﻿A new genus of minute stingless bees from Southeast Asia (Hymenoptera, Apidae)

﻿Abstract A new genus of minute stingless bees (Meliponini: Hypotrigonina) is described from Southeast Asia. Ebaiotrigona Engel & Nguyen, gen. nov., is based on the type species Lisotrigonacarpenteri Engel, recorded from Vietnam, Thailand, Laos, Cambodia, and southern China. The species was previously considered an enigmatic member of Lisotrigona Moure, but is removed to a new genus based on its unique male terminalia that differs considerably from that of Lisotrigona and instead shares resemblances with Austroplebeia Moure, and the presence of yellow maculation (also similar to that of Austroplebeia). It is possible that Ebaiotrigona is the extant sister group of Austroplebeia, but this requires confirmation by future phylogenetic analyses. Based on available biological observations, Ebaiotrigonacarpenteri could not be confirmed as lachryphagous as is well documented from the tear-drinking species of Lisotrigona and Pariotrigona Moure.


Introduction
Among the considerable diversity of stingless bees (Meliponini), several species stand out for their Lilliputian sizes, typically with body sizes under 4.1 mm. Concomitant with these minute proportions is the further reduction of the wing venation beyond that of most meliponines, lacking closed cells in the hind wing, lacking defined submarginal cells, and the disappearance of 2Rs+M or 3M without a bend apically (Michener 2001). Such diminutive bees occur independently in various lineages of Meliponini and can be found in the genera Austroplebeia Moure, Plebeia Schwarz, Proplebeia Michener, Scaura Schwarz, and Tetragonula Moure. In addition, ten genera include exclusively tiny species: Asperplebeia Engel, Exebotrigona Engel & Michener, Friesella Moure, Hypotrigona Cockerell, Kelneriapis Sakagami, Liotrigona Moure (sensu Engel et al. 2021), Liotrigonopsis Engel, Lisotrigona Moure, Pariotrigona Moure, and Trigonisca Moure (Michener 2001). Michener (2001) discussed the various features of minute Meliponini as well as traits that indicate the relationship, or lack of a close relationship, among the various groups, and considered some genera of this group as synonyms of others (e.g., Friesella as a synonym of Plebeia, Tetragonula as a synonym of Heterotrigona Schwarz in a polyphyletic Trigona Jurine).
During a revision of Lisotrigona (Engel 2000), one species stood out as remarkably distinct from its congeners. Lisotrigona carpenteri Engel, based on a type series collected in northern Vietnam, was unique among all other members of the genus for the presence of yellow maculation on the face, pronotal lobe, and sometimes the mesoscutellar apex. At that time, males for any species of the genus were unknown and L. carpenteri was interpreted to be an autapomorphic taxon based on these morphological features of the worker caste. Subsequently, males of L. furva Engel (Michener 2007a) and L. cacciae (Nurse) (under two synonymic names: Viraktamath and Sajan Jose 2017) were discovered, revealing a unique and remarkably distinctive morphology, further emphasizing the distinctiveness of Lisotrigona from other minute genera, and particularly its relatives in Asia and Australia, Pariotrigona and Austroplebeia. Moreover, it was also discovered that Lisotrigona and Pariotrigona were lachryphagous, collecting tears from the eyes of various vertebrates (Bänziger et al. 2009(Bänziger et al. , 2011Bänziger and Bänziger 2010;Bänziger 2018). While these significant revelations were brought forward, the biology and males of L. carpenteri remained elusive.
Recently, two groups have independently discovered males of L. carpenteri and noted the considerable morphological departure in the characters of the terminalia from other Lisotrigona herein). In fact, the terminalia are generally more similar to that of Austroplebeia than to any other minute Meliponini, and certainly lack the synapomorphies that otherwise characterize Lisotrigona (Michener 2007a). The male terminalia of L. carpenteri are so remarkably different from Lisotrigona that the species is here considered to belong to a separate genus. Here we provide a description of the genus and the male, and encourage melittologists to seek additional nests, immature stages, and further biological data for this unique species among the Southeast Asian bee fauna.

