A New Contradens from Laos (Bivalvia: Unionidae: Contradentini)

Ecologica Montenegrina 24: 25-31 (2019) This journal is available online at: www.biotaxa.org/em

https://zoobank.org/urn:lsid:zoobank.org:pub:8008A4E5-12DF-4E47-85DA-7F835726C997

A new Contradens from Laos (Bivalvia: Unionidae: Contradentini)

EKATERINA S. KONOPLEVA1,2*, IVAN N. BOLOTOV1,2, VITALY M. SPITSYN1,2, ALEXANDER V. KONDAKOV1,2, MIKHAIL YU. GOFAROV1,2 & ILYA V. VIKHREV1,2

1Federal Center for Integrated Arctic Research, Russian Academy of Sciences, 163000, Arkhangelsk, Russia 2Northern Arctic Federal University, 163002, Arkhangelsk, Russia *Corresponding author: [email protected]

Received 2 October 2019 │ Accepted by V. Pešić: 10 November 2019 │ Published online 18 November 2019.

Abstract Here, we describe Contradens novoselovi sp. nov. (Bivalvia: Unionidae), a new freshwater mussel species from the Mekong Basin in Laos. Phylogenetic and morphological analyses reveal that the new taxon is closely related to Contradens comptus (Deshayes & Jullien, 1874) but can be distinguished from it by a smaller size, sub-ovate shell shape, tiny wrinkles near umbo, stronger pseudocardinal and lateral teeth, and fixed nucleotide substitutions. Contradens novoselovi sp. nov. is known only from the type locality in northwestern Laos and may represent a rare endemic species with restricted range.

Key words: Freshwater bivalve, Unionida, new species, Mekong River, Southeast Asia.

Introduction

Contradens Haas 1911 is a widely distributed freshwater mussel genus, whose range covers the Mekong, Chao Phraya and Mae Klong basins, rivers of the Malay Peninsula and the Greater Sunda Islands (Zieritz et al. 2016; Bolotov et al. 2017a, 2017b; Pfeiffer et al. 2018; Konopleva et al. 2019). During recent decades some researchers attempt to revise the genus Contradens by means of an integrative taxonomic approach (Bolotov et al. 2017b; Lopes-Lima et al. 2017; Jeratthitikul et al. 2019; Konopleva et al. 2019). Based on a growing body of integrative taxonomic research, several nominal taxa were transferred from other genera to Contradens, e.g. C. eximius (Lea, 1856), C. misellus (Morelet, 1865), C. pallegoixi (Sowerby, 1867), C. peninsularis (Simpson, 1900), and C. comptus (Deshayes & Jullien, 1874) (Pfeiffer et al. 2018; Konopleva et al. 2019). Furthermore, Contradens rolfbrandti Jeratthitikul & Panha, 2019 was recently described from Thailand (Jeratthitikul et al. 2019). In summary, the genus Contradens contains at least 13 valid species (Graf and Cummings 2019). However, there are still several taxa with an uncertain status, e.g. C. fulvaster (Drouet & Chaper, 1892) and C. subcircularis Brandt, 1974 requiring further investigations using a molecular approach. In the present study, we describe a new species of Contradens from northwestern Laos based on multi-locus molecular and morphological data. Until recently, this species was treated as a separate

Ecologica Montenegrina, 24, 2019, 25-31

A NEW CONTRADENS FROM LAOS phylogenetic lineage preliminary labelled as Сontradens sp. “Vieng Phou Kha” without a valid taxonomic name (Konopleva et al. 2019).

Materials and methods

The type specimens of Contradens novoselovi sp. nov. (N = 10) were studied in the malacological collection of the Russian Museum of Biodiversity Hotspots (RMBH thereafter), Federal Center for Integrated Arctic Research of the Russian Academy of Sciences, Arkhangelsk, Russia. This sample was collected from northwestern Laos (Fig. 1). A comparative analysis of shell morphology was carried out based on shell shape, the structures of pseudo-cardinal and lateral teeth, muscle attachment scars, and umbo position (Konopleva et al. 2019). The length, height, and width of the shell (all at the maximum diameter) of each specimen were measured using dial calipers (±0.1 mm). DNA extraction, PCR, sequencing and sequence alignment were performed as described in Bolotov et al. (2017b). New sequences of the mitochondrial cytochrome c oxidase subunit I (COI), small ribosomal RNA (16S rRNA), and the nuclear large ribosomal RNA (28S rRNA) gene fragments from three specimens of the new species were generated (Table 1). Phylogenetic analysis was based on the combined data set of Contradentini and Rectidentini (COI + 16S rRNA + 28S rRNA) with 86 unique haplotypes (Appendix 1). Sequences of Pseudodon species were used as outgroup. The best-fit evolutionary models applied to each partition were calculated by ModelFinder within the IQ-Tree web-server (Chernomor et al. 2016; Kalyaanamoorthy et al. 2017), based on the Bayesian Information Criterion (BIC). The models were selected as follows: F81 (1st codon of COI), GTR + G (2nd codon of COI), TNe + G (3rd codon of COI), TIM2 + G (16S rRNA), and TIM3 + I (28S rRNA). Bayesian inference analysis was performed in MrBayes v. 3.2.6 (Ronquist et al. 2012) at the San Diego Supercomputer Center through the CIPRES Science Gateway (Miller et al. 2010). Three runs, each with three heated (temperature = 0.2) and one cold Markov chain, were conducted for 15 million generations. Trees were sampled every 1000th generation. The first 25% of trees were discarded as burn-in (pre-convergence part). The effective sample size (ESS) value for each parameter sampled from the MCMC analysis was always recorded as >1100. Mean uncorrected COI p-distance to the sister species was calculated using MEGA 7 (Kumar et al., 2016). The molecular diagnosis of the new species was designed as described in Bolotov et al. (2017b, 2019).

