Molecular phylogeny reveals a new genus of freshwater mussels from the Mekong River Basin (Bivalvia: Unionidae)

A new genus of freshwater mussels (Bivalvia: Unionidae) is described from the Mekong River Basin as Namkongnaia gen. nov. The validity of the new genus is supported by its unique conchological characteristics, namely the lack of hinge dentition and elongated shells, together with its evolutionary distinctiveness as estimated by multi-locus phylogenetic analyses (mitochondrial COI and 16S, and nuclear 28S genes). The new genus includes two lineages with deep divergence, shown by 5.10% uncorrected COI p-distance. One lineage is a type species described herein as Namkongnaia inkhavilayi gen. et sp. nov. The other is a recognized species under the name ‘Pilsbryoconcha lemeslei (Morelet, 1875)’. Molecular phylogenetic analysis further shows that the new genus belongs to the tribe Pseudodontini, and evolutionarily is closely related to the genus Monodontina Conrad, 1853. However, its conchology is similar to the genus Pilsbryoconcha Simpson, 1900. Time-calibrated phylogeny suggests that the main radiation events of the tribe Pseudodontini occurred during the Late Cretaceous to the Eocene, with the divergence between the new genus and Monodontina placed in the Miocene. The discovery of new freshwater mussel taxa in this study highlights the importance of the Mekong River Basin as one of the world’s biodiversity hotspots for freshwater fauna. European Journal of Taxonomy 775: 119–142 (2021) 120


