male eye shape. Using a cladistic analysis of morphology, Reyes (1998) raised Exoneura s. s., Brevineura and Exoneurella to generic status, while retaining Inquilina as a full genus as well. Finally, Michener (2000, 2007) retained Exoneurella as a genus but combined Exoneura s.s., Brevineura and Inquilina as subgenera of Exoneura. Michener (2007) pointed out the difficulty of separating Exoneurella from Brevineura on the basis of adult morphology alone, noting that the generic status of Exoneurella was mainly justified by larval morphology. In a molecular phylogenetic study, Chenoweth and Schwarz (2011) included an undescribed allodapine species from Western Australia that was recovered as a basal lineage in Exoneurella. The basal position of this species has the potential to help resolve the problematic relationship between Brevineura and Exoneurella, but Chenoweth and Schwarz (2011) did not report the adult or larval morphology of this species. As such, this undescribed species presents an unrealized opportunity to understand the morphological evolution associated with generic divergences in the allodapine bees. Here, we formally describe this new species of Exoneurella and adjust the diagnostic features of this genus to incorporate all five current species contained within it. Methods Bee samples were collected November 2013 from south-west Western Australia, including both nest samples and specimens from floral sweeps. Nests, found in dead stems of Kangaroo Paw (Anigozanthos spp.), were collected during early morning or late evening when all adults would be in the nest. The adults and brood present in each nest were recorded and preserved in 100% ethanol. Images were obtained using a Nikon D5100 (Japan) digital camera mounted on a Nikon SMZ1000 (Japan) stereo microscope, and manually focused to obtain 6–12 images using the software Camera Control Pro2 ver. 2.22.0 (Nikon) to produce a compiled montaged image using Helicon Focus ver. 6.4.1. (Helicon Soft Ltd). All paratypes were examined and only minimal variation was noted. Measurements presented are based on the holotype. Type specimens were deposited in the West Australian Museum (WAM) and the South Australian Museum (SAMA). An additional three females and one male were also viewed for the description. These specimens were from Denmark, Western Australia, and details on their collection and accession numbers for genetic material of the then undescribed Exoneurella Western Australia D are available in Chenoweth and Schwarz (2011). DNA Barcoding We performed DNA barcoding on two Exoneurella specimens from separate nests in order to confirm that these were indeed samples of the same undescribed species that was included in Chenoweth and Schwarz (2011). DNA was extracted using a microfiber vacuum plate method (Ivanova et al. 2006). Extracted DNA was eluted into 50-μl TLE (10-mM TRIS, 0.1-mM EDTA pH8). Polymerase chain reactions (PCRs) of 25 μl were used for DNA amplification of a 612-bp region of cytochrome c oxidase 1 (COI). Reactions contained 0.1-μl immolase as the active enzyme, 5 μl of MRT buffer, 15.4-μl water, 2.5-μl DNA, and 1 μl each of the forward and reverse primers. The forward primer is a combination of M13/pUC (Messing 1988) and LC01490 (Folmer et al. 1994; 5’-GTTTTCCCAGTCACGACCCTTTTATAATTGGAGGATTT GG -3’). The reverse primer combines the reverse M13/pUC and primer M399 designed by S. Cooper (Schwarz et al. 2004; 5’-CAGGAAACAGCTATGACTCATCTAAAAACTTTAATTCC TG-3’). The PCR cycle began with 10 min of 94°C. The annealing stage had 5 cycles consisting of 60 s at 94°C, 90 s at 45°C, and 90 s at 72°C followed by 35 cycles of 60 s at 94°C, 90 s at 50°C, and 60 s at 72°C. Elongation was 10 min at 72°C with a final 2 min at 25°C. Raw PCR products were cleaned by a vacuum plate wash with 100-μl TLE before storage in 30-μl TLE. The clean PCR product was sent to the Australian Genome Research Facility for sequencing. The sequences have been submitted to GenBank (KY292349, KY292350, HQ268578). Phylogenetic Reconstruction We used both morphological and molecular data to place E. micheneri sp. nov. within