Eight new freshwater mussels (Unionidae) from tropical Asia

Freshwater mussels are sensitive to habitat and water quality, revealing the fastest rates of human-mediated global extinction among aquatic animals. These animals are especially diverse in tropical Asia, the faunas of which are characterized by high levels of endemism. Here we describe four new species and four new subspecies of freshwater mussels from Myanmar. Leoparreysia whitteni sp. nov., the smallest representative of this genus, was discovered from the Ayeyarwady and Chindwin rivers. Radiatula myitthanensis sp. nov. and R. chindwinensis sp. nov. were recorded from the Chindwin Basin, and R. mouhoti haungthayawensis ssp. nov. has been discovered from the Haungthayaw River. Indochinella pugio has been revised with a description of three subspecies: I. pugio viridissima ssp. nov. from the Sittaung, Bilin and Bago rivers, I. pugio daweiensis ssp. nov. from the Dawei River, and I. pugio paradoxa ssp. nov. from the Haungthayaw River. Yaukthwa elongatula sp. nov., a peculiar species, conchologically resembling representatives of the genus Solenaia (Gonideinae) with ultra-elongated shell was found in the Chindwin Basin. Our records highlight that tropical Asia harbors numerous, but still overlooked local endemic lineages of freshwater bivalves, which may be on the brink of extinction due to the high anthropogenic and climate change impacts.

Freshwater bivalves contribute to or provide a plethora of ecosystem functions and services 1 . Currently, freshwater bivalves are among the most threatened groups in the world with 40% of the species being near threatened, threatened or extinct 2 . The interior basin of the USA, Central America, Yangtze Basin, India and Southeast Asia are the most species-rich hotspots of freshwater bivalves at the global scale [2][3][4] .
Myanmar differs from other tropical Asian countries by a spectacular freshwater fauna with numerous local endemic taxa at the genus and species levels [5][6][7][8][9][10] . Biogeographically, most of country's river basins, e.g. the Ayeyarwady, Bago, Sittaung, and Salween, belongs to the Western Indochina Subregion 7 . However, northwestern drainages of the Rakhine Coast, e.g. the Kaladan and Lemro rivers, seem to belong to the Indian Subregion, although the mussel fauna of those basins is still to be explored in detail 11 . Finally, the most eastern edge of the Shan State belongs to the Mekong River basin, and, hence, to the Sundaland Subregion 7 .
Bolotov et al. 6 published the first integrative revision of the freshwater mussel fauna of Myanmar, with a description of two new genera, seven new species and one new subspecies. Two additional new endemic genera, Indochinella and Yaukthwa, have recently been introduced 7,9 . However, our current knowledge of freshwater mussels of Myanmar is far from being complete, with many lineages still waiting to be described. Further taxonomic research is necessary to develop a national conservation action plan for freshwater bivalves for Myanmar. This plan is urgently needed due to the current high rates of economic development and cropland expansion, leading to the rapid degradation of freshwater habitats 12 . Species delimitation. The species delimitation analysis through the BEAST2 package STACEY with an initial grouping of taxa supported our hypothesis on 20 putative species-level lineages (probability = 0.80). Eight of these taxa are new to science and are described here (Tables 1-2; Figs 1-6). Each new taxon can be clearly distinguished from sister species or subspecies by molecular diagnosis (Table 2). Four lineages are assigned to valid species, i.e. Leoparreysia whitteni sp. nov., Radiatula myitthanensis sp. nov., R. chindwinensis sp. nov., and Yaukthwa elongatula sp. nov., because they share high levels of molecular divergence from sister clades (mean uncorrected COI p-distance = 3.2-9.3%). In contrast, four novel lineages sharing rather low molecular divergence from nearest neighbors (mean uncorrected COI p-distance = 1.4-1.6%) are introduced as the following subspecies: Indochinella pugio viridissima ssp. nov., I. pugio daweiensis ssp. nov., I. pugio paradoxa ssp. nov., and Radiatula mouhoti haungthayawensis ssp. nov. However, Indochinella pugio daweiensis ssp. nov. and I. pugio paradoxa ssp. nov. have one or two diagnostic nucleotide substitutions in the nuclear 28S rRNA gene fragment ( Table 2) that usually indicates species-level differences between the Unionidae taxa.  Diagnosis: The new species is morphologically most similar to Leoparreysia burmana (Blanford, 1869) but can be distinguished from it by a more elongated shell, and different location and size of umbo (small subinequilateral umbo in the new species vs. large strongly inequilateral umbo in L. burmana). Leoparreysia whitteni sp. nov. has a high genetic divergence from all other taxa in this genus by the COI, 16S rRNA and 28S rRNA gene fragments ( Table 2).