Introduction
The Mekong River is the longest river in Southeast Asia. The river and its tributaries cover a vast range of geographic and climatic zones that support various aquatic habitats, including the river's mainstream, mountainous streams, tributaries, fl oodplains, lakes, wetlands, and estuaries (Coates et al. 2003). As a result, the basin contains one of the most unique and diverse freshwater faunas on Earth (Köhler et al. 2012;Kang & Huang 2021). Freshwater mussels (Unionidae) are of special interest for their high species diversity in the Mekong River Basin (Graf & Cummings 2021a); furthermore, it has been hypothesized to be one of the locations of origin for mussel radiation prior to their spread worldwide (Bolotov et al. 2017a). The currently known richness of freshwater mussels in the Mekong River Basin comprises 67 species across 21 genera (Graf & Cummings 2021a;Konopleva et al. 2021). Several of these genera and a dozen of these species were discovered within the last decade (Kongim et al. 2015;Jeratthitikul et al. 2019a;Konopleva et al. 2019Konopleva et al. , 2021Bolotov et al. 2020;Pfeiffer et al. 2021). Moreover, 59 species are endemic to the basin, and fi ve genera are considered monotypic, highlighting the extraordinary endemism within the tributaries (Brandt 1974;Pfeiffer et al. 2018;Bolotov et al. 2020;Graf & Cummings 2021a;Konopleva et al. 2021). However, freshwater mussels in the Mekong River Basin are at severe risk of population decline and even extinction from the signifi cant threats of pollution, natural habitat modifi cation, urbanization, and overharvesting Ngor et al. 2018). Meanwhile, efforts to conserve these fragile animals in the region have made little progress due to the lack of essential data, especially regarding population size and distribution range. Their taxonomic classifi cation also remains uncertain Zieritz et al. 2018). Pseudodontini Frierson, 1927 represents one of the most taxonomically diverse group of freshwater mussels in Southeast Asia. At least 44 valid species are recognized, and half are distributed in the Mekong River Basin (Graf & Cummings 2021a). This family-group name was fi rst proposed as a subfamily by Frierson (1927) based on "large high (pseudocardinal) teeth, one in each valve", and included the genus Pseudodon Gould, 1844. Modell (1942) redefi ned and relocated this subfamily under Margaritiferidae Henderson, 1929 and included several genera from Asia, Europe, , and North America based on the reduced pseudocardinal teeth. Subsequent revisionary works synonymized this subfamily with either Unioninae Rafi nesque, 1820 (Haas 1969a, b) or Ambleminae Rafi nesque, 1820 (Subba Rao 1989). Later, Brandt (1974) resurrected the subfamily rank and incorporated a second genus, Pilsbryoconcha Simpson, 1900. He also expanded diagnostic characters as "hinge teeth either completely missing or reduced to a knob-like pseudocardinal in each valve". However, all of these taxonomic opinions were based mainly on shell morphology. Molecular phylogenetic studies do not accept Brandt's (1974) classifi cation, but instead reassign Pseudodon and Pilsbryoconcha to Gonideinae (Whelan et al. 2011;Pfeiffer & Graf 2015). The nominal name was recently recognized as a valid tribe, Pseudodontini, within Gonideinae Ortmann, 1916by Lopes-Lima et al. (2017 with two genera, Pseudodon and Pilsbryoconcha . The authors also suggested that the V-shaped fossette present in the inner shell at the posterior end of the hinge structure is a diagnostic character of the tribe. This classifi cation was followed and supported by several studies using various molecular markers and techniques (Zieritz et al. 2018;Pfeiffer et al. 2019;Bolotov et al. 2020;Zieritz et al. 2020;Graf & Cummings 2021a;Konopleva et al. 2021). Controversially, the subfamily rank has also been used by some molecular phylogenetic studies (Bolotov et al. 2017a(Bolotov et al. , 2017b(Bolotov et al. , 2018Huang et al. 2019).
Initially, Pseudodontini includes two genera, Pseudodon and Pilsbryoconcha (Lopes-Lima et al. 2017). Bolotov et al. (2017b) used multi-locus phylogenetic approaches to revise genus-level clades within this group. They revealed a polyphyletic relationship of at least fi ve clades within Pseudodon s. lat. , and applied two previously available names and erected three new genera. Hence, a total of seven genera are currently included in Pseudodontini Graf & Cummings 2021a). Bolotov et al. (2020) further suggested classifying Pseudodontini into a monotypic subtribe Pseudodontina and subtribe Pilsbryoconchina containing the other six genera. These two subtribes are genetically different and can be distinguished by their morphology. Shells of Pseudodontina are relatively thick, and pseudocardinal teeth are tubercle-like, rather solid, and prominent. In contrast, shells of Pilsbryoconchina are thinner, and pseudocardinal teeth are reduced or lacking (Bolotov et al. 2017b).
In the present study, we propose another new genus from the Middle Mekong River Basin belonging to Pseudodontini. This new genus has long been recognized under Pilsbryoconcha s. lat. (Haas 1920(Haas , 1969aBrandt 1974;Graf & Cummings 2007;Zieritz et al. 2018;Ng et al. 2020;Graf & Cummings 2021a). We further include two species: the new species described herein, and another previously recognized as ' Pilsbryoconcha lemeslei '. The validity of these taxa is supported by their unique conchological characteristics and their evolutionary distinctiveness as estimated through multi-locus phylogenetic analyses using sequences from both mitochondrial and nuclear genes. Furthermore, the evolutionary relationship and estimation of divergence times within Pseudodontini are also discussed.

Specimen sampling
Animal use protocol in this study was approved by the Faculty of Science, Mahidol University Animal Care and Use Committee, SCMU-ACUC (MUSC63-026-534).
Specimens were collected by hand and euthanized by a two-step method (AVMA 2020): living specimens were placed in a container fi lled with fresh water. Then, 95% (v/v) ethanol was gradually added to the container starting from approximately 5% (v/v) concentration until specimens were fully anesthetized. Specimens were moved to 70% (v/v) ethanol to complete the process and for tissue fi xation. Shells were separated from the soft body and were then gently cleaned to remove residual tissues. Tissues from the foot and/or mantle were cut and preserved in 95% (v/v) ethanol at -20°C for DNA extraction. The other remaining soft parts were preserved in 70% (v/v) ethanol for anatomical study and kept together with their shells to serve as vouchers.
Shell morphology was compared among specimens, the type series, and photographs from museum collections available on the online database (i.e., MUSSELp Database; Graf & Cummings 2021b). Shell comparisons were done based on shell shape, shell size, umbo position, teeth, and adductor muscle scars. Shell length, height, and width were measured using a digital Vernier calliper (± 0.01 mm). Anatomical characteristics, i.e., excurrent and incurrent aperture, labial palps, and gills, were examined under a stereomicroscope.