the exoneurine phylogeny. We used a subset of the molecular data from Chenoweth and Schwarz (2011), including 16 allodapine species from 14 genera, covering a wide range of the nonparasitic allodapines. The data consisted of three gene regions: mitochondrial COI, and nuclear elongation factor 1-α (EF1-α), and elongation factor 2-α (EF2-α). Ceratina speculifronsCockerell, 1920 (Apidae: Ceratinini) was used as the outgroup. Of the allodapine taxa, 10 samples were from African allodapine genera, to help root the exoneurines, and secondly to allow more informative ancestral reconstruction of morphological traits. Within the allodapines, adult female morphology is often blurred between genera (Michener 1976). Larval traits, however, show clear generic discontinuities. This is particularly important for E. micheneri sp. nov., which shows distinct Exoneurella larval morphology, but adult female morphology that is largely synapomorphic between Brevineura and Exoneurella (see Discussion). To capture these morphological patterns in our phylogeny, we assembled data on 16 morphological traits. We used the 21 characters defined by Reyes (1998) as a starting point. All character states were checked against actual specimens, or where this was not possible, information from published descriptions and diagrams was used. Only traits where the character codes were confirmed for all genera in the phylogeny were included in the analysis. This led to the exclusion of some traits from Reyes original dataset reducing the total number to 16. Morphological data were obtained from various papers describing the Allodapini and their larvae (Michener 1975a,b,c, 1976, 2007; Chenoweth et al. 2008; Kayaalp et al. 2011). A number of traits including the coding of head hairs, wing venation, and various aspects of the male genitalia were changed to address concerns raised by Schwarz et al. (2003) and more accurately represent the morphological variation among species and genera. A full list of the traits used (Supp Table 1 [online only]) and coding for each species (Supp Table 2 [online only]) is available in the supplementary material. The phylogenetic reconstruction was performed in BEAST using both the molecular and morphological data. Ancestral reconstruction for all morphological characters was run using *BEAST (Heled and Drummond 2010; Supp Table 1 [online only]). Each gene (COI, EF1-α, and EF2-α) and codon position were partitioned separately based on model comparison in JModel Test (Guindon and Gascuel 2003, Darriba et al. 2012). A Yule process tree prior with a GTR+ I+ Γ substitution model was employed and run with an uncorrelated log normal relaxed clock model. The analysis ran for 1 × 108 generations, with trees and model parameters logged every 500 generations. Stabilization of the posterior was examined in Tracer ver. 1.6.1 (Rambaut et al. 2014) and a maximum credibility tree was generated using a burnin of 5 × 106 iterations. The analysis was run a total of three times. Nomenclature This paper and the nomenclatural act it contains have been registered in Zoobank (www.zoobank.org), the official register of the International Commission on Zoological Nomenclature. The LSID (Life Science Identifier) number of the publication is urn:lsid:zoobank.org:pub:5979B6BC-B1F9-4188-AD4B-8DA8910FDF89 Results Phylogenetic Reconstruction The maximum credibility tree gives very high support for the placement of E. micheneri sp. nov. as sister species to all other Exoneurella (PP = 1.0; Fig. 1). This phylogenetic position may help explain the synapomorphies in adult morphology with Brevineura species. Inquilina and Exoneura are strongly supported as sister clades (PP = 1.0). The placement of Brevineura was not resolved, with a PP value of 0.62 grouping it with Exoneura + Inquilina. Chenoweth and Schwarz (2011) obtained moderately strong support (PP = 0.93) for this same topology using a larger taxon set, suggesting that Brevineura is closely united with these two genera. Fig. 1. View largeDownload slide Maximum credibility tree based on molecular and morphological data. C. speculifrons