Materials and methods
Material of L. carpenteri was examined from the following collections: Division of Entomology, University of Kansas Natural History Museum, Kansas (SEMC); Division of Invertebrate Zoölogy, American Museum of Natural History, New York (AMNH); and Insect Ecology Department, Institute of Ecology & Biological Resources (IEBR), Hanoi, Vietnam. Morphological terminology follows that of Engel (2001Engel ( , 2019, Michener (2007b), and Engel et al. (2021). The format for the generic description follows that of Rasmussen et al. (2017), Engel (2019), and Engel et al. (2021). Bees were sampled from sites with a series of nests and swept by net from around the nesting area. Most nests were left undisturbed, although two were exposed but contents were not sampled. Figs 1, 4 were prepared at the University of Kansas, while the remainder were prepared by the Institute of Ecology & Biological Resources.

Systematics
Ebaiotrigona Engel & Nguyen, gen. nov. http://zoobank.org/99CCDC03-E122-46B9-81F6-D8C11E432DA2 Type species. Lisotrigona carpenteri Engel, 2000. Diagnosis. The new genus resembles both Lisotrigona and the smallest individuals of Austroplebeia. The presence of yellow maculation on the face and mesosoma of the worker differentiates this caste from those of Lisotrigona (Figs 1, 5-7), while in males the clypeus can be brownish to black (refer to Description, vide). In addition, workers of Lisotrigona have distinct erect bristles on the antennal scape (lacking in Ebaiotrigona) and have minute plumose facial setae covering the entire face, including the upper frons (in Ebaiotrigona the upper frons is lacking in plumose setae and instead covered by minute, fine, simple setae). While yellow maculation is shared between workers of Ebaiotrigona and Austroplebeia, the latter has a more complete wing venation ( Fig. 1D for Ebaiotrigona venation), with 1Cu, 2Cu, and 3Cu present, at least as nebulous if not tubular veins, and thereby a more completely delimited subdiscoidal (= second cubital) cell. Additionally, Austroplebeia s.str. has 6 distal hamuli (versus 5 in Ebaiotrigona, Lisotrigona, and Anteplebeina), and Anteplebeina has 1M and 1cu-a confluent (versus 1M distad 1cu-a in Lisotrigona and Austroplebeia s.str.). Perhaps the most dramatic differences between Ebaiotrigona and other minute stingless bees are in the male terminalia. The male terminalia of Ebaiotrigona (Fig. 4) lack those distinctive features of Lisotrigona, that is, in the latter the gonocoxae are enormously expanded proximally into an incomplete ring, the slender gonostyli with apical setae are articulated at about midlength on the elongate gonocoxae, the genital capsule is schizogonal, and sterna VI and VII are entirely different (cf. Fig. 4 vs figs 4, 5 of Michener 2007a). Instead, in Ebaiotrigona the genital capsule is rectigonal, with more transverse gonocoxae in which the gonostyli articulate more distally. Furthermore, the gonostyli are uniquely modified: flattened laterally, with lamellate margins broadened proximally on both ventral and dorsal sides, and tapering apically to an acute point lacking setae. The bulb of the penis valve is enlarged and longer than broad, and abruptly tapers to a thin apical process that is shorter than the basal bulb. In Lisotrigona, the bulb is smaller and about as long as the apical process, the former gently tapering into the latter (Michener 2007a).
Description. ⚲: Minute, total length ca 3.95-4.15 mm, forewing length ca 2.96-3.10 mm; integument generally smooth and polished, some places with widely scattered minute punctures on head and mesosoma, with distinct pale yellow maculation on face, specifically undersurface of scape, supraclypeal area, and clypeus; pronotal lobe; and sometimes as small triangle on lower parocular area, as thin line on lateral margin of mesoscutum bordering tegula and as apicolateral spots on mesoscutellar apical margin (such yellow maculation absent in Lisotrigona and Pariotrigona, present but more extensive in Austroplebeia). Setae generally pale to white; those of body fine, short, and simple, face with minutely plumose setae except on upper frons with only minute simple setae (in Lisotrigona the minutely plumose setae distributed across face, including upper frons), scape with fine minute simple setae but without erect short bristles (erect short bristles present in Lisotrigona); mesoscutum with numerous erect, fine, short, simple setae; disc of mesoscutellum with similar setae to those of mesoscutum except twice as long or longer, particularly along posterior margin.