Molecular analysis
Total genomic DNA was extracted from small pieces of foot or mantle tissue using a DNA NucleoSpin ® extraction kit for animal tissue (MACHEREY-NAGEL, Germany) following the standard procedure of the manual. Fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and the 16S small ribosomal RNA (16S) genes, and the nuclear 28S ribosomal RNA gene (28S) were amplifi ed by polymerase chain reactions (PCR). Primers used for the COI gene were LCO1490 and HCO2198 (Folmer et al. 1994); for 16S gene were 16sar-L-myt and 16Sbr-H-myt (Lydeard et al. 1996), and for 28S gene were C1 and D2 (Jovelin & Justine 2001). Thermal cycling was started at 94°C for 3 min; followed by 35 cycles of 94°C for 30 s; annealing at 48°C for COI, 46°C for 16S rRNA, and 58°C for 28S rRNA, for 60 s; extension at 72°C for 90 s; then a fi nal 72°C for 5 min. Amplicons were purifi ed using a MicroSpin purifi cation kit (Qiagen, USA). Each amplicon was bi-directionally sequenced using the same primers used for amplifi cation on the ABI 3730XL DNA Analyzer (BIONEER, Republic of Korea). The derived consensus nucleotide sequences newly obtained in this study were deposited in the GenBank database under accession numbers MZ822395-MZ822416 for COI, MZ822895-MZ822916 for 16S and MZ822917-MZ822938 for 28S (Table 1).

Phylogenetic analyses
Phylogenetic analyses were based on 55 sequences covering all genera from Pseudodontini. Sequences from other tribes were used as outgroups (Table 1). The details of taxon sampling used in phylogenetic analysis are shown in Table 1. Sequence alignments were generated using the MUSCLE option as implemented in MEGA ver. 7.0.26 (Kumar et al. 2016). The fi nal concatenated alignment used in phylogenetic tree construction contained 1967 bp: 660 bp of COI, 501 of 16S, and 806 bp of 28S.
The best-fi t evolutionary model for each gene and gene partition was calculated by PartitionFinder2 ver. 2.3.4 (Lanfear et al. 2016), based on the corrected Akaike Information Criterion (AICc) and using a heuristic search algorithm. The program suggested dividing the concatenated dataset into fi ve partitions, consisting of partitions for 16S and 28S genes and for each of three codon positions of the COI gene. The best-fi t model was GTR+I+G for the fi rst codon position of COI, 16S, and 28S; F81+I for the second codon position of COI; and GTR+G for the third codon position of the COI gene. This partition setting was used in subsequent Bayesian inference (BI) analysis. The BI analysis was carried out by running two runs of 10 million generations of Metropolis-coupled Markov chain Monte Carlo (MC-MCMC) in MrBayes ver. 3.2 (Ronquist et al. 2012). Each MCMC run consisted of two sets of three heated chains and one cold chain. Each run used a random tree as starting tree and collected a sampling tree every 1000 th generations. The fi rst 25% of the obtained sampling trees were discarded as burnin. Maximum likelihood analysis (ML) was performed through 1000 ML bootstrap replicates using    (Miller et al. 2010).
Average genetic distances among taxa were estimated for the COI data set using uncorrected pairwise genetic distances as implemented in MEGA ver. 7.0.26 (Kumar et al. 2016).

Estimation of divergence times
Divergence time was estimated by constructing a time-calibrated phylogeny as implemented in BEAST ver. 2.6.1 (Bouckaert et al. 2019) through the online CIPRES Science Gateway (Miller et al. 2010).
The same multi-locus dataset as for phylogenetic analyses was used. The external COI evolutionary rate of 0.265 ± 0.06% substitutions per site per million years as previously estimated for Unionidae (Fonseca et al. 2016) was applied for tree dating. The use of evolutionary rates taken from literature without calibration by fossils or geological events may give inappropriate results and should be interpreted with caution. Nevertheless, a previous study that used this evolutionary rate showed reliable estimation results , which were largely congruent with those estimated from mitogenomic data (Fonseca et al. 2016) and those using fossil data as calibration points (Bolotov et al. 2017a). The evolutionary model for each partition was set separately, following the suggestion of jModelTest ver. 2.1.10 (Darriba et al. 2012). The evolutionary rate was implemented only for the COI partition and was set as estimated for other partitions. The analyses were run using a lognormal relaxed clock algorithm (Drummond et al. 2006). A random tree generated from the COI dataset with the Yule speciation process was used as a tree prior. Two independent MCMCs were run for 50 million generations, and tree sampling was collected every 1000 th generation. The output fi les were checked for convergence diagnostics and Effective Sample Sizes (ESS) using Tracer ver. 1.7 (Rambaut et al. 2018). The effective sample size (ESS) values were greater than 1500 for all parameters. Results from two independent runs were compiled with 25% burn-in using LogCombiner ver. 2.6.2 (Bouckaert et al. 2019) and then summarized for the maximum clade credibility (MCC) tree in TreeAnnotator ver. 2.6.2 (Bouckaert et al. 2019).