was constrained as the outgroup (black). Posterior probability indicated if less than 0.99. Colour represents the social behavior of each species; red: eusocial, orange: hierarchical social groups, yellow: casteless social groups, blue: social parasites, gray: social behavior unknown. Fig. 1. View largeDownload slide Maximum credibility tree based on molecular and morphological data. C. speculifrons was constrained as the outgroup (black). Posterior probability indicated if less than 0.99. Colour represents the social behavior of each species; red: eusocial, orange: hierarchical social groups, yellow: casteless social groups, blue: social parasites, gray: social behavior unknown. Morphological Ancestral Trait Reconstruction The BEAST traits analysis gives strong support for origins of the larval bilobed head (i.e., head has pronounced lateral bulges; PP = 0.97), bilobed labrum (PP = 0.96) and bent body shape (PP = 0.99), in the most recent common ancestor (MRCA) of the Allodapini (Supp Figs. 1–3 [online only]). The larval morphology of E. micheneri is strongly similar to that of Exoneurella species, for all traits with the singular exception that it is relatively hairless compared to all other Exoneurella larvae, instead displaying pubescence similar to the less hairy Exoneura and Inquilina (Supp Fig. 4 [online only]). All Exoneurella lack tubercles, unlike Brevineura and particularly Exoneura where these are greatly elaborated (Supp Fig. 5 [online only]). A lack of larval tubercles is also seen in Macrogalea (basal to the other allodapine genera) and in the outgroup Ceratina. Our tree was unable to resolve whether the lack of larval appendages was due to retention of the ancestral condition or secondary losses, either at the MCRA of Macrogalea (PP = 0.711), MCRA of the other allodapine genera (PP = 0.55), or the MCRA of the exoneurines (PP = 0.50). Notably, the male genital morphology of E. tridentata does not follow that of the other exoneurines (see Reyes 1998). Rather, it has a straightened and relatively elongated valve like those of Macrogalea, Allodapula, and Eucondylops. Family APIDAE Subfamily XYLOCOPINAE Tribe ALLODAPINI Genus Exoneurella Type species Exoneurella lawsoni Diagnosis The Exoneurella can be readily distinguished from other Allodapini by the unique fourth instar larval body shape, which shows a 90° bend at the fifth and sixth abdominal segments. All other allodapine genera have larval body shapes that vary from straight to curled. Exoneurella larvae also lack tubercules and appendages. Macrogalea larvae similarly lack tubercules and appendages but their bodies are curled and covered in small hook-like hairs. Adult female Exoneurella can be differentiated from most allodapine genera by the combination of a concave and upturned T6, the apex of which is not obscured with pubescence and the costal margin of the second submarginal cell, which is shorter than the first transcubital vein. However, these traits are not unique from all species of Brevineura and larval morphology or DNA analysis is needed to make a definitive diagnosis. Description Our analyses firmly place E. micheneri sp. nov. in a monophyletic clade with the four described Exoneurella. We redefine the generic description of Exoneurella here so that it incorporates all five species. Female Costal margin of the second submarginal cell is shorter than the first transcubital vein (except E. tridentata where the costal margin of the second submarginal cell is slightly longer than the first transcubital vein; Fig. 7); T6 is concave and upturned, sometimes strongly so (as in E. tridentata and E. eremophila); margin of T6 often has lateral flanges (except E. micheneri and E. lawsoni; Fig. 5); apex of T6 not obscured by pubescence. Remarks The adult female morphology of Exoneurella and Brevineura are difficult to distinguish. But these genera are clearly separated based on larval morphology and genetic data. Male Wing venation as for female; eyes bulbous; ocelli larger than antennal sockets; clypeal mark cream to yellow. Remarks Males genitalic traits may provide useful