Head as broad as mesosoma, slightly broader than long, with face narrower than compound eye length; vertex gently rounded, not produced or ridged; preoccipital area rounded; scape shorter than antennal-ocellar distance, not reaching median ocellus; ocelli near top of vertex; flagellomere I trapezoidal, longer than flagellomere II; flagellomere II about as long as flagellomere III, each slightly broader than long; middle flagellomeres about as long as or slightly longer than broad; intertorular distance slightly shorter than torulocular distance; upper torular tangent below facial midlength; gena rounded, narrower than compound eye in profile; supraäntennal area with triangular medial elevation bordered laterally by converging furrows forming distinct scapal basins; frontal carina absent, indicated on supraäntennal triangle by weakly demarcated ridge, otherwise a narrowly polished line to median ocellus; malar area nearly linear, subequal to or shorter than 0.5× flagellar diameter (similar to Lisotrigona and Austroplebeia s.str.; Pariotrigona with malar space slightly longer than flagellar diameter; Anteplebeina with malar space as long as flagellar diameter); labrum transverse, simple, apical margin rounded; mandible with apical margin slightly oblique, largely edentate except for two small teeth on upper margin of margin (thus, bidentate), teeth well defined, acute, and incised, interdental spaces distinct but not broadly incised; labial palpomeres I and II with a few, scattered, elongate, apically arched to slightly wavy setae, such setae more clustered apically and sparse elsewhere (similar to that of Lisotrigona).
Metatibia approximately triangular, approximately 2.8-3.0× as long as greatest width; retromargin gently curved with subangulate distal superior angle, retromarginal setae and superior prolateral surface setae simple; prolateral surface shallowly concave apically, with corbicula occupying slightly less than apical half; retrolateral surface with broad keirotrichiate zone and narrow superior subglabrous zone, without defined clivulus except proximally; keirotrichiate zone approximately 4× as broad as superior glabrate zone, keirotrichiate zone extending to apical margin, with keirotrichiate zone narrowing and superior glabrate zone broadening in apical-most portion of metatibia; inferior penicillum and rastellar comb present, each composed of fine soft setae. Metabasitarsus with proventral margin straight, retrodorsal margin generally paralleling proventral margin, apical margin weakly convex to comparatively straight, distal superior angle not projecting; retrolateral surface without basal sericeous area.
Metasoma subtriangular, about as wide as mesosoma, with metasomal terga smooth and shining except apical margins more matte and faintly and minutely imbricate, all terga almost glabrous, except by minute, erect, simple setae apically forming a narrow band on apical margin, such setae progressively longer, more abundant, and more spread on apical-most terga; sterna largely smooth and glabrous, with long to elongate, fine, erect, simple setae apically.
♂: Darker than worker, apparently without facial maculation of worker (Figs 2, 3), instead yellow areas of worker dark brown to black in drone [brown in Vietnamese male, wholly black in Chinese male (Li Yuran, pers. comm. and unpublished images to MSE)] [note that sometimes areas of yellow maculation start off brownish in individuals who are not fully pigmented and so brown areas of the male could eventually become fully yellow as in the worker; however, the fact that the Vietnamese male appears to be fully pigmented elsewhere on the body (Figs 2, 3) and that the Chinese male is wholly black tends to suggest that drones of Ebaiotrigona truly lack facial maculation, but this will require confirmation through extensive future sampling of males throughout the range of the species], except pronotal lobe consistently yellow. Scape shorter and broader than that of worker (Fig. 3); flagellomere I trapezoidal, shorter than pedicel or flagellomere II; flagellomere II about as long as flagellomere III, each slightly longer than broad. Metasomal sternum VI medioapically chamfered, bilobed; sternum VII medially broadly convex and slightly depressed, with shallow apicolateral concavities; genital capsule rectigonal (Fig. 4); gonobase somewhat transverse; gonocoxae broader than long, with gonostylus articulating somewhat proximally; gonostylus elongate and bladelike, flattened laterally, and broadened proximally and lamelliform mesally on dorsal and ventral sides, tapering apically to acute point with a single fine apical seta, seta simple (Fig. 4); penis valves elongate, bulb enlarged, longer than broad, well sclerotized, abruptly tapering to thin elongate apical process, process gently arched and acutely rounded apically, process slightly shorter than bulb, blub without elongate proximal apodeme, instead with short, twisted apodeme (Fig. 4).