Description: Very small mussel. Shell length 26.7-28.7 mm, height 17.6-19.5 mm, width 10.3-12.4 mm. Shell ovate, subinequilateral, slightly inflated and thick. Posterior margin broader than anterior margin, with arched bars along the slope. Strong v-shaped sculpture all over the shell surface (except for one specimen), but it is slightly visible or absent on umbo area. Periostracum yellow-green or brown-green, nacre whitish, somewhat shining. Pseudocardinal teeth strong and very indented, by two teeth in each valve, of typical Leoparreysia shape. Lateral teeth moderately short, curved, two teeth in left valve, one tooth in right valve. Anterior muscle attachment scar rounded, more or less deep. Posterior muscle attachment scar shallow.
Habitat and ecology: Silty-clay sites in large rivers (Fig. 6A). Distribution: Ayeyarwady and Chindwin rivers, Ayeyarwady Basin, Myanmar. Comments: There are only three available specimens of this species, each from a different locality. The specimen from the Chindwin River has smooth periostracum and no shell sculpture, only bars along the posterior margin. Etymology: The name of this species is derived from its type locality, the Myit Tha River. Diagnosis: This species is morphologically and genetically close to Radiatula mouhoti Vikhrev, Bolotov & Konopleva 2017, but differs from it by more elongated and rounded posterior margin, less developed posterior muscle scar and smaller umbo. Radiatula myitthanensis sp. nov. is also externally similar to R. chindwinensis sp. nov., but it differs from the latter species by a rounded posterior slope (vs. truncated posterior slope in R. chindwinensis sp. nov.) and curved ventral margin (vs. straighter ventral margin in R. chindwinensis sp. nov.). The new species also differs from all the congeners by fixed nucleotide substitutions in the COI and 16S rRNA gene fragments ( Table 2).

Tribe
Description: Shell length 35.5-45.1 mm, height 19.4-23.7 mm, width 13.8-17.2 mm. Shell ovate-elongated, subinequilateral, somewhat inflated and thick. Dorsal margin slightly curved, ventral margin straight. Anterior margin rounded; posterior slope smooth, margin covered by small wrinkles from the beak. The umbo area has poorly visible v-shaped sculpture, corrugated. Periostracum jade-green, concentrically striated; nacre yellow-whitish. Right valve with a single slightly curved lateral tooth and two pseudocardinal teeth, anterior tooth small and lamellar; posterior tooth rectangular and more or less indented. Left valve with two slightly curved lateral teeth and two pseudocardinal teeth, anterior tooth rectangular and ribbed, posterior tooth smaller and pyramidal. Anterior muscle attachment scar ovate, well pronounced. Posterior muscle attachment scar less well marked.
Etymology: The name of this subspecies refers to the Haungthayaw River, its type locality. Diagnosis: The new subspecies is genetically close to Radiatula mouhoti Vikhrev, Bolotov & Konopleva, 2017, but is morphologically more similar to R. chaudhurii (Preston, 1912). Radiatula mouhoti haungthayawensis differs from the latter species by possessing a smoother periostracum, a stronger inflation, and a curved ventral margin. The new taxon also differs from the nominative subspecies by two diagnostic nucleotide substitutions in the COI gene fragment (Table 2). Anterior end rounded, ventral and dorsal margin curved; posterior side truncated, covered by small wrinkles. The umbo corrugated, with w-shaped sculpture, continued along all over the shell surface. Periostracum olive-green with pinkish patches, concentrically striated; nacre blue-whitish with yellow umbo cavity. Right valve with a single slightly curved lateral tooth and two pseudocardinal teeth, anterior tooth reduced; posterior tooth pyramidal or more rectangular and can be slightly indented. Left valve with two slightly curved lateral teeth and two pseudocardinal teeth, anterior tooth rectangular, posterior tooth smaller and pyramidal. Anterior muscle attachment scar ovate and well-marked. Posterior muscle attachment scar rounded and less pronounced.