Description of the new species

Family Unionidae Rafinesque, 1820 Subfamily Rectidentinae Modell, 1942 Tribe Contradentini Modell, 1942 Type genus: Contradens Haas, 1911 (by original designation) Genus Contradens Haas, 1911 Type species: Unio contradens Lea, 1838 (by original designation)

Contradens novoselovi sp. nov. Contradens sp. “Vieng Phou Kha” Konopleva et al. (2019): 2. Figs. 1-3, Table 1

Type material. Holotype RMBH biv 203_4: LAOS: Mekong River basin, a tributary of Nam Fa River near Vieng Phou Kha village, 20.682°N, 101.0794°E, 24.v.2016, Spitsyn and local villagers leg.; paratypes: same locality and date, 9 specimens [RMBH biv 202_2 and biv 203_5 are sequenced; biv 202_3, biv 202_4, biv 202_5, biv 202_6, biv 203_1, biv 203_2, biv 203_3], Spitsyn and local villagers leg.

Etymology. This new species is dedicated to Dr. Alexander P. Novoselov, a prominent Russian ichthyologist and our colleague.

KONOPLEVA ET AL.

Figure 1. Type locality of Contradens novoselovi sp. nov.: A) map of the type locality; B) general view of the type locality, a tributary of the Nam Fa River near Vieng Phou Kha village, Mekong Basin, Laos. (Map: M. Yu. Gofarov; photo: Vitaly M. Spitsyn).

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A NEW CONTRADENS FROM LAOS

Differential diagnosis. The new species is morphologically similar to C. comptus but can be distinguished from it by a smaller size, sub-ovate shell shape (vs. trapezoidal), broadly rounded anterior margin (vs. narrower and more elongated anterior margin), tiny wrinkles only near umbo (vs. along all dorsal margin and posterior slope), and strong lamellar pseudocardinal and lateral teeth.

Molecular diagnosis. Our multi-locus phylogeny (3 codons of COI + 16S rRNA + 28S rRNA) reveals that Contradens novoselovi sp. nov. forms a highly supported clade within the genus Contradens (BPP = 1.00; Fig. 3). Phylogenetically, the new species is most closely related to Contradens comptus (mean uncorrected COI p-distance = 3.6±0.7%) but can be distinguished from it by six fixed substitutions in the COI gene fragment as follows: 122 T, 164 C, 185 C, 257 A, 338 C, 434 A.

Figure 2. Shell and soft body morphology of Contradens novoselovi sp. nov.: A) lateral view of the shell: inner side of the left valve and outer side of the right valve [holotype RMBH biv 203_4]; B) lateral view of the shell: vice versa; C) dorsal view of the shell; D) soft body morphology (right valve and corresponding mantle tissue were removed) [paratype RMBH biv 202_2]. Scale bars = 10 mm. (Photos: Ekaterina S. Konopleva and Ilya V. Vikhrev).

Description. Small mussel: shell length 23.0–41.1 mm, shell height 13.1–25.1 mm, shell width 8.1–16.1 mm (Table 1). Shell sub-ovate, moderately thick and inflated, anterior margin broadly rounded, ventral margin straight or slightly curved, dorsal margin elevated posteriorly. Posterior margin covered by small wrinkles elongated from the umbo. Posterior slope gradual. Periostracum smooth, brown with darker areas. Nacre bluish-white. Umbo small, slightly elevated above hinge line, eroded. Pseudocardinal teeth strong, two on each valve. On the right valve, anterior tooth thin and lamellar, posterior tooth strong, indented. On the left valve, two strong lamellar indented teeth. Lateral teeth pronounced, thin, slightly curved, two teeth on the left valve, one tooth on the right valve. Anterior adductor muscle scars drop-like or oval-shaped, contiguous with pedal retractor scars. Posterior adductor muscle scars rounded and shallow. Mantle and its edges creamy. Gills creamy or light-yellow, elongated and slightly ribbed. Anterior margin of outer gills slightly longer and wider than that of inner gills. Foot medium-sized, light-brown. Small epithelial fold divides exhalant and inhalant siphons. Papillae of the inhalant siphon represent monodactylous outgrowths,

KONOPLEVA ET AL. decreasing in height in the direction of the ventral margin. Ascending lamella of the inner demibranchs attached to the visceral mass only anteriorly.

Table 1. Shell measurements and reference DNA sequences for the type series of Contradens novoselovi sp. nov.

