A new genus and tribe of freshwater mussel (Unionidae) from Southeast Asia

The freshwater mussel genus Oxynaia Haas, 1911 is thought to be comprised of two geographically disjunct and morphologically variable species groups but the monophyly of this taxon has yet to be tested in any modern cladistic sense. This generic hypothesis has important systematic and biogeographic implications as Oxynaia is the type genus of the currently recognized tribe Oxynaiini (Parreysiinae) and is one of the few genera thought to cross several biogeographically important barriers in Southeast Asia. Morphological and molecular data clearly demonstrate that Oxynaia is not monophyletic, and the type species and its allies (O. jourdyi group) belong to the Unioninae, and more specifically as members of the genus Nodularia Conrad, 1853. Therefore, neither Oxynaia syn. nov. nor Oxynaiini Starobogatov, 1970 are applicable to the Parreysiinae and in the absence of an available name, Indochinella gen. nov. and Indochinellini trib. nov. are described. Several combinations are proposed as follows: Indochinella pugio (Benson, 1862) gen. et comb. nov., Nodularia jourdyi (Morlet, 1886) comb. res., N. gladiator (Ancey, 1881) comb. res., N. diespiter (Mabille, 1887) comb. res. and N. micheloti (Morlet, 1886) comb. res. Finally, we provide an updated freshwater biogeographic division of Southeast Asia.

Integrative taxonomic studies are of substantial practical importance to conservation stakeholders as accurate information on the systematics and distributions of biodiversity forms the foundation of taxon-and habitat-based conservation efforts. The application of basic systematic and phylogenetic research has played a critical role in the conservation of freshwater mussels (Bivalvia: Unionidae), which are among the most threatened groups of animals worldwide 1 . However, the vast majority of recent systematic research has focused on the North American and European fauna, while the comparatively diverse tropical lineages have received disproportionately less attention 2 . Although, several recent systematic efforts focused on Asian lineages have dramatically improved our understanding of the classification, morphological evolution, and biogeography of many tropical freshwater mussel clades [3][4][5][6][7][8][9] , many biographically interesting and systematically important taxa remain poorly understood from a phylogenetic perspective. This is particularly true of the genus Oxynaia, which has an unusual disjunct geographic distribution in Myanmar and northern Vietnam and is the type genus of the tribe Oxynaiini Starobogatov, 1970.
Whelan et al. 10 recently transferred the tribe Oxynaiini from the subfamily Ambleminae to the Parreysiinae on the basis of recovering Oxynaia pugio (Benson, 1862) among the latter subfamily in a molecular phylogeny. Whereas, traditional morphological classifications consistently consider Oxynaia a member of the subfamily Unioninae (in its various usages [11][12][13][14]. However, these seemingly irreconcilable subfamily-level classifications of Oxynaia (Unioninae vs Parreysiinae) have relied primarily on only one of the two geographically disjunct species groups, suggesting that Oxynaia may not be monophyletic.
Our objective herein is to test the monophyly of the genus Oxynaia, evaluate the morphological traits of the resultant suprageneric clades containing Oxynaia species, and to make the appropriate taxonomic changes to more accurately reflect our hypotheses of evolutionary history.

Results
Phylogenetic analyses. Our family-level phylogenetic analyses based on mitochondrial and nuclear markers (five partitions: three codons of COI + 16S rRNA + 28S rRNA) reveals that Oxynaia is not monophyletic (Fig. 2). Oxynaia jourdyi is well supported as a member of the subfamily Unioninae, whereas O. pugio is recovered in the distantly related subfamily Parreysiinae. Oxynaia jourdyi is resolved in a shallow and strongly supported clade comprised of representatives of the genus Nodularia Conrad, 1853 (BS/BPP = 100). The Oxynaia pugio is recovered in a well-supported clade with the genus Radiatula Simpson, 1900. The genus Indonaia Prashad, 1918