Status of Specimen
Habitat and ecology: This subspecies was collected from a single location, an upstream pool site of the river with sandy-clay substrate (Fig. 6C Etymology: The new species is named after the Chindwin River, the largest tributary of the Ayeyarwady Basin, from which the species was collected. Diagnosis: The new species is conchologically similar to Radiatula mouhoti and R. myitthanensis sp. nov., but can be distinguished from these taxa by more straight dorsal margin and smooth pseudocardinal teeth without marked sculpture (vs. more curved dorsal margin and strongly sculptured pseudocardinal teeth in R. mouhoti and R. myitthanensis sp. nov.). The new species also differs from all the congeners by fixed nucleotide substitutions in the COI and 16S rRNA gene fragments ( Table 2).
Description: Small mussel. Shell length 28.7-34.5 mm, height 16.7-19.5 mm, width 10.2-15.0 mm. Shell elongate-ovate, rather thin, nearly equilateral in some specimens, not inflated. Anterior end rounded, slightly shifted upward at some specimens, ventral and dorsal margin straight or slightly curved; posterior side truncated, covered by small arched bars from umbo along the slope. Umbo corrugated, with unpronounced v-shaped sculpture, continued until the middle or ventral margin of the shell. Periostracum olive-green with yellow and dark regions, concentrically striated; nacre blue-whitish. Two lateral teeth on the left valve and a single tooth on the right valve, straight or slightly curved. Right valve with two pseudocardinal teeth, anterior tooth reduced; posterior tooth pyramidal or trapezoidal. Left valve with two pseudocardinal teeth, anterior tooth pyramidal or rectangular, posterior tooth smaller and pyramidal. Anterior muscle attachment scar ovate, rather deep. Posterior muscle attachment scar rounded and shallow.  Etymology: The name of this subspecies refers to its olive-green periostracum. Diagnosis: This subspecies is conchologically similar to Indochinella pugio pugio (Benson, 1862), but differs from it by shorter and higher shell, more pronounced and curved lateral teeth, moderately strong sculpture on shell disc, and a diagnostic nucleotide substitution in the COI gene fragment ( Etymology: This local subspecies is named after Dawei River, in which the type series was collected. Diagnosis: This taxon differs from the nominative subspecies in having oval-shaped shell, a more rounded posterior ridge and a more gradual posterior slope, distinct zigzag ridges across the shell disc (Fig. 5B), and fixed nucleotide substitutions in the COI, 16S rRNA and 28S rRNA gene fragments (Table 2). Perhaps, it is the most conchologically peculiar taxon within the genus.
Description: Very small mussel. Shell length 26.1-28.0 mm, height 11.5-20.4 mm, width 8.2-16.8 mm. Shell shape obovate, inequilateral, rather thick. Posterior ridge broader than the anterior ridge, oblique. Shell sculpture very strong, with distinct zigzag ridges across the shell disc. Periostracum sandy-brown with numerous dark-green zigzag ridges, nacre yellow-whitish. Umbo prominent, slightly corrugated, beak sculpture not very strong. Left valve with two short lateral teeth and two pseudocardinal teeth. Right valve with a single lateral tooth and blunt pseudocardinal tooth, anterior tooth not developed. Umbo cavity shallow and open. Anterior adductor scar marked, rounded or oval-shaped. Posterior adductor scar not pronounced, obovate.