Phylogenetic analysis and divergence time estimation
Both ML and BI analyses returned similar topologies. Therefore, only the tree topology from ML is presented in Fig.1. All of the genera in Pseudodontini are recovered as highly supported clades, with 95-100% bootstrap support (BS) values for ML and Bayesian posterior probability (BPP) of 1 for BI, except Sundadontina Bolotov et al., 2020, which shows poor support (BS = 32%, BPP = 0.79). The pairwise distance analysis also reveals a large genetic distance among these genera, with 10.65-13.59% uncorrected COI p-distance (Table 2).
Among available clades, there is one highly supported clade that is distantly separated from other genera, with pairwise uncorrected COI p-distance ranging from 11.02% to 12.42%. This clade shows unique morphological characteristics distinguishable from other genera. Therefore, it is described herein as Namkongnaia gen. nov. The new genus further includes two well-defi ned clades (BS = 99-100%, BPP = 0.99), with a deep divergence of 5.10% uncorrected COI p-distance. Both clades are morphologically distinct from each other. One clade has long been recognized under the name ' Pilsbryoconcha lemeslei ' and the other is described herein. Interestingly, a sequence recognized as Pilsbryoconcha lemeslei sensu Bolotov et al. (2020) fails to group with our specimens; instead, it is placed within Pilsbryoconcha s. str.
The phylogenetic relationship among genera within Pseudodontini is uncertain and with poor support. Nevertheless, the phylogenetic tree reveals a monophyletic relationship among six genera in Pilsbryoconchina (BS = 96%, BPP = 0.99) and separates them from Pseudodontina (only the genus Pseudodon currently belongs to this subtribe). The average genetic distance between these subtribes is 12.5% uncorrected COI p-distance. In addition, the new genus is suggested as a sister clade with Monodontina , although with only 0.97 BPP support from BI.
A reconstructed time-calibrated phylogeny (based on 0.265 ± 0.06% substitutions per site per million years of COI evolutionary rate) is shown in Fig. 2

Taxonomy
Given the results of the morphological examination and multiple lines of molecular evidence mentioned above, we thus propose describing the novel discovered clade in Pilsbryoconchina as Namkongnaia gen. nov., together with a new species from Laos. We also transfer Anodonta lemeslei Morelet, 1875 to this new genus.

Differential diagnosis
The new genus is distinguished from other genera in Pseudodontini by having a narrow, elongated, and less infl ated shell. It is also represented as a distinct clade in multi-locus phylogenetic analyses.

Description
Shell Medium-sized, thin, narrow and elongated, rather compressed, very inequilateral; anteriorly round; posteriorly round or somewhat pointed; umbonal area not elevated and usually eroded. Ligament very narrow. Hinge without dentition, posterior end of the hinge structure with V-shaped fossette. Anterior adductor muscle scar shallow, ovate, and fused with pedal retractor muscle scars; posterior adductor muscle scar very shallow. Excurrent aperture smooth, shorter than incurrent; incurrent with 1-2 rows of conical papillae. Gills elongated and slightly ribbed; anterior margin of inner gills slightly longer and wider than outer gills.

Etymology
The generic name " Namkongnaia " is from the word "Namkong" , a name for the Mekong River used by Thai and Lao peoples, and Greek word " naiad " meaning freshwater mussels. The name of this genus thus means "freshwater mussels from Mekong River".

