www.nature.com/scientificreports OPEN Received: 31 July 2018 Accepted: 18 October 2018 Published: xx xx xxxx Discovery of Novaculina myanmarensis sp. nov. (Bivalvia: Pharidae: Pharellinae) closes the freshwater razor clams range disjunction in Southeast Asia Ivan N. Bolotov 1,2, Ilya V. Vikhrev1,2, Manuel Lopes-Lima3,4, Zau Lunn5, Nyein Chan5, Than Win 6, Olga V. Aksenova1,2, Mikhail Yu. Gofarov1,2, Alexander V. Kondakov1,2, Ekaterina S. Konopleva1,2 & Sakboworn Tumpeesuwan7 The razor clam genus Novaculina represents an example of a marine-derived, secondary freshwater group. It was thought to comprise three species: N. gangetica (Ganges and smaller basins in Bangladesh and northwestern Myanmar), N. siamensis (Bang Pakong and Pasak rivers in Thailand and Mekong River in Vietnam), and N. chinensis (lower Yangtze River, China). Here we describe Novaculina myanmarensis sp. nov., an additional species from the Ayeyarwady and Salween basins representing a divergent lineage that appears to be sister to N. gangetica. This new record closes a Novaculina range disjunction between northwestern Myanmar and Thailand. The populations of this novel species share a shallow molecular divergence from each other indicating potential dispersal events between the two distant freshwater basins during the Late Pleistocene. Our ancestral area modeling suggests that the MRCA of Novaculina crown group was a salt-tolerant freshwater species. The recent Novaculina species most likely originated via allopatric speciation. Our findings highlight that generalist estuarine species could have played the role as a source for bivalve expansions into freshwater and that western Indochina is a separate biogeographic subregion, which is clearly distinct from India. A new synonymy is proposed as follows: Pharellinae Stoliczka, 1870 = Novaculininae Ghosh, 1920 syn. nov. Freshwater bivalves are a taxonomically diverse ecological group, which includes representatives of at least 19 families1,2. Unionida is the only strictly freshwater order among Bivalvia representing a monophyletic entity with six families, i.e. Unionidae, Margaritiferidae, Etheriidae, Iridinidae, and Mulleriidae2,3. However, several other orders have small to large radiations in freshwater, e.g. Venerida, which includes families such as Cyrenidae, Dreissenidae, Sphaeriidae, and Pharidae1. Pharidae is a primary marine family4, but it contains a single typically freshwater genus, Novaculina that was thought to include three species: N. gangetica, N. siamensis, and N. chinensis. This genus belongs to the subfamily Novaculininae, which also comprises a second genus with two species, Sinonovacula constricta5 and S. mollis. Annandale6 suggested that Novaculina is a relict marine-derived freshwater lineage, and this hypothesis has recently been supported by multi-locus phylogenetic analyses4. Novaculina gangetica was considered an endemic species of the Ganges River system in India and Bangladesh7,8, but it was recently discovered in the Kaladan and Lemro rivers in northwestern Myanmar 4. Novaculina siamensis was known from the Bang Pakong and Pasak rivers in Thailand9,10, but Sayenko et al.11 1 Northern Arctic Federal University, Arkhangelsk, Russia. 2Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia. 3CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Matosinhos, Portugal. 4CIBIO/InBIO - Research Center in Biodiversity and Genetic Resources, University of Porto, Vairão, Portugal. 5Fauna & Flora International – Myanmar Program, Yangon, Myanmar. 6Department of Zoology, Hpa-An University, Hpa-An, Kayin State, Myanmar. 7Department of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham, Thailand. Correspondence and requests for materials should be addressed to I.N.B. (email: inepras@yandex.ru) SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 1 www.nature.com/scientificreports/ Figure 1. Distribution range of the genus Novaculina Benson, 1830 based on available georeferenced records (Supplementary Table 2). The map was created using ESRI ArcGIS 10 software (www.esri.com/arcgis); the topographic base of the map was created with Natural Earth Free Vector and Raster Map Data (www. naturalearthdata.com) and Global Self-consistent Hierarchical High-resolution Geography, GSHHG (http:// www.soest.hawaii.edu/wessel/gshhg/). (Map: Mikhail Yu. Gofarov). found this species in the Mekong Delta in Vietnam. Finally, Novaculina chinensis was described from the Taihu Lake, a large floodplain water body in the