Habitat and ecology: The subspecies is known only from its type series, which was collected from river sites with clay and gravel bottom substrate (Fig. 6F) www.nature.com/scientificreports www.nature.com/scientificreports/ almost straight or slightly curved, parallel to each other at some specimens. Posterior end biangular, from umbo to upper angle covered by ridges. Umbo slightly elevated with w-shaped sculpture; umbo area well-separated. Periostracum green, concentrically striated, laminiferous, with dark-green zigzag ridges in lower half of the shell; nacre white-yellow. Lateral teeth lamellar, thin, straight or slightly curved, a single tooth on right valve and two teeth on left valve. Two pseudocardinal teeth on right valve, anterior tooth lamellar, posterior tooth trapezoidal; two pseudocardinal teeth on left valve, anterior tooth rectangular and ribbed, anterior tooth small and pyramidal. Anterior muscle attachment scar rounded, well-visible. Posterior muscle attachment scar shallow.
Habitat and ecology: This species is known from the same locality as R. mouhoti haungthayawensis ssp. nov. (Fig. 6G).   Etymology: The name of this species refers to its elongated shell shape. Diagnosis: The new species remotely resembles Yaukthwa nesemanni, but differs from it by much more elongated shell, broader posterior margin, narrower anterior margin, reduced pseudocardinal teeth, and fixed nucleotide substitutions in the COI, 16S rRNA and 28S rRNA gene fragments (Table 2).
Description: Medium-sized mussel. Shell length 35.1-64.3 mm, height 16.4-28.1 mm, width 8.2-14.9 mm. Shell trapezoidal, elongated, inequilateral, thin, not inflated. Anterior end rounded, very narrow at some specimens, dorsal side curved, ventral margin slightly concaved. Posterior end broader than anterior end, truncated. Umbo not projected, slightly elevated, strongly corrugated at some specimens; corrugation may cover almost entire shell. Periostracum from light-to dark-brown, nacre white-bluish with yellow regions. Lateral teeth very  www.nature.com/scientificreports www.nature.com/scientificreports/ Discussion taxonomic implications. Here, we introduce eight new mussel taxa belonging to the genera Leoparreysia, Radiatula, Indochinella, and Yaukthwa. Leoparreysia was established by us for a group of species from the Western Indochina Subregion that was previously placed within the Indian genus Parreysia because of their external similarity 6 . However, we found that the Leoparreysia members represent a separate phylogenetic clade of the Parreysiinae, which is distantly related to the true Parreysiini 6 . Leoparreysia whitteni sp. nov. appears to be the smallest representative of this group. This species was recorded from the Ayeyarwady Basin, which seems to be the primary evolutionary hotspot of Leoparreysiini diversity, with at least five valid species 6 .
Radiatula appears to be another endemic genus of Western Indochina 6,7 , while its sister groups, e.g. Scabies Haas, 1911, Unionetta Haas, 1955, and Harmandia Rochebrune, 1881, are distributed in the Sundaland Subregion 14 . In this study, we describe three additional members of Radiatula, i.e. R. myitthanensis sp. nov., R. chindwinensis sp. nov., and R. mouhoti haungthayawensis ssp. nov. Various Radiatula species are abundant throughout Myanmar 6 , while R. crispisulcata, the type species of this genus, is known only from old museum lots.
Indochinella was introduced by us for the Oxynaia pugio group as a monotypic genus, endemic to Western Indochina 7 . The pugio-group is characterized by a high genetic and morphological variability, indicating the presence of additional taxa 6,7 . In this study, we introduce three more subspecies-level lineages: Indochinella pugio viridissima ssp. nov. from the Sittaung, Bilin and Bago rivers, I. pugio daweiensis ssp. nov. from the Dawei River, and I. pugio paradoxa ssp. nov. from the Haungthayaw River (Fig. 2). The two latter subspecies differ from other taxa by one or two diagnostic substitutions in the nuclear 28S rRNA gene fragment ( Table 2). This feature is remarkable, because such differences in the slowly evolving nuclear genes for freshwater mussels often correspond to species-level divergence 16 . Furthermore, these two subspecies are conchologically more similar to the Scabies taxa than to the nominative subspecies of I. pugio.