traits for generic determination, as they provide clear species level delineation within Exoneurella (Figs. 8 and 9). The comparative variation among and within other exoneurine genera is not currently known but may prove useful for future systematic studies of this group. Larvae (Fig. 10). Body bent at an approximately 90° angle at the fifth and sixth abdominal segment, forming an angulate shape; antenna tapering to a sharp point; lateral areas of head with bulging lobes (bilobed head shape); body lacks tubercles and appendages. Remarks This generic description does not mention the long tapering hairs on the ventro-lateral lobes of the head, referred to in the previous generic description by Michener (2000). These hairs are absent in E. micheneri sp. nov. and are relatively short and blunt in E. tridentata. This variability means it is no longer a defining trait of the genus. Exoneurella micheneri sp. nov. Zoobank registration—urn:lsid:zoobank.org:pub:5979B6BC-B1F9- 4188-AD4B-8DA8910FDF89 HOLOTYPE: AUSTRALIA: ♀, Western Australia: Gardner State Forest, 34° 46ʹ 36.6ʺ S, 116° 10ʹ 59.1ʺ E, 20-XI-2013, M. Stevens, WAM E-95910. PARATYPES: 2♂, same data, SAMA 32-035318, WAM E-95909; 1♀, Northcliffe, 34° 35ʹ 24.8ʺ S, 116° 4ʹ 39.7ʺ E, 16-XI-2013, R. Dew and M. Stevens, in dead and broken stems of kangaroo paw (Anigozanthos sp.), SAMA 32-035315, Genbank accession KY292349; 1♀, same data, WAM 95913, GenBank KY292350; 2♂, 2♀, same data, males: SAMA 32-035316, WAM E-95911, females: SAMA 32-035317, WAM E-95912. Additional specimens examined AUSTRALIA: Western Australia: 2♀, Northcliffe, XI-2013, M. Stevens; 3 ♀, Denmark, IX-2008, L. Chenoweth. Etymology Named in tribute to the late C.D. ‘Mich’ Michener who did so much and inspired so many about the wonders of bees. Mich provided the first revision of Australian allodapines and the first to realize their utility for understanding social evolution. His magnum opus, Bees of the World (2000, 2007), stands as a truly monumental resource to all researchers whose work involves bees. Female (Fig. 2A–D) Dimensions. Body length ~5 mm; wing length ~2.5 mm; head width ~1.20 mm; relative head measurements: width 50, length 48, lower interocular distance 24, upper interocular distance 32, interantennal distance 8, antennocular distance 15, interocellar distance 9, ocellocular distance 9. Colouration Live specimen integument glossy dark brown to black (Fig. 2A); clypeal marking pale yellow varying from stripe to T-shape; antenna dark brown at base, with gradient to light brown at tip of flagellum; wing venation and stigma dark brown (N.B. Metasoma, leg, antenna and wing colouration lightens to a yellowy brown over time in storage, e.g., Fig. 5B). Fig. 2. View largeDownload slide Female of E. micheneri sp. nov.: (A) lateral view; (B) head; (C) dorsal view of head and propodeum; and (D) female and larvae in nest (photo: Cyrille D’Haese). Fig. 2. View largeDownload slide Female of E. micheneri sp. nov.: (A) lateral view; (B) head; (C) dorsal view of head and propodeum; and (D) female and larvae in nest (photo: Cyrille D’Haese). Pubescence Margin between the scutum and pronotum has dense ring of white plumose setae; setae of the same colour, length and plumosity are spartanly dispersed on ventral side of thorax; mesoscutum and mesoscutellum with short sparse setae; dorsal surface of the metapostnotum and upper propodeum with isolated long feathered setae; vertex of head and clypeus with short setae; T1–T3 of metasoma has short sparse golden barb-like setae, with longer finer setae toward the sides of the metosomsa; T4–T6 has longer and more closely spaced setae, but not so dense as to obscure the integument or apex of T6; thick bands of setae at the posterior margins of S3–S5; profemur has feathered white setae; mesotibia, metatibia and basitarsus on all legs with long stout golden scopa. Structure Face circular when viewed anteriorly; clypeus extends almost to the antennal sockets; mandible tridentate; frontal line strongly raised; costal margin of second submarginal cell of forewing half as long as the first transcubital