lower Yangtze River basin12, and later reported from two additional lakes and a river in the same region13–15. A large Novaculina range disjunction was situated in central and eastern Myanmar (Ayeyarwady and Salween river basins) (Fig. 1). However, we have discovered an additional species in this genus during a recent field trip to Myanmar. The present study aims to describe a new species, Novaculina myanmarensis sp. nov., to provide a brief taxonomic overview of all Novaculina species, and to discuss the putative origin of freshwater lineages in estuarine bivalves within a broad phylogenetic and biogeographic context. Results Multi-locus phylogeny of the Pharidae. Our multi-locus phylogeny (five partitions: three codons of COI, 16S rRNA, and 28S rRNA) indicates that Novaculina myanmarensis sp. nov. and N. gangetica represent phylogenetically distant lineages belonging to a separate, fully-supported subclade (Fig. 2). The mean p-distances (±standard error estimates) between the new species and Novaculina gangetica are as follows: COI = 8.0 ± 1.0%, and 16S rRNA = 1.9 ± 0.6%. There is a single nucleotide substitution in the nuclear 28S rRNA gene between these species. The Novaculina subclade is fully supported by both Bayesian and maximum likelihood models, and it appears to be closely related to another Pharidae subclade, which includes representatives of Sinonovacula, Pharella javanica, and Cultellus attenuatus. Pharella javanica belongs to the Sinonovacula subclade, and this pattern is strongly supported by our models, indicating the synonymy of Pharellinae Stoliczka, 1870 and Novaculininae Ghosh, 1920. Divergence times. Our fossil-calibrated phylogeny suggests that the crown group of the Pharidae has been originated in the mid-Cretaceous (mean age = 103 Ma, 95% HPD 100–113 Ma) (Fig. 2). The Pharellinae (=Novaculininae) clade most likely originated in the Paleocene (mean age = 61 Ma, 95% HPD 48–77 Ma). The origin of the Novaculina crown group placed in the Miocene (mean age = 8 Ma, 95% HPD 5–12 Ma). Finally, the crown group of Pharella + Sinonovacula clade most likely originated in the Oligocene (mean age = 27 Ma, 95% HPD 19–36 Ma). Ancestral areas. The ancestral area modeling indicates that the most recent common ancestor (MRCA) of the Pharellinae (=Novaculininae) clade was an estuarine species (probability 92.1% by integrative model, 100% by S-DIVA model, 87.5% by DEC model, and 88.8% by S-DEC model). The MRCA of the Novaculina crown group was most likely a salt-tolerant freshwater species like its recent descendants (probability 56.9% by SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 2 www.nature.com/scientificreports/ Figure 2. Fossil-calibrated chronogram of the Pharidae computed under a lognormal relaxed clock model and a Yule process speciation implemented in BEAST 1.8.4 and obtained for the complete data set of mitochondrial and nuclear sequences (five partitions: three codons of COI + 16S rRNA + 28S rRNA). Bars indicate 95% confidence intervals of the estimated divergence times between lineages (Ma). Black numbers near nodes are BPP values of BEAST model/BPP values of MrBayes model/BS of RAxML model. Blue numbers near nodes are mean ages (Ma). Stratigraphic chart according to the International Commission on Stratigraphy, 2018. The node pies indicate ancestral area reconstructions (probability of each area combination) in accordance with the combined biogeographic model (combination of the S-DIVA + DEC + S-DEC models). Age values for weakly supported nodes are not shown. Species Locality Myanmar: Lemro River N. gangetica Benson, 1830 Myanmar: Kaladan River N. myanmarensis sp. nov. Myanmar: Donthami River Myanmar: Ayeyarwady River Acc. numbers of reference sequences Voucher no. COI haplotype code COI 16S rRNA 28S rRNA Reference biv_150_1 L1 MF958986 MF958997 MF959011 4 biv_150_2 L2 MF958987 MF958998 MF959012 4 biv_150_3 K1 MF958988 MF958999 MF959013 4 biv_151_1 K1 MF958989 MF959000 MF959014 4 biv_151_2 K2 MF958990 MF959001 MF959015 4 biv_151_3 K3 MF958991 MF959002 MF959016 4 biv_369_1 D1 MH670876 MH670886 MH664920 This study biv_369_2 D2 MH670877 MH670887 MH664921 This study biv_369_3 D3 MH670878 MH670888 MH664922 This study biv_369_4 D4 MH670879 MH670889 MH664923 This study biv_369_5 D3 MH670880 MH670890 MH664924 This study biv_420_1 A1 MH670881 MH670891 MH664925 This study biv_420_3 A1 MH670882 MH670892 MH664926 This study biv_420_4 A1 MH670883 MH670893 MH664927 This study biv_420_5 A1 MH670884 MH670894 MH664928 