Distribution
Mekong River and its tributaries in Laos, Cambodia, and Thailand ( Fig. 3; Brandt 1974;Ng et al. 2020). The distribution range may include the Mae Klong River Basin in Thailand (Brandt 1974), although the taxonomic status of the specimens recorded in Brandt (1974) needs to be confi rmed.

Remarks
The new genus is currently composed of two species, which are confi rmed by our multi-locus molecular data. Other potential species are probably among those previously recognized under the name ' Pilsbryoconcha lemeslei ' by Brandt (1974). This includes the specimens from 'Bang Pae', Mae Klong River Basin, Ratchaburi Province (USNM-786217, SMF-BR2663, and ANSP-H19041). These specimens show elongate and narrow shell, which are diagnostic characters of the genus, but differ from other species by having a minute concave ventral margin, and truncated posterior margin with a pointed posterior end. Further examination of fresh materials and DNA investigation would confi rm the taxonomic status of these populations.  Tables 1, 3 Pilsbryoconcha lemeslei -Brandt 1974: 263, pl. 18 fi g. 22 (in part, only records from "Nang Rong (Prov. Burirum)", Thailand) [non Anodonta lemeslei Morelet, 1875].

Etymology
This new species is dedicated to our colleague and distinguished malacologist, Dr Khamla Inkhavilay, from the National University of Laos.

Description
Shell medium-sized, length 80.9-102.2 mm, height 26.2-37.2 mm, width 13.6-19.1 mm (Table 3). Shell rather thin, narrow and elongated (H/L ratio = 0.32-0.36), very inequilateral, compressed. Dorsal margin straight, anterior at the same level as posterior. Umbonal area eroded, not elevated. Anterior margin round; posterior margin elongated and rounded. Ventral margin almost straight, slightly curved upward posteriorly. Posterior ridges low, wide and obtuse, not prominent. Periostracum thin, greenish to dark brown, the eroded part coppery-brown. Shell surface with fi ne growth lines. Ligament very narrow. Hinge without dentition, posterior end of the hinge structure with V-shaped fossette. Anterior adductor muscle scar placed relatively far from the dorsal margin, shallow, ovate, fused with pedal retractor muscle scars; posterior adductor muscle scars very shallow, almost invisible. Pallial line very faint. Nacre whitish with cream tint near the umbo. Excurrent aperture smooth, shorter than incurrent. Incurrent with 1-2 rows of conical papillae, varying in length. Small epithelial folds form a fused bridge separating excurrent and incurrent aperture. Gills elongated and slightly ribbed. Anterior margin of inner gills slightly longer and wider than that of inner outer gills. Glochidia unknown.

Distribution
This new species appears to occur in the Lower Mekong watershed. In Laos, it was recorded from several locations near Vientiane, Kammoune, and Champasak Provinces. In Thailand, it is known from the Songkram and Mun river basins in the northeast region (Brandt 1974). Habitat Specimens of the new species were bought on a local market near the Xe Bangfai River. However, specimens from the Thuai and Songkhram Rivers in Thailand were found buried in the mud substrate of still water. Brandt (1974) also reported ponds as a general habitat of this species.

Remarks
The specimens recorded by Brandt (1974) from Nang Rong, Buriram, Thailand (SMF-220822) show a shorter but wider shell and a relative rounded ventral margin. In addition, although the hinge area of this species has no dentition, the trace of a rudimentary pseudocardinal tooth is seen as a very tiny tubercle in some individuals.

Differential diagnosis
This species resembles the type species, but it can be distinguished by the relatively smaller size with a shell length of 67.0-81.0 mm (vs 80.9-102.2 mm), anterior adductor muscle scar placed near the dorsal margin (vs relatively far from the dorsal margin), adductor muscle scar relatively deep for the thin shell (vs shallower, although thicker shell), nacre yellowish (vs whitish), anterior margin rounded and little shouldered (vs wider and not shouldered), and the anterior portion of the dorsal margin somewhat lower than the posterior (vs at the same level in both anterior and posterior portions).