vein; Cu1 short, second recurrent vein absent; hindwing with five hamuli; basitibial plate present; Inner hind tibial spur simple, with very fine serration; apicoventral notch on S6; viewed laterally T5–T6 slope steeply down to the apex, where there is a slight upturn; viewed dorsally margin of T6 curves smoothly to apex. Male (Fig. 3A–C) Dimensions. Wing length ~2.2 mm; head width ~1.2 mm; relative head measurements: width 50, length 49, clypeal length 22, lower interocular distance 18, upper interocular distance 30, interantennal distance 9, antennocular distance 11, interocellar distance 9, ocellocular distance 7. Fig. 3. View largeDownload slide Male of E. micheneri sp. nov.: (A) head; (B) dorsal view; and (C) lateral view. Fig. 3. View largeDownload slide Male of E. micheneri sp. nov.: (A) head; (B) dorsal view; and (C) lateral view. Colouration As for female, except entire clypeus plus the lower paraocular area is pale yellow. Pubescence. As for female but lacking scopa, reduced and minimal setae on the metasoma except for long setae on T6 and T7. Structure As for female, with expected sexual differentiation; malus of protibial spur forks toward the end into two tines of equal length; genitalia with transparent flap-like ventroapical plate curved upward to ¾ the length of the penis valve; minute ventral gonostylus, apex reaching ½ the length of the penis valve, apex with brush-like setae; penis valve greatly broadened to midlength; lateral margin with 10–15 robust setae. Larvae, 4th instar (Fig. 4A–C) Dimensions Body width ~0.85 mm; Body length ~3.0 mm; Head width ~0.48 mm; Head length ~0.56 mm; Antenna length ~0.19 mm. Fig. 4. View largeDownload slide Larvae of E. micheneri sp. nov.: (A) lateral view; (B) head, showing lobe seta; and (C) head, showing mandible. Details indicated by arrows. Fig. 4. View largeDownload slide Larvae of E. micheneri sp. nov.: (A) lateral view; (B) head, showing lobe seta; and (C) head, showing mandible. Details indicated by arrows. Pubescence Head with very short fine setae on dorsal surface; Lateral lobes of head each with one blunt seta, about 2/3 antennal length; dorsal surface of head and first segment with fine setae 2/3 antennal; distal edges of segments with shorter slightly more robust setae; lateral and ventral surfaces setae is absent. Structure Body bent at an approximately 90° angle at the fifth and sixth abdominal segments forming an angulate shape; ventrolateral region of head swollen into pronounced lobe, forming an overall bi-lobed head shape; antenna thin and tapering to acute point; strong intersegmental lines; labrum does not extend beyond margin of head or over mandibles; mandibles simple, slender apically; body lacking tubercles or appendages; dorsal surface of segments 2 and 3 raised into wave shaped structures. Diagnosis Adult females of E. micheneri sp. nov. can be distinguished from other Exoneurella by the presence of the basitibial plate, not seen in any other Exoneurella, and by the smooth lateral margins of T6, which lead to a simple apex, without the additional dentation or flanges seen in most other species with the exception of E. lawsoni (Fig. 5B). Males are easily identified by the clypeal mark, which covers the entire clypeus, plus the lower paraocular area (Fig. 6A). Males of the other Exoneurella have their own distinctive face marking. The gonostylus is characterised by transparent flap-like ventroapical plates curved upward to ¾ the length of the penis valve, not seen in the other Exoneurella. There is also a minute ventral gonostyli that reaches ½ the length of the penis valve that is topped with brush-like setae, only present in E. micheneri and E. tridentata (Figs 8A and B). Fig. 5. View largeDownload slide Dorsal view of T6: (A) Brevineura elongata; (B) E. micheneri sp. nov.; (C) E. tridentata; (D) E. eremophila; (E) E. setosa; and (F) E. lawsoni. Fig. 5. View largeDownload slide Dorsal view of T6: (A) Brevineura elongata; (B) E. micheneri sp. nov.; (C) E. tridentata; (D) E. eremophila; (E) E. setosa; and (F) E. lawsoni. Fig. 6. View largeDownload slide Male heads: (A) E. micheneri sp. nov.; (B) E. tridentata; (C) E. eremophila; (D) E. setosa; and (E) E. lawsoni. Fig. 6. View largeDownload slide Male heads: (A) E. micheneri sp. nov.; (B) E. tridentata; (C) E. eremophila; (D) E. setosa; and (E) E. lawsoni. Larvae with short setae on the distal edges of segments, lacking the large spines of E. lawsoni, E. setosa and E. eremophila larvae (Fig. 4A). There is a single blunt seta on each lateral lobe of the head (Fig. 4B), other species having multiple setae. Dorsal surface of segments 2 and 3 raised into wave shaped structures (Fig. 4C); setae not present on ventral surface of body. Species Keys Females 1. (1) Lateral margins of T6 with projections or flanges (Fig. 5C–E).………………………………..……………………… 2 - (2) Lateral margins of T6 without projections or flanges (Fig. 5B and F)……………………………………….….………… 4 2 (1) Second submarginal cell costal margin is slightly longer than the first transcubital vein (Fig. 7C)……………….. E. tridentataHouston 1976 - (2) Second submarginal cell costal margin is shorter than the 1st transcubital vein (Fig. 7B and D–F)…………………… 3 3. (1) Metasoma of individuals varies from mottled yellow to brown with large irregularly shaped cream stripes toward distal edges of terga; T6 with pointed lateral flanges (Fig. 5D)…………...……………… E. eremophilaHouston 1976 - (2) Metasoma of individuals varies from predominantly dark brown to black, with thin (often faint) cream bands at distal edges of terga; T6 with blunt lateral flanges (Fig. 5E).………………………………… E. setosaHouston 1976 4 (1) T6 apex simple (Fig. 5F); basitibial plate absent.………………………………… E. lawsoniRayment, 1946 - (2) T6 apex simple (Fig. 5B); basitibial plate present.……………………………………………………..…E. micheneri sp. nov. Fig. 7. View largeDownload slide Forewings: (A) B. elongata; (B) E. micheneri sp. nov.; (C) E. tridentata; (D) E. eremophila; (E) E. setosa; and (F) E. lawsoni. Gray arrows: costal margins of the second submarginal cell; black arrows: first transcubital veins. Fig. 7. View largeDownload slide Forewings: (A) B. elongata; (B) E. micheneri sp. nov.; (C) E. tridentata; (D) E. eremophila; (E) E. setosa; and (F) E. lawsoni. Gray arrows: costal margins of the second submarginal cell; black arrows: first transcubital veins. Males 1. (1) Compound eyes greatly enlarged, bulging inward to lateral margin of antennal sockets; ocelli enlarged, greater in circumference than antennal socket; clypeal mark T-shaped, cream to pale yellow (Fig. 6B); penis valve straightened and scoop-like, with medial and lateral margins subparallel, slightly wider toward the apices (Figs. 8B and 9B). ………….. E. tridentataHouston, 1976 - (2) Compound eyes not greatly enlarged, at least an antenna width between compound eye and lateral margin of antennal socket; ocelli approximately the same circumference of the antennal socket; clypeus entirely cream to pale yellow (Fig. 6A and C–E); penis valve with lateral margin strongly convex, dorsal surface of valve appearing triangular (Fig. 8A and C–E).…...……………………………………….………...….…… 2 2. (1) Face mark extends from the paraocular area to the lower paraocular area (Fig. 6C and E).……………..………………… 3 (2) Face mark does not extend from the paraocular area to the lower paraocular area (Fig. 6A and D).………………………………… 4 3. (1) Metasoma with large cream blotches on lateral faces of terga, extending onto sides of dorsal surface; ventroapical plate laterally raised; inner flanks of penis valve membrane absent (Fig. 8C).…………………..……… E. eremophilaHouston, 1976 - (2) Metasoma without cream colouration or blotches; ventroapical plate flat; setae proceed along almost entire length of penis valve flanks; penis valve inner flanks with transparent membrane (Fig. 9E).……….……...……………… E. lawsoniRayment, 1946 4. (1) Face mark covering entire clypeus with additional dots on either side of the clypeus restricted to the paraocular area (Fig. 6D); flat ventroapical plate; setae proceed at least three-quarters down penis valve flanks (Fig. 9D).…………………………………………………….............