Morphological analyses.
Comparisons of the soft anatomy and larval morphology of representatives of the major freshwater mussel clades in the Oriental Region clearly demonstrate the polyphyly of Oxynaia s. lato ( Table 1). The Oxynaia jourdyi group is unambiguously placed in the subfamily Unioninae by the combination of ectobranchous brooding condition and hooked larvae with basal spines, known synapomorphies of the Unioninae 3,5,16 . The Oxynaia pugio group (=Indochinella gen. nov.) is united with all other members of the subfamily Parreysiinae on the basis of possessing a tetragenous brooding condition (sans Lamellidentini Modell, 1942 = ectobranchous), unhooked glochidia, as well as having the ascending lamella of the inner demibranchs attached to the visceral mass across their entire length (vs. attached only anteriorly in the Unioninae). This combination of traits is a novel method of recognizing the Parreysiinae (sans Lamellidentini). Several shell characters also unite the Oxynaia jourdyi group with representatives of the Unioninae, especially Nodularia, including the position, elevation, and sculpturing of the umbo, as well as several features of the dentition (Table 2 and Fig. 3). Nodularia differs from Indochinella gen. nov. by having a very pronounced and elevated umbo (vs. not pronounced and not elevated), umbo location in the first half of the shell (vs. in the first third), nodulose wrinkles umbo sculpture (vs. v-shaped), and a rectangular and sharp anterior pseudocardinal tooth and a thick and pyramidal posterior pseudocardinal tooth in the left valve (vs. two separated ribbed teeth placed in parallel line with one another).
The combination of the molecular phylogeny, soft anatomy, larval characters, and shell morphology clearly demonstrate the polyphyly of Oxynaia s. lato. The type species and its allies (i.e. the Oxynaia jourdyi group) are unambiguously placed in the subfamily Unioninae, rendering both Oxynaia and Oxynaiini inapplicable to the Parreysiinae. The tribe Oxynaiini Starobogatov, 1970 is herein recognized as an available family-group level name of the Unioninae Rafinesque, 1820. In the absence of an available name for the Oxynaia pugio species group and the larger Parreysiinae clade including the O. pugio group, Radiatula, and Indonaia (i.e. the former Oxynaiini), the genus Indochinella gen. nov and tribe Indochinellini trib. nov. are described here.
Range disjunction. The two species groups of Oxynaia have clearly distinct ranges (Fig. 1). All the reliable records (mostly type localities, see Taxonomic Account) of the Oxynaia jourdyi species group are concentrated within the Red, Cả and Cầu River drainage basins of northern Vietnam. The Oxynaia pugio species group is known from the Irrawaddy, Sittaung and Tavoy River drainages. Neither species group occurs in the drainages situated between eastern Myanmar and northern Vietnam (i.e., Salween, Mae Klong, Chao Phraya, and Mekong). Zieritz et al. 17    Description: Adults small (22 mm) to medium (54 mm) sized for family. Shell outline narrow, elongate, strongly inequilateral, always with a straight or convex ventral margin. Moderately inflated, posterior ridge rounded with moderately to very steep posterior slope. Umbo only slightly elevated above hinge line usually with v-shaped umbo sculpture. Green zigzag sculpturing on shell disc common, but absent in some individuals and taxa, e.g., Indonaia caerulea (Lea, 1831), Radiatula humilis (Lea, 1856), R. pilata (Lea, 1866), and Indochinella pugio gen. et comb. nov. Shells moderately thick. Pseudocardinals erect and stumpy to long and bladelike; two in left valve (may become one in blade-like teeth) and one in right valve occasionally with a second rudimentary anterior tooth. Laterals are moderately short and diverging; two in left, one in right. Unhooked glochidia brooded in all four gills. Mantle margin ventral to incurrent aperture with many prominent simple papillae. Incurrent aperture papilose, excurrent and supra-anal apertures smooth. Ascending lamella of inner demibranch attached to visceral mass for its entire length.   25 . Figure 3a, Tables 1 and 2 Etymology: The name of this genus is derived from the greater Indochinese Peninsula. Diagnosis: The genus is distinguished from Nodularia by the presence of tetragenous brooding of unhooked glochidia (vs. ectobranchous brooding of hooked glochidia), as well as complete fusion of the ascending lamella of the inner demibranchs to the visceral mass (vs. only anterior fusion) ( Table 1). Indochinella gen. nov. can also be distinguished from Nodularia by the umbo being only slightly elevated above the hinge line (vs. strongly elevated), fine v-shaped beak sculpture (vs. wrinkled and nodular), triangular posterior pseudocardinal (vs. trapezoidal or rectangular), and moderately short lateral teeth (vs. elongate) ( Table 2 and Fig. 3). Adult Indochinella (with exception of a lineage from the Tavoy River) can be distinguished from all other representatives of the tribe by the presence of a sharp posterior ridge (vs. rounded) and very steep posterior slope (vs. gradual). Pseudocardinal teeth in Indochinella also tend to be more strongly developed than in other Indochinellini.
Description: Shell moderately thick; elongate with rounded anterior end and strongly pointed posterior end. Posterior ridge sharp. Posterior slope steep. Umbo hardly elevated above hinge line, fine v-shaped umbo sculpture, shell disc smooth to strongly sculptured. Moderately inflated. Pseudocardinal teeth strong, two in the left Distribution: The genus is primarily known from Myanmar in the Irrawaddy, Sittaung, and Tavoy River drainages. The genus may also inhabit several other river basins in Myanmar, e.g., the Great Tenasserim, Salween, and some coastal rivers of the Bay of Bengal 14,26-28 . A few records from India (e.g., Assam) 14 are in need of future studies because these specimens could have been collected within the Irrawaddy Basin.
Comments: We assigned a single described species to the genus, although the divergent molecular lineages from the Sittaung and Tavoy River drainage basins may be worthy of formal taxonomic recognition. (Benson, 1862)    Fine v-shaped sculpture on umbo, umbo only slightly elevated above hinge line. Periostracum smooth, grey-brown to yellow-green, with dark parts along the radial lines; nacre whitish. Left valve with two parallel rather short lateral teeth and two ribbed parallel pseudocardinal teeth. Right valve with a single slightly curved lateral tooth and two pseudocardinal teeth, anterior tooth reduced, posterior tooth high, ribbed and strong. Umbo cavity not very deep, nacre in umbo cavity commonly tinted peach to golden-brown. Anterior adductor scar well pronounced, funneled; posterior adductor scar rounded. The Sittaung lineage differs from the Irrawaddy lineage in having a shorter and higher shell, more pronounced and curved lateral teeth, and a moderately strong sculpture on shell disc. The Tavoy lineage differs from the two other lineages in having an oval-shaped shell, more rounded posterior ridge, more gradual posterior slope, and distinct zigzag ridges across shell disc.