Description
Shell medium-sized, rather thin, narrow and elongated (H/L ratio = 0.39), very inequilateral, compressed. Dorsal margin straight; somewhat lower anteriorly than posteriorly. Umbonal area eroded, not elevated. Anterior margin round, little shouldered; posterior margin elongated, somewhat pointed. Ventral margin almost straight, or minutely concave in the middle in old specimens, posteriorly slightly curved upward. Posterior ridges low, wide and obtuse, not prominent. Periostracum thin, yellowish to dark brown, the eroded part coppery-brown. Shell surface with fi ne growth lines. Ligament very narrow. Hinge without dentition, posterior end of the hinge structure with V-shaped fossette. Anterior adductor muscle scar placed near the dorsal margin, relatively deep for the thin shell, ovate, fused with pedal retractor muscle scars; posterior adductor muscle scars very shallow, almost invisible. Pallial line very faint. Nacre yellowish with creamy tint near the umbo.

Additional description
Examination of the soft body of newly collected specimens in this study yielded additional description of the animal: excurrent aperture smooth, shorter than incurrent; incurrent with 1-2 rows of conical papillae, similar in length; small epithelial folds form a fused bridge separating excurrent and incurrent aperture; gills elongated and slightly ribbed; anterior margin of inner gills slightly longer and wider than that of outer gills. However, no brooding specimens were available to examine glochidia.

Distribution
This species is restricted to Tonle Sap Lake and its tributaries in Cambodia. Its distribution probably reaches the headwater of Tonle Sap basin in eastern Thailand (Brandt 1974).

Habitat
This species was recorded in mud substrate in still sections of rivers or in lentic habitat, i.e., ponds and lakes (Morelet 1875;Morlet 1889;Brandt 1974).

Remarks
This species has been noted for its rarity (Brandt 1974;Ng et al. 2020). Only a few lots are available in museum collections. We obtained additional fresh materials from the Kampong Kdei River, Siem Reap Province. These specimens resemble the syntype MNHN-MP-3150 (Fig. 5D). The only detected variations are less erosion of the umbo area and more yellowish-brown colour of the periostracum. Morelet (1875) described Anodonta lemeslei based on specimens collected by M. Le Mesle. The fi gured specimen is eroded, with a concave ventral margin ( Fig. 5B-C), while another shell has a relatively parallel shell and a straight ventral margin (Fig. 5D). Morelet (1875) identifi ed the ventrally concave specimens as fully-grown individuals, while the ventrally straight specimens were considered to be young individuals. The concavity trait is quite unusual for species that live in stagnant water habitats. Nevertheless, some specimens from Tonle Sap Lake (MNHN-MP-3156) also show the characteristic of a concave ventral margin. However, all of our specimens have a straight ventral margin. This raises some suspicions about the existence of both variations. In this study, we retain both variations as the same species until topotype specimens with concave ventral margin are available for DNA analysis.
Apart from a ventral margin, other morphological variations are also observed. The posterior ends of some specimens are more or less rounded than others. Crosse & Fischer (1876) noted that some specimens from Cambodia are narrower and more elongated than the type fi gure. The posterior dorsal margin in some specimens is also somewhat higher. Morelet (1875) noted that one of his type specimens is undoubtedly similar to Anodonta schomburgki Martens, 1860[= Pilsbryoconcha compressa (Martens, 1860]. However, it is more or less narrow, and the high posterior cannot be considered a posterior wing as in Anodonta schomburgki Martens, 1860 . Several records and specimens previously identifi ed as Pilsbryoconcha lemeslei have been examined and listed in this species. However, a specimen from Khanh Hoa, Vietnam, is conchologically different and does not belong to this species (Thach 2007;Do et al. 2018); rather, it is probably a species of Pilsbryoconcha . This specimen shows more or less curved ventral margin and relatively wider shell.
Similarly, the DNA sequence of specimen assigned to Pilsbryoconcha lemeslei by Bolotov et al. (2020) and Konopleva et al. (2021) is recovered as monophyletic within the Pilsbryoconcha , together with specimens of Pilsbryoconcha exilis from Java, Indonesia (type species) and Pilsbryoconcha linguaeformis , another sympatric species with Namkongnaia lemeslei gen. et comb. nov., from Tonle Sap Lake, in our phylogenetic analyses. However, without examination of the specimen mentioned in Bolotov et al. (2020) and Konopleva et al. (2021), it can only be classifi ed to the genus Pilsbryoconcha .