….... E. setosaHouston, 1976 - (2) Face mark covering entire clypeus with additional dots in the lower paraocular area (Fig. 6A); ventroapical plate transparent, flap-like, curved upward to ¾ the length of the penis valve (Fig. 9A); setae proceed at most halfway down penis valve flanks (viewed ventrally, Fig. 8A).…........……..… E. micheneri sp. nov. Fig. 8. View largeDownload slide Ventral view of male genitalia: (A) E. micheneri sp. nov.; (B) E. tridentata; (C) E. eremophila; (D) E. setosa; and (E) E. lawsoni. Black arrows: ventral-apical plates; gray arrows: penis valve spines; white arrows: dorsal gonostyli. Fig. 8. View largeDownload slide Ventral view of male genitalia: (A) E. micheneri sp. nov.; (B) E. tridentata; (C) E. eremophila; (D) E. setosa; and (E) E. lawsoni. Black arrows: ventral-apical plates; gray arrows: penis valve spines; white arrows: dorsal gonostyli. Fig. 9. View largeDownload slide Dorsal view of male genitalia: (A) E. micheneri sp. nov.; (B) E. tridentata; (C) E. eremophila; (D) E. setosa; and (E) E. lawsoni. Black arrows: ventral-apical plates; white arrows: dorsal gonostyli; gray arrows: penis valve inner flank membranes. Fig. 9. View largeDownload slide Dorsal view of male genitalia: (A) E. micheneri sp. nov.; (B) E. tridentata; (C) E. eremophila; (D) E. setosa; and (E) E. lawsoni. Black arrows: ventral-apical plates; white arrows: dorsal gonostyli; gray arrows: penis valve inner flank membranes. Larvae (Fourth Instar) 1 (1) Head lateral lobe with a single thick blunt seta (Fig. 5B); dorsal surface of segments 2 and 3 raised into wave shaped structures; ventral surface of body hair absent (Fig. 10B).…………………………………… E. micheneri sp. nov. - (2) Head with multiple long setae on lateral lobes; dorsal surface of segments 2 and 3 unmodified; hair present on ventral surface of body (Fig. 10C–F).…………………………………..…… 2 2. (1). Dorsal surface of abdominal segments 5 and 6 (at bend in body) with large obtusely pointed ridges; lateral lobe of head with thick blunt seta; antennae short, less than ¼ the height of the head (from dorsal to ventral surface, Fig. 10C).………………….……….. E. tridentataHouston 1976 - (2) Dorsal surface of segment 6 unmodified; lateral lobe of head with long setae, tapering to an acute point; antennae long, ¼ head height or more (Fig. 10D–F).….…..………………….…... 3 3 (1) Dorsal surface of segment 5 bulging (Fig. 10E).……………………………… E. setosaHouston, 1976 - (2) Dorsal surface of segment 5 unmodified (Fig. 10D–F). ……………………………….…………………………………….. 4 4 (1) Antenna very long straight, more than ½ head height; lateral lobe of head with very long setae, about ½ head height (Fig. 10F).……………….…………… E. lawsoniRayment, 1946 - (2) Antennae about ¼ head height, slightly hooked at apex; lateral lobes of head with setae just longer than antennae (Fig. 10D).……..…………..…….. E. eremophilaHouston, 1976 Fig. 10. View largeDownload slide Comparison of larvae: (A) B. elongata; (B) E. micheneri sp. nov.; (C) E. tridentata; (D) E. eremophila; (E) E. setosa; and (F) E. lawsoni. Fig. 10. View largeDownload slide Comparison of larvae: (A) B. elongata; (B) E. micheneri sp. nov.; (C) E. tridentata; (D) E. eremophila; (E) E. setosa; and (F) E. lawsoni. Discussion The placement of E. micheneri sp. nov. in Exoneurella is confirmed by both molecular phylogenetic reconstruction and morphological considerations. The most informative morphological characters were E. micheneri’s larval traits, including its bent body shape, bilobed head, and lack of tubercles, all occurring in other Exoneurella species but absent in all other exoneurines. Brevineura and Exoneurella are difficult to discriminate from each other based on adult morphology alone. Both have oscillated between generic and subgeneric status in successive taxonomic treatments but their distinctive larval morphologies clearly distinguish them (Michener 1963, 1965, 2000, 2007; Reyes 1998). The description of Exoneurella micheneri sp. nov. and re-examination of the other Exoneurella species further blurs the morphological distinctiveness of adult female morphology between the two genera. Exoneurella micheneri sp. nov. females lack the lateral flanges or apical teeth on the metosomal T6 found in other Exoneurella