Indochinella pugio
Distribution: Irrawaddy, Sittaung and Tavoy River basins. In the Irrawaddy River, it is known as far north as Mya Taung and as far south as Hinthada. A few records from India (e.g., Assam) 14 are in need of future studies because these specimens could have been collected within the Irrawaddy Basin. The lineages from the Sittaung and Tavoy River catchment areas may represent separate species-or subspecies-level taxa but requires further systematic research.
Habitat and ecology: The species seems to be rather a habitat generalist, and it is known from the mainstream of large, medium-sized and small rivers, as well as from their floodplain lakes.
Comments: Unio digitiformis Sowerby, 1868 from India is not a synonym of Indochinella pugio gen. et comb. nov 29 . but a separate species, the generic placement of which is unclear. Haas 13 noted that the location of this form is certainly not India and that it most likely belong to the Lanceolaria Conrad, 1853.

Discussion
Taxonomic implications. Our integrative molecular and morphological approach has determined that the genus Oxynaia is polyphyletic. This result has clear implications regarding the higher-level classification of the Unionidae, the morphological characteristics of the Parreysiinae, and corroborates broader biogeographic patterns in Southeast Asia. Molecular and morphological data reject the monophyly of Oxynaia with its former constituents being recovered in phylogenetically divergent and morphologically diagnosable clades. The Oxynaia jourdyi group is unambiguously placed in the Unioninae on the basis of our molecular phylogeny and several morphological synapomorphies and is herein considered a junior synonym of Nodularia, rendering the tribe name Oxynaiini inapplicable to the Parreysiinae. This taxonomic rearrangement required the description of a new genus for the "Oxynaia" pugio group (=Indochinella gen. nov.) and a new tribe (=Indochinellini trib. nov.) to recognize these morphologically cohesive clades of Parreysiinae.
Freshwater biogeography of Southeast Asia. Previous to this study, Oxynaia was thought to be one of the few genera distributed across much of Southeast Asia from central Myanmar to northeastern Vietnam 17 . This distribution was thought to be unusual in that it crossed two important biogeographic barriers in Southeast Asia: (1) the Salween/Mekong river drainage divide separating the Western Indochinese freshwater mussel assemblage from the Sundaland assemblage, and (2) the Mekong/northern Vietnamese drainage divides separating the Sundaland assemblage from the East Asian fauna. However, the seemingly large distribution of Oxynaia s. lato is discovered here to be spurious and based on incorrect interpretations of common ancestry. The geographic distributions of Nodularia and Indochinella closely follow these two influential biogeographic barriers. These two biogeographic barriers divide eastern Asia into three faunistically distinct subregions (Fig. 5), each of which is briefly discussed below. probably corresponding to the gigantic paleo-Mekong River basin [6][7][8] . The fauna of the Greater Sunda Islands (Sumatra, West Java, northern and western Borneo) appears similar to that of mainland Southeast Asia and may also belong to this subregion, as suggested by the molecular data for northern Borneo 9 and by the putative connections of paleo-drainages during the Pleistocene 30 31,32 occurs in the region, although, it has likely been introduced. Two large endemic monophyletic radiations of freshwater mussels, i.e., the tribes Pilsbryoconchini and Rectidentini, were recorded in this region 6,7 . The highest levels of diversity of these clades occur within the Mekong River basin, with a few representatives inhabiting the Chao Phraya River, the Malay Peninsula, and the Greater Sunda Islands [6][7][8][9] . The Indochinellini is the only tribe of the Parreysiinae distributed in the Sundaland Subregion, whose constituents belong to a subclade Radiatula that appears to have significant levels of cryptic diversity (Fig. 2). Several characteristic elements of the fauna of western Indochina are lacking in Sundanese assemblage, such as the members of Lamellidentini, Leoparreysiini, and Pseudodontini. (3) East Asian Subregion. The Red and Ca River drainage basins, and numerous coastal rivers of Vietnam comprise the East Asian Subregion, whose