Shell Shell Shell NCBI’s GenBank acc. nos. Status of Specimen Voucher length, height, width, specimen No.* COI 16S rRNA 28S rRNA mm mm mm Holotype RMBH biv 203_4 40.8 25.1 15.8 KY561632 KY561647 KY561664 Paratype RMBH biv 202_2 37.0 21.9 14.7 KY561630 KY561645 KY561662 Paratype RMBH biv 203_5 32.6 20.5 13.7 KY561631 KY561646 KY561663 Paratype RMBH biv 202_5 35.3 20.5 14.1 n/a n/a n/a Paratype RMBH biv 202_3 29.2 16.6 10.2 n/a n/a n/a Paratype RMBH biv 202_4 26.5 15.2 9.2 n/a n/a n/a Paratype RMBH biv 202_6 23.0 13.1 8.1 n/a n/a n/a Paratype RMBH biv 203_1 35.7 21.6 13.8 n/a n/a n/a Paratype RMBH biv 203_2 39.7 24.7 15.3 n/a n/a n/a Paratype RMBH biv 203_3 41.1 25.0 16.1 n/a n/a n/a

*RMBH – Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia. n/a – not available.

Distribution. This species is known only from its type locality in northwestern Laos (Fig. 1).

Habitat. The new species was recorded in a mixed substrate of tree roots, silty and clay, near the banks on the slow flowing sections of the stream. Bineurus mouhotii (Lea, 1863) (Bivalvia: Unionidae: Pseudodontini) was found in sympatry with the new species.

Discussion Although Southeast Asian fauna of freshwater bivalves has been subject of intense research efforts during the last five years (e.g. Zieritz et al. 2016; Bolotov et al. 2017a, 2017b, 2019; Lopes-Lima et al. 2017; Jeratthitikul et al. 2019; Konopleva et al. 2019), the fauna of Laos is still poorly known (Zieritz et al. 2018). For example, modern information on the population status and ecology is only available for a single species, Gibbosula laosensis (Lea, 1863) (Bivalvia: Margaritiferidae) (Bolotov et al. 2014). Nevertheless, molecular data on freshwater mussels from Laos is presented in a few works (Pfeiffer and Graf 2015; Bolotov et al. 2017a, 2017b; Lopes-Lima et al. 2017) but the taxonomic placement of those sequences is still uncertain. Here, a freshwater mussel sample from Laos was recovered as the new species, Contradens novoselovi sp. nov. Phylogenetic analyses, diagnostic substitutions and the level of genetic divergence clearly demonstrate that Contradens novoselovi sp. nov. represents a separate lineage of this genus which is sister to C. comptus. The type series of Contradens novoselovi sp. nov. was collected from a single locality in northwestern Laos, what may point to a rather restricted distribution of this species. However, this species requires further surveys for the evaluation of its distribution and conservation status. Unfortunately, Northern Laos is under high anthropogenic pressure leading to habitat degradation, river pollution and damming, fish and mussel overharvesting, and intense soil erosion (Bolotov et al. 2014). Therefore, Contradens novoselovi sp. nov. may be under severe threat as a prospective local endemic species with restricted range and need a long-term population monitoring and conservation efforts. The Upper Mekong Basin in northern Laos is a well-known biodiversity hotspot, especially for birds, butterflies and fishes (Pierre 2004; Tordoff et al. 2011). However, the levels of species richness and endemism of freshwater mussels in northern Laos are virtually unknown due to the absence of reliable data (Zieritz et al. 2018). The Annamite Range harboring numerous endemic animal and plant species (Tordoff et al. 2011) seems to be a potential high-priority area for future field surveys of freshwater mussel diversity in Laos.

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Figure 3. Phylogenetic tree recovered from Bayesian inference analysis of the combined data set of mitochondrial and nuclear sequences of the Rectidentini (collapsed) and Contradentini (five partitions: three codons of COI + 16S rRNA + 28S rRNA). Pseudodon species were used as outgroup (not shown). Scale bar indicates the branch lengths. Black numbers near nodes are Bayesian Posterior Probabilities (BPP); asterisk (*) indicates high BPP values ≥ 0.95. Contradens novoselovi sp. nov. is colored red, and its sister species, C. comptus is colored blue. (Photos: Ekaterina S. Konopleva).

Acknowledgements This study was supported by the Ministry of Science and Higher Education of Russia, the Ministry of Europe and Foreign Affairs of France (MEAE), and the Ministry of Higher Education, Research and Innovation of France (MESRI) under project No. 05.616.21.0114 of the Hubert Curien Partnership (PHC) for the Franco-Russian Cooperation for Science and Technology (PHC Kolmogorov 2019). The work was carried out with the support and equipment of the Russian Museum of Biodiversity Hotspots, N. Laverov Federal Center for Integrated Arctic Research, Russian Academy of Sciences, an Unique scientific setting (www.ckp-rf.ru/usu/352654). We are very grateful to Dr. Manuel Lopes-Lima and Dr. Maxim V. Vinarski for their suggestions and valuable comments which helped to improve earlier versions of this paper.

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