This study biv_420_6 A1 MH670885 MH670895 MH664929 This study Table 1. List of Novaculina (Bivalvia: Pharidae) sequences used in this study. integrative model, 85.0% by DEC, and 85.6% by S-DEC model), although S-DIVA model assumes that it might be an estuarine species (probability 100%). Phylogeography. A unique COI haplotype of Novaculina myanmarensis sp. nov. has been found in the Ayeyarwady River, and four unique COI haplotypes were recorded in the Salween Basin (Table 1). The mean COI p-distance (±standard error estimates) between these groups is 0.3 ± 0.1%. Almost all specimens from both rivers share a single 16S rRNA haplotype, with exception of a specimen from the Salween Basin having another haplotype with a single nucleotide substitution (239 G). The 28S rRNA sequences were identical among the samples. SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 3 www.nature.com/scientificreports/ Figure 3. Shells of Novaculina spp. (A) N. myanmarensis sp. nov., holotype RMBH no. biv0420_8, Ayeyarwady River near Thin Baw Kone village, Pakokku Region, Myanmar. (B) N. myanmarensis sp. nov., paratype RMBH no. biv0369_3, Donthami River, Salween River basin, Myanmar. (C) N. gangetica, RMBH no. biv0150_24, Lemro River, Myanmar. Scale bar = 2 cm. (Photos: Ekaterina S. Konopleva). Taxonomy Family Pharidae H. Adams & A. Adams, 1856 Subfamily Pharellinae Stoliczka, 1870 Type genus: Pharella Gray, 1854 = Novaculininae Ghosh, 1920 syn. nov. Type genus: Novaculina Benson, 1830 Genus Novaculina Benson, 1830 Type species: Novaculina gangetica Benson, 1830 (by monotypy). Novaculina myanmarensis sp. nov. Type locality. 95.0591°E]. Figures 3A,B, 4A, 5 and 6A, Tables 1 and 2. Myanmar: Ayeyarwady River, near Thin Baw Kone village (Pakokku Region) [21.3146°N, Holotype RMBH Biv420_8. Myanmar: Ayeyarwady River, near Thin Baw Kone village (Pakokku Region), clay bottom near the river shore, 21.3146°N, 95.0591°E, 2 March 2018, Bolotov, Vikhrev, Zau Lunn, Nyein Chan, and locals leg. Paratypes. Myanmar: Type locality, same label data, 47 specimens [RMBH Biv0420]; downstream of Donthami River, hard gravel-clay bottom, 16.6935°N, 97.5819°E, 11 February 2018, 5 specimens, local collector leg. [RMBH Biv0369]; Magway Division, Ayeyarwady River, large sandbar 1/2 mi SE of Nyaung-U, 21.2066°N, 94.9062°E, November 2009, 3 specimens, C. N. Piotrowski leg. [CAS 180843]; Ayeyarwady River, near Minbu, 20.1911°N, 94.8788°E, 29 April 2018, 4 specimens, Nyein Chan leg. [FFI]. Etymology. The name of this species is derived from the country of Myanmar. Conchological diagnosis. Shell length 20.5–46.5 mm, shell height 7.9–17.5 mm, shell width 4.5–13.3 mm (N = 54, Table 2). This species has an elongated shell, and is closely related to N. gangetica and N. chinensis, but it can be distinguished from these taxa by a more rectangular shell shape with truncated posterior end (vs more oval shell shape with rounded posterior end). Molecular diagnosis. The new species differs from N. gangetica by the fixed nucleotide substitutions: 49 substitutions in the COI gene fragment [29 G, 38 A, 53 G, 59 A, 92 A, 128 C, 134 C, 161 G, 170 T, 173 A, 182 A, 185 A, SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 4 www.nature.com/scientificreports/ Figure 4. Soft body morphology (right valve and corresponding mantle tissue were removed) and hinge structure of Novaculina spp. (A) N. myanmarensis sp. nov. (holotype RMBH biv0420_8), including (a1) soft body (scale bar = 10 mm), (a2) pseudocardinal teeth on the left valve, and (a3) pseudocardinal teeth on the right valve (scale bars = 0.4 mm). (B) N. gangetica (RMBH biv0150_24), including (b1) soft body (scale bar = 10 mm), (b2) pseudocardinal teeth on the left valve, and (b3) pseudocardinal teeth on the right valve (scale bars = 1 mm). (Photos: Ekaterina S. Konopleva and Ilya V. Vikhrev). 