New freshwater mussels from two Southeast Asian genera Bineurus and Thaiconcha (Pseudodontini, Gonideinae, Unionidae)

The Mekong and Chao Phraya rivers harbor a species-rich freshwater mussel assemblage containing a large radiation of the Pseudodontini species. Members of the genera Bineurus Simpson 1900 and Thaiconcha Bolotov et al., 2020 primarily inhabit small and medium-sized tributaries of these rivers. Here, we present an integrative taxonomic review of these genus-level clades. We show that Bineurus contains four species: B. mouhotii (Lea, 1863), B. exilis (Morelet, 1866) stat. rev., B. anodontinum (Rochebrune, 1882) stat. rev., and B. loeiensis sp. nov. In its turn, Thaiconcha comprises three species: T. callifera (Martens, 1860), T. munelliptica sp. nov., and T. thaiensis sp. nov. Two species, Pseudodon ovalis Morlet, 1889 and P. thomsoni Morlet, 1884, are considered here as questionable taxa. These findings further highlight that Southeast Asia represents a significant evolutionary hotspot of freshwater mussels, which requires further international collaborative research and conservation efforts.

Diagnosis: Bineurus can be distinguished from its sister genera by an elongated rhomboid/kidney-shaped, inequilateral, compressed, and rather thin shell, usually with straight or concave ventral margin. Hinge plate narrow, with a small, tubercle-like pseudocardinal tooth in each valve. Muscle scars are usually more or less developed.
Distribution: Mekong River basin. Comments: This genus contains four species (Table 2), one of which is new to science and described here. Distribution: Mekong River basin in western and northern Laos (including Nam Ou River) and northern Thailand.
Diagnosis: This new taxon differs from its sister species Thaiconcha callifera by having a massive, more oblong, and inflated shell, and less prominent pseudocardinal teeth. It can also be distinguished from the sister taxon by fixed nucleotide substitutions in the COI and 16S rRNA gene fragments (Table 1)  Etymology: The name of this species is derived from the country of Thailand, in which its type locality is situated.
Diagnosis: This taxon is conchologically similar to Thaiconcha califera and T. munelliptica but it may be distinguished from these taxa by its more yellow periostracum (especially in young individuals), more broadly rounded posterior margin, and less prominent posterior wing. It can also be distinguished from the sister taxon by fixed nucleotide substitutions in the COI gene fragment (Table 1).
Description: Specimens small to large: length 34.0-93.8 mm; height 20.5-53.9 mm; width 9.1-28.9 mm. Shell elongate-ovate, inequilateral, moderately thick, somewhat compressed; anterior and posterior margins rounded, dorsal and ventral margins curved. Smaller specimens thinner shelled and more lightly colored, and broadly rounded posteriorly. Larger specimens thicker shelled, darker, and more pointed posteriorly. Radial striola occasionally present, faint. Nacre faintly purple to blue, often with brown stains, strongly reflective. Umbones The representatives of the genus Thaiconcha are rather variable morphologically and mainly differed by shell shape, especially within T. callifera. Such shape plasticity could be driven by environmental conditions and hydrology which may influence the conchological characteristics [43][44][45] . Bineurus and Thaiconcha species were mainly collected from small and medium sized rivers, as well as at the confluence of small tributaries of the Mekong proper, usually with soft (clay, sandy, or somewhat silty) substrate. Less specimens were found in the mainstreams of the Mekong and Chao Phraya.
Our analyses demonstrated a well-resolved phylogeny with high and moderate supports for all available taxa under discussion. Three species new to science, i.e. Bineurus loeiensis sp. nov., Thaiconcha munelliptica sp. nov. and T. thaiensis sp. nov. were recognized and described under the framework of this study. The minimum level of genetic divergence was 2.4% (Table 1), what is generally appropriate for the species delimitation 41,46 . The lineage of Bineurus mouhotii from a Mekong's tributary around Nong Khai (catalog No. UF 507756) is rather divergent phylogenetically (based on the COI gene p-distance = 2.2%), and it may belong to a separate taxon (Fig. 5). However, the morphological similarity and the lack of sequences for 16S rRNA and 28S rRNA gene fragments preclude any final solution on the taxonomic status of this lineage (Supplementary Table 1).
Based on conchological analyses of the type specimens, Pseudodon ovalis and P. thomsoni have been transferred to the group of questionable taxa. Features such as winged and circular shell, small umbo, and weakly developed pseudocardinal teeth are not specific for Thaiconcha members. However, the type of Pseudodon thomsoni represents a young individual that complicates morphological studies, because small specimens of Thaiconcha spp. can often be winged. The taxonomic status of both species is yet to be confirmed based on DNA sequence data.
Distributional and biogeographic notes. The genera Bineurus and Thaiconcha are primarily restricted to the Mekong basin but T. thainesis sp. nov. has also been found in the Upper Chao Phraya basin. This disjunct population suggests a historical stream capture event between the Mekong and Upper Chao Phraya drainages. The hypothesis of past connections between upper parts of these large river basins was supported by a wide array of papers concerning mussels 7 and fishes 47,48 , as well as summarized in several biogeographic works 49,50 . Genetically related snakehead populations of Channa striata (Bloch, 1793) were observed in the Mekong (Chiang Rai) and Chao Phraya (Chiang Mai, Lumphun, Sukhothai and others in the northern and central parts of Thailand) 47 . The cyprinid fish Garra theunensis Kottelat, 1998, known from the Mekong basin, was found in the Upper Nan River belonging to the Chao Phraya drainage 48 . Among freshwater mussels, Lens contradens (Lea, 1838) represents an opposite example to Thaiconcha thaiensis as it primarily ranged throughout the Chao Phraya, with a few geographically disjunctive populations in the Mekong (Ing, Kok, and Loei) 7 .