Yaukthwa is a species-rich genus in the subfamily Rectidentinae 9 . Historically, all Yaukthwa taxa were placed within the genus Trapezoideus Simpson, 1900 that was thought to comprise several widespread species, e.g. T. exolescens (Gould, 1843) [17][18][19] . However, based on a multi-locus phylogeny, Trapezoideus exolescens was found to be a member of the Parreysiinae 13 , and it has subsequently been transferred to its own genus, Trapezidens Bolotov, Vikhrev & Konopleva, 2017 6 . A taxonomic revision of the tribe Contradentini revealed that the genus Trapezoideus is a monotypic taxon with a single species, T. foliaceus 9 . Several species from Western Indochina previously assigned to Trapezoideus 6 were transferred to Yaukthwa 9 . This genus seems to be an endemic clade of Western Indochina, which includes a variety of endemic species with restricted distribution ranges, mostly in headwaters of rivers and streams 6,9 . Yaukthwa elongatula sp. nov. has a remarkable shell shape, which resembles the ultra-elongate representatives of the genus Solenaia (Gonideinae). This new species inhabits the Chindwin Basin, and it is associated with specific rocky and hard clay bottom sites.
While several recent integrative works 6,7,9,14,15,[20][21][22] have greatly improved our knowledge on the taxonomy and biogeography of freshwater mussels in Southeast Asia, many gaps in these fields are still to be filled. This novel study contributes to the further expansion and redefinition of the Oriental Unionidae. However, many nominal species of high importance for taxonomic and phylogenetic research, e.g. Pseudodon crebristriatus (Anthony, 1865) and Modellnaia siamensis Brandt, 1974, were not rediscovered by extensive field surveys during the last seven years 6,7,9,14,15 , and such taxa are in need of future research efforts. patterns of endemism. Previously, we have considered every large or medium-sized freshwater basin in the Oriental Region to be a separate evolutionary hotspot of the Unionidae fauna 6,15 . Pfeiffer et al. 14 noted that this point of view does not consider the intra-basin heterogeneity, when several evolutionary hotspots can be recorded within a single drainage basin. Additionally, it was suggested that the proportion of single-drainage endemics proposed by Bolotov et al. 15 may have been overestimated that underestimated the role of inter-basin faunal exchanges in shaping distribution patterns of freshwater mussels in Southeast Asia 14 . Based on the newly obtained results, we partly agree with those comments and propose an updated classification scheme for endemic freshwater mussel taxa from Southeast Asia as follows: (1) Subregion endemics: widespread taxa, the range of which encompasses several freshwater drainages within a single biogeographic subregion. This group seems to contain a rather small number of taxa from Western Indochina, e.g. Lamellidens savadiensis (Nevill, 1877), Indochinella pugio viridissima ssp. nov., and Leoparreysia tavoyensis (Gould, 1843). A few examples of species with rather broad ranges are known from the Sundaland Subregion, e.g. Scabies crispata (Gould, 1843), S. phaselus (Lea, 1856), and S. mandarinus (Morelet, 1864) 14 . Several freshwater mussel species are widespread in rivers of the Malay Peninsula and northern Borneo 20,21 . Such multi-drainage distribution patterns were likely originated by dispersal events via direct connections between freshwater basins during the Late Pleistocene 6,14,15 .
(3) Intra-drainage endemics: taxa with local ranges corresponding to a certain section within a freshwater basin. Currently, members of this group from Western Indochina are poorly known, because our previous field surveys were primarily focused on sampling taxa from separate freshwater drainages 6,15 . Currently, a few species can be assigned to this group, e.g. Radiatula myitthanensis sp. nov., R. chindwinensis sp. nov., Yaukthwa elongatula sp. nov., Y. inlenensis Konopleva et al., 2019, and Y. paiensis Konopleva et al. 9 . However, a number of local endemics from other sites may be overlooked.