197 A, 200 A, 212 G, 213 C, 215 T, 230 A, 243 T, 245 A, 254 A, 299 A, 302 A, 308 T, 311 C, 314 G, 329 G, 347 T, 362 G, 371 G, 377 C, 380 A, 404 G, 413 G, 464 T, 467 G, 470 G, 485 G, 491 G, 497 T, 521 G, 545 A, 551 G, 560 G, 569 A, 572 A, 590 T, 617 C, 644 A], 8 substitutions in the 16S rRNA gene fragment [24 G, 74 G, 237 A, 239 G, 242 A, 287 G, 288 T, 305 A], and one substitution in the nuclear 28S rRNA gene fragment [429 A]. Description. Shell shape from rectangular to oval-elongated, dorsal and ventral margin are almost parallel to each other (Fig. 3A,B). Shell thin or moderately thick, not inflated. Periostracum from light yellow to brown; nacre whitish, shining. Umbo more or less prominent, in the first half of the shell. Pseudocardinal teeth small and distant from each other, two on the right valve and three on the left valve. Anterior muscle scar pyriform, posterior muscle scar shallow and with rounded shape. The mantle and its edges colored in light yellow. The gills elongated and ribbed (Fig. 4A). The anterior margin of inner gills slightly longer and wider than the outer gills. Foot stumpy, slightly dilated at the end and somewhat truncated; branchial siphon stouter than the anal one, almost the same length, surface of siphon ribbed (Figs 4A and 6A). Intraspecific conchological variability. Specimens from the Donthami and Ayeyarwady rivers are rather different from each other conchologically (compare Fig. 3A,B). The first ones are stronger and thicker, have more truncated posterior end, slightly concave dorsal margin, more developed umbo and hinge. The specimens from the Ayeyarwady River are characterized by more elliptical and very thin shell with light-yellow and smoother periostracum. At first glance, these conchological differences may reflect an environment-induced variability, because the populations were recorded from sites with different bottom substrate (i.e. soft clay substrate in the Ayeyarwady River vs hard gravel-clay substrate in the Donthami River). SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 5 www.nature.com/scientificreports/ Figure 5. Type locality and habitats of Novaculina myanmarensis sp. nov. (A) Habitat in the downstream of the Donthami River, 16.6935°N, 97.5819°E. (B) Type locality: the middle section of the Ayeyarwady River near Thin Baw Kone village (Pakokku Region), 21.3146°N, 95.0591°E. (C) Сlay bottom substrate with clam burrows, the type locality. (D) A clam in its burrow, the type locality. (Photos: Ilya V. Vikhrev). Figure 6. Live Novaculina clams with protruded siphons. (A) N. myanmarensis sp. nov., Donthami River. (B) N. gangetica, Lemro River. Scale bar = 10 mm. (Photos: Ilya V. Vikhrev). Distribution. Donthami (Salween River basin) and Ayeyarwady rivers in Myanmar. Habitat. Downstream and middle section of large rivers, in fresh water (Fig. 5A,B). This species inhabits gravel-clay and clay bottom, in which it makes deep vertical holes (Fig. 5C,D). Comments. Local villagers harvest N. myanmarensis sp. nov. from the downstream section of the Donthami River (food for consumption). In contrast, this species seems to be unutilized in the Ayeyarwady River. SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 6 www.nature.com/scientificreports/ Locality Status of specimen Voucher no.* Shell length, mm Shell height, mm Shell width, mm Myanmar: Ayeyarwady River, Pakokku Region, near Thin Baw Kone village Holotype 40.6 Myanmar: downstream of Donthami River Biv0420_8 15.2 9.8 Paratype Biv0420_1 34.5 12.8 7.9 Paratype Biv0420_2 38.1 13.9 8.7 Paratype Biv0420_3 36.4 12.9 8.7 Paratype Biv0420_4 34.9 11.8 7.8 Paratype Biv0420_5 34.1 12.3 7.7 Paratype Biv0420_6 38.5 13.9 8.5 Paratype Biv0420_7 33.7 12.2 7.4 Paratype Biv0420_9 40.6 15.2 9.8 Paratype Biv0420_10 30.3 11.1 6.4 Paratype Biv0420_11 31.4 11.9 7.0 Paratype Biv0420_12 35.2 12.4 8.1 Paratype Biv0420_13 27.6 10.0 5.8 Paratype Biv0420_14 35.4 12.9 7.9 Paratype Biv0420_15 33.5 12.2 7.3 Paratype Biv0420_16 27.9 10.9 6.1 Paratype Biv0420_17 30.3 11.0 6.7 Paratype Biv0420_18 36.3 12.5 8.0 Paratype Biv0420_19 29.6 11.0 7.0 Paratype Biv0420_20 36.4 12.2 7.5 Paratype Biv0420_21 30.9 11.3 6.5 Paratype Biv0420_22 34.1 12.7 8.0 Paratype Biv0420_23 33.4 12.0 7.0 Paratype Biv0420_24 33.1 11.4 7.1 Paratype Biv0420_25 29.8 10.5 6.7 Paratype Biv0420_26 30.2 11.0 6.5 Paratype Biv0420_27 29.1 10.8 6.8 Paratype Biv0420_28 32.3 11.8 7.0 Paratype Biv0420_29 25.4 9.0 6.0 Paratype Biv0420_30 28.3 10.1 6.3 Paratype Biv0420_31 26.4 9.7 6.2 Paratype Biv0420_32 27.1 10.5 6.1 Paratype Biv0420_33 24.3 9.3 5.4 Paratype Biv0420_34 24.1 9.5 4.9 Paratype Biv0420_35 26.2 10.2 6.3 Paratype Biv0420_36 21.6 8.1 4.9 Paratype Biv0420_37 28.5 11.6 5.6 Paratype Biv0420_38 20.5 7.9 4.5 Paratype Biv0420_39 25.0 9.5 5.8 Paratype Biv0420_40 38.1 14.2 9.0 Paratype Biv0420_41 40.4 15.0 9.3 Paratype Biv0420_42 35.6 12.8 7.9 Paratype Biv0420_43 34.3 12.5 7.6 Paratype Biv0420_44 33.5 11.4 7.1 Paratype Biv0420_45 33.1 11.4 6.9 Paratype Biv0420_46 32.3 12.0 7.5 Paratype Biv0420_47 36.8 12.7 7.9 Paratype Biv0420_48 26.5 10.0 5.6 Paratype Biv0369_1 46.5 17.0 12.0 Paratype Biv0369_2 41.9 16.2 11.0 Paratype Biv0369_3 43.9 17.0 13.3 