species, though it has the same concave and upturned dorsal surface. It also has a basitibial plate, a feature of Brevineura not present in any other Exoneurella. This may be associated with the basal position of E. micheneri sp. nov. in the phylogeny of Exoneurella, such that it has retained some features present in the common ancestor to Brevineura and Exoneurella, now absent in the more distal Exoneurella lineages. One of the most striking features of Exoneurella is the lack of larval tubercles. The only other allodapine genus to lack tubercles is Macrogalea, and no tubercles occur in the Xylocopinae outgroup. It is unclear from our analysis if Exoneurella has simply retained an ancestral condition or has lost tubercles that are otherwise present in all other exoneurines (Supp Fig. 5 [online only]). An increased sampling of African taxa in the phylogeny would likely be able to resolve this. This paper presents the first comparison of male Exoneurella genitalia. All Exoneurella species show variation in genitalic structure but the morphology of the penis valve in E. tridentata is strikingly unique from not only other Exoneurella but all other exoneurines (Figs. 8B and 9B). The penis valve of E. tridentata is straightened with scoop-like sides, not forming the triangulated shape of the other exoneurines. While unique to the Australian taxa, this shape is seen in Macrogalea, Allodapula, and Eucondylops. The dramatically different genitalic morphology of E. tridentata is suggestive of strong selective pressure. E. tridentata is the only highly eusocial exoneurine, colonies consisting of only one to two reproductives (queens) per colony, the other being nonreproductive workers (Hurst 2001). One possible explanation is that the high competition among males for mating success drove rapid morphological evolution. Another possibility is that the relatively long and straight valves in E. tridentata are associated with the highly unusual metasomal morphology of queens, where the terminal external tergite is greatly extended both dorsally and laterally, and where internal musculature of the metasoma is extremely well developed. Implications for Social Evolution Research The recognition of E. micheneri sp. nov. as a member of Exoneurella has important implications for future studies of social behavior. This species is the sister species to all of the other Exoneurella. While E. tridentata is eusocial, the three previously known species of Exoneurella are facultatively social, with social groups that are casteless (Michener 1964; Hurst 2001; Dew et al. 2016, 2017a,b). Determining the social behavior of E. micheneri sp. nov. will provide critical data on the evolution of sociality across the genus. Understanding how morphological traits have moved with or facilitated changes in social behavior between species is a key part of this process. Acknowledgments We would like to thank Cyrille D’Hease for use of photographic material and assistance with field work. We are extremely grateful for logistic and/or financial support that contributed in part to this study from the BoEM laboratory, a Holsworth Wildlife Research Endowment, a Lirabenda Endowment Fund, a Nature Foundation of South Australia Scholarship Grant, the Sir Mark Mitchell Foundation and CaFoTrop. References Cited Chenoweth L. B. and M. P. Schwarz. 2011. Biogeographical origins and diversification of the exoneurine allodapine bees of Australia (Hymenoptera, Apidae). J. Biogeogr . 38: 1471– 1483. Google Scholar CrossRef Search ADS Chenoweth L. B., S. Fuller, S. M. Tierney, Y. C. Park, and M. P. Schwarz. 2008. Hasinamelissa: a new genus of allodapine bee from Madagascar revealed by larval morphology and DNA sequence data. Syst. Entomol . 33: 700– 710. Google Scholar CrossRef Search ADS Cockerell T. D. A. 1920. XXII. On South African bees, chiefly collected in natal. Ann. Durban Muse . 2: 247– 262. Darriba D., G. L. Taboada, R. Doallo, and D. 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Insect Systematics and Diversity – Oxford University Press
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