fauna appears to be more closely allied to the Palearctic Region than to the Oriental Region (Fig. 5). This large biogeographic subregion extends north to Japan and the Far East of Russia. This freshwater mussel fauna is entirely different from those of Western Indochina and Sundaland, and it has strong biogeographic affinities to the Pearl, Yangtze and Huang He river drainage basins 33 . With respect to available paleontological data 34  This biogeographic division of Southeast Asia largely corresponds with that of Graf and Cummings 33 suggesting four freshwater biogeographic subregions, i.e. (1) Yangtze-Huang, from the Pei south to the Qiantang and Taiwan; (2) Indochina, including southern China and the Mekong west to the Salween; (3) India-Burma, from the Indus to the Irrawaddy; and (4) Sunda Islands-Philippines. However, our new scheme (Fig. 5) reveals that the Salween, Irrawaddy and Sittaung unionid faunas are close to each other and should belong to the separate Western Indochina Subregion and that the Yangtze-Huang (=East Asian) Subregion comprises the drainage basins in northern Vietnam and appears to be a part of the Palearctic Region. Additionally, we suggest that Sumatra, West Java, northern and western Borneo may belong to the Sundaland Subregion, but this preliminary hypothesis is in need of future confirmation based on an expanded molecular dataset.
Zieritz et al. 17 distinguished two major hotspots ("epicentres") of the subfamily-level diversity and endemism of the Unionidae in Asia, i.e. (1) Southeast Asian Hotspot harboring the highest diversity of the Rectidentinae, Gonideinae (+Pseudodontinae), Parreysiinae, and Modellnaiinae, and (2) Chinese Hotspot dominated by the Unioninae (+Anodontini) lineages. Our biogeographic division is largely congruent with this diversity-based model, i.e., the Western Indochinese and Sundaland subregions correspond to the Southeast Asian diversity hotspot and the East Asian Subregion correlates with the Chinese diversity hotspot.

Methods
Nomenclatural acts. The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank (http://zoobank.org), the online registration system for the ICZN. The LSID for this publication is: urn:lsid:zoobank.org:pub:C585DACD-6AB1-4692-B68A-50ABA47940A4. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.

Morphological methods. Soft anatomy and larval characters were scored for representatives of both
Oxynaia species groups (Table 1). These three characters were chosen as they have been previously demonstrated to be useful in diagnosing suprageneric clades of freshwater mussels 16 . Character states were observed using a Leica M27s dissecting scope and a Leica DM LB2 compound microscope. Representative taxa relevant to previous classifications of Oxynaia were also included, as were other major lineages present in the region (Table 1). Parreysia corrugata was scored using literature sources 35,36 , as no soft anatomy for this species was available. A more detailed comparison of the shell characters that distinguish the three most commonly confused genera are provided in Table 2. Umbo character states follow Zieritz et al. 37 .
Phylogenetic analyses. We included novel Oxynaia jourdyi sequences in the recent phylogenetic data set of Bolotov et al. 7 to test the monophyly of Oxynaia. This data set was simplified to include only one haplotype of each species, with exception of the Oxynaia pugio sequences (Supplementary Table 1). Additionally, we excluded several taxa that were represented only by sequences of the COI gene, but Parreysia spp. and Indonaia spp. from India were left as the members of the Parreysiinae. The sequence alignment of COI, 16S rRNA and 28S rRNA gene fragments was performed separately using the Muscle algorithm implemented in MEGA6 38 . The alignment data sets were joined in a multi-locus alignment. Lacking sites were treated as missing data. We performed maximum likelihood and Bayesian inference phylogenetic analyses using RAxML v. 8.2.6 HPC Black Box 39 and MrBayes v. 3.2.6 40 , respectively. The settings of the analyses were as described in Bolotov et al. 7 . The phylogenetic models were calculated at the San Diego Supercomputer Center through the CIPRES Science Gateway 41 .
Data availability. The sequences used in this study are available from GenBank. Accession numbers for each specimen are presented in Supplementary Table 1.