Paratype Biv0369_4 43.3 17.3 12.9 Paratype Biv0369_5 42.6 17.5 12.3 Mean ± s.e.m. 32.91 ± 0.81 12.12 ± 0.31 7.58 ± 0.27 Table 2. Measurements of the type series of Novaculina myanmarensis sp. nov. *RMBH – Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research, Russian Academy of Sciences (Arkhangelsk, Russia). SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 7 www.nature.com/scientificreports/ Novaculina gangetica Benson, 1830. Novaculina gangetica Benson16: p. 63; Subba Rao7: p. 223; Graf2: p. 152. Figures 3C, 4B, and 6B. Type locality. Ganges, Calcutta [India, approximately 22.6°N, 88.3°E]. Type series. The University Museum of Zoology, Cambridge, UK [UMZC I.102125: eleven syntypes from the Robert McAndrew collection, labeled “Bens. Coll., Ganges, Calcutta”]. Conchological diagnosis. Shell length 28.1–39.7 mm, shell height 12.9–17.5 mm, shell width 8.1–12.7 mm (N = 24). This species has an elongated shell, and is closely related to N. myanmarensis sp. nov. and N. chinensis. It could be distinguished from N. myanmarensis sp. nov. by a more ovate shell shape with rounded posterior end (vs more rectangular shell shape with truncated posterior end). N. gangetica differs from N. chinensis by somewhat higher and shorter shell with slightly convex ventral margin (vs more elongated shell with straight ventral margin). Intraspecific conchological variability. posterior end4. Some specimens have somewhat trapezoidal shell, with slightly expanded Distribution. Ganges River and its tributaries in India and Bangladesh ranging from the delta to at least 1,500 km upstream7,8,17,18, Buriganga and Pashur river systems in Bangladesh19,20, and Kaladan and Lemro rivers in Myanmar4. Habitat. The species inhabits downstream and middle sections of large rivers, in fresh or slightly brackish water4,8. N. gangetica prefers clay bottom, in which it makes cylindrical holes4,16,17, but it was also recorded in soft sand and silt bottom8. In the Kaladan River, this species also inhabits submerged rocks, in which it was recorded from the vacant borings of Lignopholas fluminalis, filled with clay4. Benson16 noted that this species rarely occurs from holes in rocks in the Jumna and Gumti rivers, and that the specimens from such a habitat have an asymmetrical shell. Comments. Local villagers harvest N. gangetica from the Kaladan and Lemro rivers in Myanmar (food for consumption and local market trade), but it seems to be unutilized in India7. Novaculina siamensis Morlet, 1889. Novaculina siamensis Morlet10: p. 172, 198; Brandt9: p. 303; Graf2: p. 152; Sayenko et al.11: p. 182. Type locality. Marais de Chantakam (Siam)10 [M. Chantakam, rainfall station on a tributary of the Phra Prong River21,22, Thailand, approximately 14.0°N, 102.0°E]. Type series. Whereabouts unknown. Morlet’s collection of shells from Indochina went to P. Dautzenberg and is in the Royal Belgian Institute of Natural History, Brussels, Belgium. However, the type series of N. siamensis seems to be lacking in this collection (Thierry Backeljau, pers. comm., 2018). Conchological diagnosis. Shell length 30–38 mm, shell height 13–18 mm, shell width 10–15 mm9. This species could be distinguished from all the other Novaculina taxa by its much shorter and higher shell, less prominent umbo, clear sculpture with concentric growth lines, and dark yellow periostracum. Intraspecific conchological variability: Some shells in the Mekong Delta population are asymmetrical and torsed11. Distribution. Bang Pakong and Pa Sak River basins in Thailand9,10, and the Mekong Delta in Vietnam11. We assume that a population of N. cf. siamensis from a tidal creek in the Trang Province of Thailand23 belongs to another species, because this creek empties into the Andaman Sea. Habitat. Upstream section of medium-sized rivers, in fresh water, probably on clay bottom substrate. However, it was found in a slightly brackish, tidal channel in the Mekong Delta11. Comments. This species seems to be unutilized in Thailand. Novaculina chinensis Liu & Zhang, 1979. 