Discussion
Our morphological and molecular analyses strongly suggest that freshwater mussels that have long been recognized as ' Pilsbryoconcha lemeslei ' from the Mekong River Basin represent a new genus in Pseudodontini . These mussels are morphologically distinct from all other genera by having narrow, elongated, and less infl ated shell. Additionally, the ancient divergence (approximately 49.3 Mya; Fig. 2) and large genetic distances from other genera (11.02-12.42% uncorrected COI p-distance; Table 2) also support it as a distinct genus. The discovery of a new mussel genus from Southeast Asia, and especially from the Mekong River Basin, is not unexpected since several new genera and a dozen new species were discovered during the last decade (Kongim et al. 2015;Jeratthitikul et al. 2019a;Konopleva et al. 2019Konopleva et al. , 2021Bolotov et al. 2020;Pfeiffer et al. 2021).
The new genus shares shell characteristics with other genera in Pseudodontini by having a V-shaped fossette at the posterior end of the hinge structure, and absent or reduced pseudocardinal teeth (Brandt 1974;Lopes-Lima et al. 2017). Another distinct morphological character of the tribe is a double-looped or W-shaped sculpture on the umbonal area (Lopes-Lima et al. 2017). Unfortunately, the umbonal area of all specimens from either the type species or specimens of Namkongnaia lemeslei gen. et comb. nov. is heavily eroded, even in young specimens. Therefore, we are unable to confi rm this character for the new genus. In the same way, we were unable to examine the glochidia of the new genus, since no brooding individuals were collected in this study. Nevertheless, the glochidia of the new genus are most likely present as an unhooked and semi-elliptical structure, which is the general shape of glochidia among the Gonideinae (Pfeiffer & Graf 2015;Lopes-Lima et al. 2017).