Etymology. The new generic name is a combination of the Ancient Greek adjective ēbaiós (ἠβαιός, meaning "small") and the generic name Trigona Jurine. The gender of the name is feminine.
Included species. Currently, the genus includes only the type species, Ebaiotrigona carpenteri (Engel), new combination.
The following modifications to the key of Rasmussen et al. (2017) and Engel et al. (2018) allow for the incorporation of Ebaiotrigona. Remark. The worker of this species has been described by Engel (2000). We do not repeat that material here but instead provide descriptive details for the hitherto undocumented male. Descriptive notes. ♂: Body length 3.5 mm; forewing length 2.9 mm; head width 1.29 mm; head length (lower margin of clypeus to anterior margin of median ocellus and to summit of head) 1.04 mm, 1.19 mm, respectively; compound eye length, upper and lower interorbital distances 0.92 mm, 0.53 mm, 0.45 mm, respectively.
Compound strongly converging below, inner orbits only feebly concave above; compound eye length greater than upper interocular distance; antennae arising below lower third of face so that distance from lower clypeal margin to lower margin of antennal torulus is about one-half distance from upper margin of torulus to lower margin of median ocellus; clypeus about twice as broad as long, lower margin scarcely below lower ocular tangent, upper margin separated from antennal torulus by about one-half torular diameter; head surface in vicinity of antennal toruli depressed, not visible in profile; interocellar distance over twice ocellocular distance, almost 3× ocellar diameter; ocelli on submit of vertex, head surface declivitous immediately behind lateral ocelli with no ridge or carina; genal area much narrower than compound eye in profile; malar area linear (mandibular base almost in contact with lower compound eye margin); mandibles comparatively long, conspicuously crossing one another, mandibular apex slender, apical margin oblique with lower apical angle pointed, upper portion of apical margin faintly sinuous and simulating a faint preapical bump (scarcely large enough to denote as a "tooth") (Fig. 3B); antennal scape short, its length less than half distance from antennal torulus to median ocellus; flagellum slightly tapering, covered with short, dense setae; flagellomere I shorter than pedicel, nearly 3× as broad as long; flagellomere II about 1.25× as broad as long, flagellomere III about as broad as long, subsequent flagellomeres progressively shorter so that flagellomeres IV-VI are shorter than broad, flagellomeres VII-X progressively longer, longer than broad, flagellomere XI with broadly rounded apex. Male terminalia as in Fig. 4.