25 2 Novaculina chinensis Liu & Zhang12: p. 356; Qin24: p. 305; He 26 & Zhuang : p. 128; Graf : p. 152; Chen et al. : p. 4. Type locality. Wuxi, Jiangsu Province [Lake Taihu, approximately 31.4402°N, 120.3143°E]12. Type series. National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing, China [holotype NZMC KS 747703, paratypes NZMC FM00855]25. Conchological diagnosis. Shell length 34–46 mm, shell height 11–16 mm, shell width 8–10 mm12. This species is closely related to N. myanmarensis sp. nov. and N. gangetica by an elongated shell shape, but could be distinguished from these species by more prominent, somewhat acute umbo. SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 8 www.nature.com/scientificreports/ Intraspecific conchological variability. Not known. Distribution. Downstream of the Yangtze River, China, most records from Lake Taihu12,24,27, Lake Hongze14, and Lake Chaohu13. Habitat. Large floodplain lakes, in fresh water12–14,24,27. A single record from the Shangqing River15. Comments. This species seems to be unutilized in China. A parasitic mite species, Unionicola imamurai Hevers, 1978, has been reported from N. chinensis15. Discussion Taxonomic conclusions. Our results reveal that the genus Novaculina comprises four species: N. gangetica from India, Bangladesh and northwestern Myanmar, N. myanmarensis sp. nov. from central and eastern Myanmar, N. siamensis from Thailand and southern Vietnam, and N. chinensis from southeastern China (Fig. 1). An additional species, Novaculina andamanensis, was described from the Andaman Islands but without a precise locality28 (holotype no. ZSI M4060/1, paratype no. ZSI 20765/4 [Subba-Rao7 considered the latter specimen to be the holotype], malacological collection of the Zoological Survey of India, Kolkata, India29). However, this species has been considered a junior synonym of the marine bivalve species Azorinus coarctatus29,30. We agree with that taxonomic conclusion, because, according to the original description and figure of the type specimen28, this species has somewhat trapezoidal shell with concave ventral margin as seen in Azorinus coarctatus. Records of Novaculina are still lacking from the downstream sections of several large and medium-sized Southeast and East Asian rivers such as the Pearl River in China and Red River in Vietnam. Taking into account a poor knowledge of freshwater fauna in these basins, further records of new Novaculina taxa could not be ruled out. An occasional record of Novaculina cf. siamensis from a small creek in southern Thailand23 suggests that the members of this genus could also establish permanent populations in small-sized freshwater basins, the fauna of which is almost unknown. Two pharid genera, i.e. Sinonovacula and Novaculina, were hitherto placed in the Novaculininae5. This subfamily was established for Novaculina gangetica31, but, later, Sinonovacula constricta had also been placed within it32. However, we found that Pharella javanica belongs to a well-supported clade together with Sinonovacula and Novaculina species (Fig. 2), as it was also shown by another study4. According to this, we propose the Novaculininae as a junior synonym of the Pharellinae. A close similarity between Pharella and Sinonovacula has also been recorded by a functional morphology, particularly in the presence of crescentric anterior and posterior pedal protractor muscles in both taxa5. Biogeographic implications. Discovery of the new Novaculina species from the Salween and Ayeyarwady river drainage basins in Myanmar indicates that the range of this genus is rather continuous and extends along the continental margin of Asia from the Ganges River to the Yangtze River (Fig. 1). Unfortunately, the phylogenetic affinities of the two eastern species, i.e. Novaculina siamensis and N. chinensis, are still unknown because of the lack of available molecular data. However, they may represent a divergent clade, because the Thai–Malay Peninsula is a significant biogeographic barrier to longitudinal dispersal of aquatic animals33. This barrier could have existed during most of the Cenozoic Epoch34, although it may have occasionally been incised, but not breached, at the Isthmus of Kra33. Our statistical biogeographic modeling strongly supports the hypothesis4,6,18 that the genus Novaculina is a relict, marine-derived freshwater clade. Similar examples of such secondary freshwater lineages are known among a variety of other taxa, e.g. in fishes, gastropods, polychaetes, and crustaceans4,35. The high level of molecular divergence between the two western species, i.e. Novaculina gangetica and N. myanmarensis sp. nov., supports a new freshwater biogeographic division of Southeast Asia that has been developed on the basis of unionid mussel research36–38. According to this model, the drainages of the Arakan coast of Myanmar, the Ayeyarwady, Bago, Sittaung, and Bilin river basins, and east to the Salween River and drainages of