Scientific Reports
| (2021) 11:8244 | https://doi.org/10.1038/s41598-021-87633-w www.nature.com/scientificreports/ The rearrangements of Southeast Asian river systems such as separations, connections, and stream captures repeatedly occurred in the past mainly caused by tectonic processes and sea level fluctuations [51][52][53] . The Wang River represents a part of the Northern Chao Phraya drainage, raising from the Phi Pan Nam Mountain Range and flowing through Chiang Rai and Lumpang to the Tak Province, where it joins the Ping River 54 . The tributaries starting from the northern slopes of this range belong to the Mekong basin, while tributaries of the western slopes fell to the Salween River 55 . The Phi Pan Nam Mountains serve as a geographic barrier separating these large river basins. Hence, a disjunctive range of Thaiconcha thainesis sp. nov. may reflect past rearrangements of the northern paleo-Chao Phraya and the paleo-Mekong. A small genetic divergence between the COI haplotypes of Thaiconcha thaiensis sp. nov. from the Wang River and a tributary of the Upper Mekong indicated that these drainage rearrangements could have been triggered by relatively recent geological events (e.g. Pliocene to Pleistocene) 56 . It was found that Lens contradens also shares shallow molecular divergence between its populations from the Mekong and Chao Phraya 7 .
In any case, a more extensive sampling is required for these remote water bodies to reconstruct the history of past drainage rearrangements and to exclude a possibility of recent human-mediated introduction events for given taxa.

Methods
Data sampling. The specimens of Bineurus and Thaiconcha species were collected from different water bodies in Thailand, Laos, and Cambodia (Supplementary Table 1 DNA sequence data and phylogenetic analyses. Molecular analysis was based on three molecular markers, i.e. the COI, 16S rRNA, and 28S rRNA gene fragments. It was carried out using PCR primers and laboratory protocols as described in our earlier papers 10,17,57 . The newly generated sequences were checked using a sequence alignment editor (BioEdit v. 7.2.5 58 ) and aligned through MUSCLE algorithm in MEGA7 59 . The alignments of three gene fragments were joined to the combined dataset using an online fasta sequence toolbox FaBox v. 1.41 60 .
Phylogenetic analyses were based on a five-partition dataset (3 codons of COI + 16S rRNA + 28S rRNA) of 147 sequences. Four representatives of the subfamily Gonideinae were used as outgroup, i.e. Gonidea angulata (Lea, 1838), Potomida littoralis (Cuvier, 1798), Leguminaia wheatleyi (Lea, 1862), and Lamprotula leaii (Gray in Griffith & Pidgeon, 1833) (Supplementary Table 1). The COI sequence of B. exilis (ICH-00734) was deleted from the dataset due to having some doubtful regions. The maximum likelihood (ML) analysis was performed through IQ-TREE (W-IQ-TREE) server 61 using an automatic identification of most appropriate evolutionary models 62 and an ultrafast bootstrap (UFBoot) algorithm with 5000 replicates 63 . Models of sequence evolution for each partition calculated through Model Finder 64 based on Bayesian Information Criterion (BIC) were following: 1st codon COI − F81 + I; 2nd codon COI − TPM2u + G + I; 3rd codon COI − TN + G + I; 28S − HKY + G + I; TIM2e + G. Bayesian inference analysis (BI) was carried out in MrBayes v. 3.2.7 65 with four runs, each with three heated (temperature = 0.1) and one cold Markov chain, during 25 million generations and sampling every 1000th generation. The first 15% of trees were discarded as burn-in. The calculation was performed at San Diego Supercomputer Center through the CIPRES Science Gateway 66 . A trace analysis tool (Tracer v. 1.7) was used to check a convergence of the MCMC chains to a stationary distribution 67 Table 3.  1 n/a n/a n/a