Integument of head and mesosoma black except basal one-third of scape (grading into black distal half ), mandible, labrum, clypeus, spot between antennae, mesoscutellum, metanotum, propodeum, legs except coxae and femora, and all metasomal segments, brown or suffused with brown ranging to black (darkest in Chinese male: Li Yuran, pers. comm. and image to MSE); pronotal lobe markedly yellow; protibia, meso-and metatibial apices, and tarsi light yellowish brown; tegulae yellowish brown; wings clear, veins light brown; stigma translucent, light brownish. Integument shining, smooth, head and mesosoma with well separated, fine, small punctures, punctures on lateral part of mesoscutum denser; punctures on mesoscutellum much stronger and denser than those on mesoscutum, separated by 2-3× a puncture width; mesepisternum centrally with coarse punctures separated by 2-3× a puncture width, otherwise integument between punctures smooth; basal area of propodeum shining, minutely tessellate, glabrous; posterior surface smooth, shining. Metasomal terga smooth except minute punctures on narrow posterior margins.  Pubescence mostly exceptionally short, mostly yellowish white, setae of lower half of face white and conspicuously plumose, other setae simple or nearly so. Posterior part of mesoscutellum with zone of upcurved setae, longest on body except for some on apical margin of clypeus and mandible, about 2.5× as long as median ocellar diameter, otherwise long setae (nearly 2× ocellar diameter) sparse on apical margin of clypeus, vertex, coxae, and trochanters, rather numerous on mandibular lower margin. Setae of mesoscutum short, some setae at apicolateral corner longer, about as long as ocellar diameter. Metasomal terga I-IV glabrous except for minute erect setae on posterior margins. Variation. Workers exhibit noticeable variation in overall coloration, and can loosely fall into a lighter and dark morph, although there are individuals who seemingly intergrade and so these morphs are not discrete (Figs 1, 5-7). Generally, areas of maculation on the clypeus, ventral surface of scape, mesoscutal lateral borders, axilla, apicolaterally  on mesoscutellum, foreleg, and in isolated areas on the mid-and hind legs can vary from pale to vivid yellow, while the males the tarsi are more consistently yellowish brown to yellow and the podites basal to the tarsi are brown to black (darkest in the Chinese male: Li Yuran, pers. comm. and image to MSE). In addition, the width of the marks on the mes-oscutum and mesoscutellum can be exceptionally narrow (mesoscutum) or faint (mesoscutellum), such that they can appear superficially absent. Most noticeably, areas of black on the mesosoma and legs can vary to dark brown (e.g., cf. Figs 1, 5, 6). The metasoma can range from being largely black to dark reddish brown, with lighter brown on the first tergum and basal sterna, to the same pattern but from pallid yellow on the basal sterna and first tergum, with ferruginous on the remaining terga, but blending from light proximally to dark apically, and within a given tergum darkening slightly toward each marginal zone. It should be noted that the holotype worker (AMNH) is of the darker morph.
New material examined. Comments. More than 10 nests were observed in a rocky wall in Yên Thủy, Hòa Bình Province, while six or seven were seen in one locality in limestone cliffs in Bắc Kạn Province (Figs 8,9). The bees appear to prefer nesting in cavities between stones, either natural limestone cliffs or even amid the rocks of human-built walls, much like that of Pariotrigona (Bänziger et al. 2011). The bees were quite a nuisance and frequented human skin where they were lapping sweat, and attempted to approach the eyes of the collectors (Truong pers. obs.), although it is unclear if this was to collect tears or merely as a timid form of defense from a perceived threat near the nests. Similar observations on the behavior of the bees were made in southern China (Li et al. in press). It is possible that the bees rely on tears (lachryphagous) in the same manner as Pariotrigona and Lisotrigona, although no tear collection could be confirmed nor were bees observed visiting the eyes of cattle or other vertebrates. The bees and nests have a strong foul odor, and future work on the chemistry of their nests and stores is needed to determine their composition and the potential derivation of these smells. Future work will more fully explore the nesting biology, nest architecture, and immature stages of E. carpenteri.

Discussion
While E. carpenteri was always a unique species as the only minute stingless bee in Southeast Asia with yellow maculation, it has not been until the discovery of the male that its real distinction was revealed. The male terminalia are clearly not of the structure so typical for Lisotrigona. Specifically, E. carpenteri lacks the enormous proximal extensions of the gonocoxae unique to species of Lisotrigona (Michener 2007a). While this form is wholly unique among Meliponini, the result of the almost proximally ringlike gonocoxae approximates the schizogonal condition as the proximal fossae of the gono- Figure 8. Nests and habitat of Ebaiotrigona carpenteri (Engel), comb. nov., at Bắc Kạn Province, Vietnam A general habitat B nest entrance in limestone cliff C exposed nest from between limestone slabs. coxae open mesally (Michener 1990). Conversely, in Ebaiotrigona, Pariotrigona, and Austroplebeia, the gonocoxae are not so modified, and the opening of the gonocoxae are directed proximally (Michener 2001;Dollin et al. 2010), and thus of the rectigonal configuration with the sole exception of Pariotrigona, which is more amphigonal. In addition, in Lisotrigona the gonostyli articulate with the gonocoxae about midlength, whereas in the others, including Ebaiotrigona, the gonostyli articulate more distally. Unlike all of the other genera, the gonostyli of Ebaiotrigona are broadened proximally as lamellae, elongate, and almost bladelike. This form is unique to the genus.