southern Myanmar belong to the Western Indochina Subregion of the Oriental Region38. This subregion has high levels of faunal endemism and is separated well from the Indian and Sundaland subregions38,39. However, our new study reveals that the northern drainages of the Aracan coast such as the Kaladan and Lemro rivers seem to be a rather marginal part of the Indian Subregion that has already been shown by another research4. Anyway, the presence of sister but highly divergent species in the Ganges and Ayeyarwady rivers even in salt-tolerant freshwater taxa such as Novaculina strongly indicates that these basins were separated at least since the Miocene (Fig. 2). In contrast, a shallow genetic divergence between populations of Novaculina myanmarensis sp. nov. from the Salween and Ayeyarwady river basins in Myanmar suggests that there were relatively recent (i.e. Late Pleistocene) dispersal events in this species among the downstream sections of these large river drainages. The phylogeography of freshwater mussels (Unionidae) partly reflects this pattern, e.g. the distribution range of Leoparreysia tavoyensis crosses numerous freshwater drainages from the Tavoy (north of the Thai–Malay Peninsula) to the Ayeyarwady36,37. However, the majority of unionid species in Myanmar appear to be restricted to certain drainage basins or their tributaries36,37. There are several widespread salt-tolerant estuarine and freshwater species, e.g. a polychaete, Neanthes meggitti (Nereididae), and a pholadid bivalve, Lignopholas fluminalis (Pholadidae)4, the range of which encompasses the downstream sections of the Ganges and Ayeyarwady rivers. Such taxa were described from the delta of Ayeyarwady and later have been discovered from the Ganges, or vice versa, and they are ecologically associated with the typical Novaculina habitats4. The discovery of a new Novaculina species in Myanmar indicates that such taxa with broad distribution may actually represent cryptic species complexes, although this preliminary assumption is in need of future molecular research with extensive field surveys in South and Southeast Asia. SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 9 www.nature.com/scientificreports/ Methods Data sampling and mapping. The samples of Novaculina myanmarensis sp. nov. were collected from two localities during a field trip to Myanmar in 2018. Additional materials were investigated in the collections of the Fauna & Flora International – Myanmar Program [FFI] (Yangon, Myanmar) and California Academy of Sciences [CAS] (San Francisco, USA). We processed new COI, 16S rRNA and 28S rRNA sequences from ten specimens of Novaculina myanmarensis sp. nov. (Table 1) using the standard approach as described in our previous work4. Sequences of Novaculina gangetica and other Pharidae taxa were obtained from GenBank (Table 1 and Supplementary Table 1). We collected a dataset of reliable georeferenced records of Novaculina species from published sources and museum specimens (Supplementary Table 2). The map was created using ESRI ArcGIS 10 software (www.esri.com/arcgis). Morphological study. The samples were studied using a stereomicroscope (Leica M165C, Leica Microsystems, Germany). The comparative analysis of taxa was performed according to the standard conchological patterns, i.e. the shape of shell, hinge structure, muscle attachment scars, and position of umbo. Sequence alignment, saturation analyses and congruence of phylogenetic signals. We aligned each gene data set using the MUSCLE algorithm in MEGA640. We performed the saturation test of Xia et al.41 with DAMBE v. 5.3.10842, but we found no evidence of substitution saturation (P < 0.001). A partition homogeneity test was calculated in PAUP* v. 4.0a151 to confirm the congruence of phylogenetic signals among sequence data sets43. This test revealed that the signals among the data sets are consistent (P > 0.1 in all the combinations). Phylogenetic analyses. We computed maximum likelihood and Bayesian inference phylogenetic models with RAxML v. 8.2.6 HPC Black Box44 and MrBayes v. 3.2.645, respectively. The settings of analyses were as described in Bolotov et al.4. The best substitution models that were used for the Bayesian analyses are listed in Supplementary Table 3. The phylogenetic analysis was done at the San Diego Supercomputer Center through the CIPRES Science Gateway46. Divergence time modeling. A time-calibrated phylogenetic model has been calculated with BEAST 1.8.447 using the same substitution