Phylogenetic analyses in this study reveal a close relationship between the new genus and
Monodontina , although with moderate support by only the BI analysis (BPP = 0.97; Fig. 1). As far as we know, the only shared feature among members of the new genus and Monodontina is the presence of a V-shaped fossette at the posterior end of the hinge structure on the inner surface of the shell.
Monodontina differs from the new genus by its ovate to obovate shell with obtuse pseudocardinal teeth (Conrad 1853). However, some species have relatively weak and fl attened pseudocardinal teeth, although never absent . Future investigation to confi rm the sister relationship between them should include longer sequences and additional gene fragments, or incorporate more informative molecular markers, such as mitogenomic or nuclear genomic data, which have been successfully used in the phylogenetic study of freshwater mussels (Pfeiffer et al. 2019;Froufe et al. 2020;Zieritz et al. 2020).
On the contrary, the new genus is similar in conchology to Pilsbryoconcha , another widespread genus in rivers of Indochina . Both genera share an elongated and laterally compressed shell and completely absent or very reduced pseudocardinal teeth (Brandt 1974). They also have a rounded anterior end with a straight dorsal margin, while other genera in Pseudodontini have a somewhat concave dorsal margin around the position of pseudocardinal teeth. Because of several shared shell characters, the new genus thus has been hidden in the name " Pilsbryoconcha lemeslei (Morelet, 1875)" for more than a hundred years (Simpson 1914;Haas 1969a;Brandt 1974;Graf & Cummings 2007, 2021aZieritz et al. 2018;Ng et al. 2020). The present molecular phylogenetic analysis recovers both genera as a distinct clade (Figs 1-2) and with a deep divergence of 12.41% uncorrected COI p-distance (Table  2). Our phylogenetic analysis included the type species, Pilsbryoconcha exilis , from Java, Indonesia, which is assumed to be the the type locality (Graf & Cummings 2021b). In addition, both genera are distinctly separated by several shell characters. The new genus has a narrower shell, with a lower posterior dorsal margin and an almost straight ventral margin. In contrast, Pilsbryoconcha s. str. is wider, elongate linguiform shaped, with a higher dorsal margin and slightly arched ventral margin (Brandt 1974). Some species of Pilsbryoconcha exhibit a high dorsal margin, creating a posterior wing, such as Pilsbryoconcha linguaeformis (Morelet, 1875) from Tonle Sap Lake (Ng et al. 2020).
Pseudodontini is divided into two subtribes, Pseudodontina and Pilsbryoconchina. These two subtribes exhibit a deep genetic divergence from each other, with an average genetic distance of 12.5% uncorrected COI p-distance and estimated at about 91.9 Mya (95% HPD = 67.8-116.9 Mya) (Fig. 6). In the present-day sense (i.e., Graf & Cummings 2021a), Pseudodontina is a monotypic clade consisting of thirteen valid species from western Indochina rivers, the Yangtze River Basin in China, and northern Vietnam (Graf & Cummings 2021b). In contrast, Pilsbryoconchina comprises at least six genera and is distributed mainly in the Mekong River and Chao Phraya River basins, the Malay Peninsula, and southern Sunda islands (Graf & Cummings 2021b). Such a deep divergence is consistent with the division of freshwater biogeography of mainland Southeast Asia into the western Indochina and Sundaland subregions . The separation of two subtribes and later a rapid diversifi cation of genera within Pilsbryoconchina may be a consequence of the palaeo-drainage rearrangement of the large rivers in East Asia as a result of the Cenozoic uplift of the Tibetan Plateau (Wang et al. 2012;Zhang et al. 2019), and in concert with favourable climatic and hydrological conditions in the early Palaeogene (Carmichael et al. 2017), followed by range fragmentation during the abrupt climate change at the Eocene-Oligocene boundary (Buerki et al. 2013). This coincides with the divergence time estimation in this study, which suggests rapid radiation events of the Pilsbryoconchina genera approximately during the Late Cretaceous to Eocene times (mean age = 70.8-49.3 Mya). The common ancestor of the palaeo-Mekong Gonideinae may have separated from the palaeo-Yangtze drainage in the Albian-Cenomanian times , and had subsequent radiations in the palaeo-Mekong River Basin in the Late Cretaceous or Paleocene (Bolotov et al. 2017a(Bolotov et al. , 2017bZieritz et al. 2020).
The new genus consists of two well-defi ned clades with a deep divergence of 5.10% uncorrected COI p-distance, and are recognized here as two distinct species. These two species are separated by a deep genetic divergence and are distributed in different geographical drainages. Namkongnaia inkhavilayi gen. et sp. nov. is distributed in tributaries in the Middle Mekong River Basin in Laos and the Korat Plateau, Thailand, while N . lemeslei gen. et comb. nov. is found in Tonle Sap Lake and its tributaries in Cambodia (Fig. 3). The separation of closely related taxa between the Middle Mekong River and Lower Mekong River basins is well documented in other unionid mussels. Almost all the examined genera display this biogeographic pattern Muanta et al. 2019), suggesting a shared biogeographic history among these taxa.
This study included museum specimens collected from Huai Tadjek, Nang Rong and, Mun River basins on Korat Plateau, Thailand (SMF-220822) for N . inkhavilayi gen. et sp. nov. These specimens agree well with our new species (Brandt 1974: pl. 18 fi g. 22). However, they show some variation by having a rather shorter but wider shell and a relatively rounded ventral margin. Unfortunately, we failed to obtain fresh material from this population, and therefore cannot test for its genetic and phylogenetic entity. Nevertheless, freshwater mussels from the Mun River Basin generally show a deep genetic divergence from other parts of the Mekong River Basin (i.e., Songkhram River Basin in the northern Korat Plateau and Tonle Sap Basin in the Lower Mekong River Basin). Usually, they are represented as distinct species Jeratthitikul et al. 2019b;Muanta et al. 2019). Therefore, there is a possibility that the population from the Mun River Basin is a different species from the currently assigned N . inkhavilayi gen. et sp. nov.
Similarly, several specimens in the museum collection were collected from the Chao Phraya River basin and provisionally identifi ed as ' Pilsbryoconcha lemeslei ' (i.e., USNM-786217, SMF-BR2663, and ANSP-H19041). These specimens have an elongated and narrow shell which is the diagnostic characteristic of this new genus, but possess some differentiated characters by having a minute concave ventral margin and truncated posterior margin with a pointed posterior end. These specimens are also waiting further confi rmation. Therefore, after DNA examination of the two populations in question, the number of members in the new genus will likely increase.