Methods
Data sampling. Samples of freshwater mussels were collected by hand from different water bodies throughout Myanmar using a rapid bioassessment approach of Cummings et al. 23 . A series of each mussel morphospecies from every study site has been collected. Soft tissue snips for DNA analyses were preserved in 96% ethanol immediately after collection.

Molecular data and phylogenetic analyses.
New COI, 16S rRNA and 28S rRNA gene sequences were generated from 52 freshwater mussel specimens using a standard approach following published works 6,9,15 . The sequence data set and outgroup taxa are presented in Supplementary Table 1. The sequence alignment of each gene fragments was performed separately using the Muscle algorithm of MEGA7 24 . The aligned data sets were concatenated into a multi-locus alignment. Absent sites were coded as missing data. Five partitions, i.e. three codons of COI + 16S rRNA + 28S rRNA, were used for phylogenetic analysis. We carried out maximum likelihood phylogenetic searches using web interface and server for IQ-TREE (W-IQ-TREE) [25][26][27] with an automatic identification of the best-fit substitution model for each partition (Supplementary Table 2). To estimate the probability of internal branches, an ultrafast bootstrap (UFBoot) algorithm 28 with 10,000 replicates was applied. Bayesian models were implemented in MrBayes v. 3.2.6 29 with two runs, each with three heated (temperature = 0.1) and one cold Markov chain (30,000,000 generations with sampling every 1000th generation) at the San Diego Supercomputer Center through the CIPRES Science Gateway 30 . The first 15% of trees were discarded as burn-in. Tracer v. 1.6 31 was used to check a convergence of the MCMC chains to a stationary distribution.

Species delimitation and diagnostics of new taxa.
For preliminary delimitation of the putative species-level clades, we used BEAST2 v.2.5.1 32 with STACEY v.1.2.4 33 package. Each sequence in the multi-locus alignment (see above) was initially assigned to a prospective species unit. Nitia teretiuscula and Coelatura aff. aegyptiaca were used as outgroup (Supplementary Table 1). Five partitions (three codons of COI + 16S rRNA + 28S rRNA) were analyzed under a HKY substitution model and lognormal relaxed clock algorithm. The priors for the Birth Death Model were applied as follows: CollapseHeight = 0.001, Relative Death rate = 0.5, and Relative Collapse Weight = 0.5 using a beta prior. All parameters were estimated. Each run was conducted for 100,000,000 generations with sampling every 5000th tree and 10% burn-in. The calculations were performed at the San Diego Supercomputer Center through the CIPRES Science Gateway 30 . A matrix of putative species-level clusters based on SMC-trees generated by STACEY was constructed using SpeciesDelimitationAnalyser with initial 10% burn-in (www.indriid.com/software.html). An uncorrected COI mean p-distance to the nearest neighbor of each lineage was calculated in MEGA7 24 . To check the putative subspecies-level units having a rather low molecular distance from nearest neighbors, we additionally used a network-based approach using Network v. 4.6.1.3 software with default settings 34 that can reveal less prominent genetic differences between prospective taxa.
After a phylogenetic species delimitation, we estimated morphological differences between a new taxon and closely related (congeneric) taxa. The comparative analysis of the shell morphology was carried out with a special focus to the shell shape, structure of pseudocardinal and lateral teeth, shape of muscle attachment scars, and umbo position 6,7,9,13 . Three shell dimensions of each specimen, included in the type series of new taxa, i.e., the length, height, and width of the shell (all at the maximum diameter), were measured using calipers (±0.1 mm).
Finally, the molecular diagnosis of every new taxon was designed using fixed nucleotide substitutions, which were estimated for each gene separately using a Toggle Conserved Sites tool of MEGA7 24 at 50% level. For each new taxon, an alignment of congeneric haplotype sequences was performed using the Muscle algorithm implemented in MEGA7 24 . All deleterious mutations were retained for the analyses. 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:B017A518-0FBE-40F5-A2A4-8C13AC7E80BC. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.

Data Availability
The type series of the new species are available in the RMBH -Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia. The sequences generated in this study are available from GenBank. GenBank accession number and collecting locality for each specimen are presented in Supplementary Table 1.