models as for the MrBayes analyses (Supplementary Table 3). A lognormal relaxed clock and Yule speciation process with continuous quantile parametrization were assigned as model’s priors. To timing the phylogeny, we used the following new crown fossil calibration: †Leptosolen otterensis Cragin (1894)48. Diagnosis and phylogenetic placement: Shell thin, elongated, moderately convex, inequilateral, compressed anteriorly, with anterior fold and angular growth lines around anterior and posterior margins. This species seems to be the oldest member of the genus49, and may represent an ancestral lineage of the Pharidae. Stratigraphic horizon and locality: dark-gray shale of Kiowa Formation (Albian) in central Kansas48. Absolute age estimate: Lower Cretaceous, upper Albian boundary, 100.5 Ma, based on stratigraphy48; 95% soft upper bound 113.0 Ma (lower Albian boundary). Prior setting: exponential distribution, mean (lambda) = 3.4, MRCA: Novaculina gangetica – Siliqua radiata. Five independent runs of 30,000,000 generations were processed, with sampling every 1,000 generation. The resulting tree sets were combined using LogCombiner 1.8.447. An appropriate burn-in was chosen for each tree set with Tracer v. 1.650. A maximum clade credibility tree has been computed with TreeAnnotator 1.8.4 with an additional resampling every 10,000 generation47. Ancestral area modeling. Ancestral area reconstruction has been performed on the basis of three algorithms, i.e., Statistical Dispersal-Vicariance Analysis (S-DIVA), Dispersal-Extinction Cladogenesis (DEC), and Statistical Dispersal-Extinction Cladogenesis (S-DEC) implemented in RASP v. 3.251 as described in Bolotov et al.4. We assigned two possible ancestral areas of the in-group species, i.e., (a) estuarine and (ab) freshwater to estuarine. The three primary models were combined into an integrative model using the Combine Results option of RASP v. 3.251. Molecular diagnoses. To test the molecular differences between N. myanmarensis sp. nov. and N. gangetica, we used an approach of Bolotov et al.37. The mean p-distances and number of fixed nucleotide substitutions were accessed using MEGA640. 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:19E34605-30C2-4DAB-B81F-53A1FDB324DB. 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 sequences used in this study are available from GenBank. 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Acknowledgements We are grateful to the Editor and two anonymous reviewers for their important comments which improved an initial version of the manuscript. This work was partly funded by grants from the Russian Ministry of Education and Science (project no. 6.2343.2017/4.6), Federal Agency for Scientific Organizations (project no. 0409-2015-0143), Russian Foundation for Basic Research (project nos. 16-34-00638 and 18-44-292001), National Geographic Society (project no. NGS-274R-18), and Northern Arctic Federal University. M.L.-L. was funded by FCT – Foundation for Science and Technology under grant no. SFRH/BD/115728/2016. We are grateful to Dr. Thierry Backeljau (Royal Belgian Institute of Natural History, Brussels, Belgium), the late Dr. Tony Whitten (Fauna & Flora International – Asia-Pacific), Mr. Frank Momberg (Fauna & Flora International – Myanmar Program, Myanmar), staff of the Department of Fisheries of the Ministry of Agriculture, Livestock and Irrigation of Myanmar, and Khin Lin Lin Kyaw (Department of Zoology, Hpa-An University, Myanmar) for their great help during this study. Our research has been performed under the survey permission no. 5/6000/LFR(210/2018) dated on 23 January 2018 issued by the Ministry of Agriculture, Livestock and Irrigation of Myanmar and the export permission no. NWCD/CITES/9/5666/2018 dated on 28 June 2018 issued by the Forest Department of the Ministry of Environmental Conservation and Forestry of Myanmar. Author Contributions I.N.B. developed the concept of the study. I.V.V., I.N.B., M.L.-L., Z.L., N.C. and T.W. collected samples. A.V.K. designed and processed molecular analyses. E.S.K. performed morphological and anatomical research. M.Y.G. created the map. I.N.B. performed phylogenetic modeling and wrote the paper, with input from E.S.K., I.V.V., M.L.-L., Z.L., N.C., A.V.K., M.Y.G., O.V.A., S.T. and T.W. All authors discussed the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-34491-8. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCIENTIFIC REPORTS | (2018) 8:16325 | DOI:10.1038/s41598-018-34491-8 12