In several respects Ebaiotrigona appears more similar to Austroplebeia, and this is borne out by the form of the male genitalia. In both genera the basal bulb of the penis valve is quite enlarged and sclerotized, and tapers apically to an elongate, thin, clawlike process. Note that these extensions in Austroplebeia and Ebaiotrigona, although well sclerotized and often melanized, are easily broken near their base (e.g., from the photograph of   fig. 2d), and in additional images of this male sent to MSE these are broken off in the Chinese male reported therein; nonetheless, the Chinese populations should be further explored in the future as to whether or not they represent a species distinct from E. carpenteri). In Pariotrigona the basal bulb is smaller and somewhat transverse, and is abruptly narrowed to the thin elongate process. In Lisotrigona, the penis valves are comparatively short relative to the overall size of the genitalia, with a broad basal bulb and tapering to a comparatively short process that is scarcely longer than the bulb. While the Asian and Australian minute species lack a Figure 9. Nests of Ebaiotrigona carpenteri (Engel), comb. nov., at Bắc Kạn Province, Vietnam A nest entrance B exposed nest from between limestone slabs. spatha, Pariotrigona is an outlier in that the spatha is present as a large membranous structure. The form of each of these genera is seemingly apomorphic when compared to putative outgroups among related members of the Hypotrigona group of genera (Table 1: sensu Engel et al. 2021). Liotrigona has another wholly unique form of genitalia. The capsule is elongate and permanently schizogonal, with the gonocoxae much longer than broad and fused ventrally, while the gonostyli articulate apically (Michener 1990). By comparison, the genitalia of Hypotrigona are comparatively drab and more generalized with conditions observed broadly across Meliponini. In Hypotrigona the capsule is rectigonal, but apomorphically the opening opens dorsally, while the gonocoxae are short and transverse (Michener 1990). The basal bulb of the penis valve is membranous.
Another unique feature of Ebaiotrigona is the shape of the sixth metasomal sternum, which is apically bilobed, versus the single medial process of Lisotrigona, Pariotrigona, and Austroplebeia. The seventh sternum also differs from these genera and therefore highlights the distinctiveness of the new genus among Old World Meliponini.
Given the similar male terminalia and the presence of yellow facial maculation, it leads one to wonder if Ebaiotrigona could be the extant sister group of Austroplebeia, diverging from this genus prior to the Miocene and when the latter still had a presence in mainland Asia (Engel et al. 2021). Naturally, future phylogenetic analyses of morphological and molecular data need to include a broader sampling of Asian species, and certainly including samples of E. carpenteri.  The nests and the biology of E. carpenteri remain to be explored in greater detail, work that the authors hope to undertake in the forthcoming years. Limited observations on nests were presented by Chinh et al. (2005), who reported nests in tree trunks, rock crevices, and human-made structures. Chinh et al. (2005) reported amorphous brood clusters, like those observed in here (Figs 8,9). The contents of the honey pots were not explored. Therefore, for the moment one must wonder whether E. carpenteri is actually lachryphagous like Lisotrigona and Pariotrigona, or a typical floral visitor as in Austroplebeia. Certainly current observations suggest that while the species laps sweat, like many bees, E. carpenteri may not actually imbibe tears (Li et al. in press; herein). A further elaboration of the nests and biology of E. carpenteri will be of considerable significance for understanding the distribution and occurrence of lachyrphagy in Asiatic Meliponini. If true, lachryphagy is restricted to Lisotrigona and Pariotrigona, then it may be that this is a biological synapomorphy for these two genera, with Ebaiotrigona more closely allied to Austroplebeia. Ideally, once the biology has been more fully elucidated for E. carpenteri, an analysis of morphological and molecular data can be undertaken for the Hypotrigona group (Table 1) to explore not only phylogenetic relationships but also patterns of biogeographic and biological significance. Fortunately, a large number of nests are available at localities in northern Vietnam for future investigation.