13urn:lsid:arphahub.com:pub:C9EFD5EB-E909-52A5-90B8-2C7119603A4Eurn:lsid:zoobank.org:pub:ED34F394-2E4C-49D6-8300-0DC18F233E6CZoosystematics and EvolutionZSE1435-19351860-0743Pensoft Publishers10.3897/zse.99.9078490784Research ArticleBivalviaInvertebrataMolluscaUnionidaeUnionoidaMolecular systematicsPhylogenySystematicsTaxonomyCenozoicLaosA freshwater mussel species reflects a Miocene stream capture between the Mekong Basin and East Asian riversKonoplevaEkaterina S.es.konopleva@gmail.comhttps://orcid.org/0000-0002-4820-49211BolotovIvan N.https://orcid.org/0000-0002-3878-419212VikhrevIlya V.https://orcid.org/0000-0002-8612-773612InkhavilayKhamla3GofarovMikhail Yu.https://orcid.org/0000-0002-8532-03071KondakovAlexander V.https://orcid.org/0000-0002-6305-64961TomilovaAlena A.https://orcid.org/0000-0001-5550-53621ChapurinaYulia E.https://orcid.org/0000-0001-9494-75931Van DoTuhttps://orcid.org/0000-0003-2881-152945PfeifferJohn M.6Lopes-LimaManuelhttps://orcid.org/0000-0002-2761-7962278BoganArthur E.https://orcid.org/0000-0003-4042-77069N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, RussiaN. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of SciencesArkhangelskRussiaSSC/IUCN – Mollusc Specialist Group, Species Survival Commission, International Union for Conservation of Nature, Cambridge, UKSSC/IUCN – Mollusc Specialist GroupCambridgeUnited KingdomNUOL – Research Academic and Service Office, National University of Laos, Vientiane, Lao People's Democratic RepublicNational University of LaosVientianeLao People's Democratic RepublicInstitute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Nghia Do, Cau Giay, Ha Noi, VietnamVietnam Academy of Science and TechnologyHa NoiVietnamGraduate University of Science and Technology (GUST), Vietnam Academy of Science and Technology (VAST), Ha Noi, VietnamGraduate University of Science and TechnologyHa NoiVietnamNational Museum of Natural History, Smithsonian Institution, Washington, DC, USANational Museum of Natural History, Smithsonian InstitutionWashington, DCUnited States of AmericaBIOPOLIS Program in Genomics, Biodiversity and Ecosystems, CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Campus de Vairão, Universidade do Porto, 4485-661 Vairão, PortugalUniversidade do PortoVairãoPortugalCIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Matosinhos, PortugalUniversity of PortoMatosinhosPortugalResearch Laboratory, North Carolina Museum of Natural Sciences, Raleigh, USANorth Carolina Museum of Natural SciencesRaleighUnited States of America
Corresponding author: Ekaterina S. Konopleva (es.konopleva@gmail.com)
Academic editor: Frank Köhler
2023060120239912943115C8288-FE36-5332-B58E-5A0E673659BA878687B7-9E5C-4CDF-AE4F-CFC6C9C377A075134872507202227092022Ekaterina S. Konopleva, Ivan N. Bolotov, Ilya V. Vikhrev, Khamla Inkhavilay, Mikhail Yu. Gofarov, Alexander V. Kondakov, Alena A. Tomilova, Yulia E. Chapurina, Tu Van Do, John M. Pfeiffer, Manuel Lopes-Lima, Arthur E. BoganThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/878687B7-9E5C-4CDF-AE4F-CFC6C9C377A0
Freshwater mussels belonging to the genus Cristaria Schumacher, 1817 (Bivalvia: Unionidae) are widespread from Mongolia to Indochina while the range of one species, C.plicata (Leach, 1814), covers two biogeographic subregions, i.e., East Asian (Amur River to Vietnam) and Sundaland (Mekong River basin). We present here a taxonomic revision of the nominal taxon Anodontabellua Morelet, 1866 which was described from the Mekong (Lake Tonle-Sap, Cambodia) but is currently considered a synonym of C.plicata. We obtained molecular data for newly collected Cristaria representatives from the Mekong’s tributaries in Laos, which were found as a divergent species-level phylogenetic clade within the genus that is distant from C.plicata. Nevertheless, comparative morphological and morphometric studies did not reveal any significant differences between these two congeners. Our time-calibrated biogeographic modeling reveals that the split between Cristariabellua (Mekong) and C.clessini (East Asia) probably occurred in the mid-Miocene (15.8 Ma) and may reflect an ancient stream capture between the Mekong Basin and East Asian rivers.
AnodontabelluaCristariaEast AsiaLaosMekongMiocenestream captureSundalandThis study was partly supported by the Russian Ministry of Science and Higher Education (project No. FSRU-2020-0007) and the Russian Science Foundation (grant No. 21-17-00126 to I.N.B. and E.S.K.). The Portuguese Foundation for Science and Technology (FCT) funded M.L.L. (2020.03608.CEECIND) under the Stimulus of Scientific Employment CEEC Individual 2020.Introduction
The freshwater mussel genus Cristaria Schumacher, 1817 (Bivalvia: Unionidae) represents a widespread taxon, which commonly occurs in water bodies throughout East Asia and Indochina (Brandt 1974; He and Zhuang 2013; Dang and Ho 2017; Bolotov et al. 2020; Lopes-Lima et al. 2020; Graf and Cummings 2021). According to the current taxonomy, it comprises five species, i.e., Cristariaplicata (Leach, 1814), C.clessini (Kobelt, 1879), C.beirensis Liu & Zhang, 1982, C.radiata Simpson, 1900, and C.truncata Dang, 1980 (Graf and Cummings 2021). Cristariaclessini is endemic to the western parts of Honshu Island, Japan (Lopes-Lima et al. 2020); C.beirensis inhabits the Amur Basin, as well as streams draining to the Sea of Japan and the Yellow Sea; the range of C.radiata crosses China (Graf and Cummings 2021); and C.truncata is distributed in Northern Vietnam (Do et al. 2018). Cristariaplicata is considered to be the most widespread and abundant taxon within the genus, the range of which extends throughout the Amur (Russia, northeastern China, and Mongolia) and Yangtze (eastern China) basins, water bodies of central Sakhalin, Japan, South Korea, and northern Vietnam (Prozorova et al. 2004; Klishko et al. 2014, 2016; Do et al. 2018; Wu et al. 2018; Bolotov et al. 2020; Lopes-Lima et al. 2020) as well as being known to occur in the Mekong Basin in Indochina (Haas 1969; Brandt 1974; Nahok et al. 2017; Goncalves et al. 2022). Consequently, the range of Cristaria, including C.plicata, covers the East Asian and Sundaland subregions, being a unique example of a unionid genus crossing the drainage divide between the Mekong and East Asian rivers (Bolotov et al. 2018, 2020; Goncalves et al. 2022).
Cristariaplicata differs from other species by its large, well recognizable elliptical-rhomboidal shell, which is usually winged posteriorly, as well as by its strong hinge plate with more or less developed lateral teeth (Brandt 1974; He and Zhuang 2013). This species has high economic importance, especially in China, and is actively used for agriculture, the food industry, pearl production, and medical purposes (Patnaik et al. 2016). Under this taxonomic name, a plethora of species was earlier synonymized (Klishko et al. 2014, 2016). In particular, among them there is a nominal taxon, Anodontabellua described by Morelet (1866), from Lake Tonle-Sap, Cambodia (‘in lacu Touli-Sap, Cambogensi’). Interestingly, this is the only nominal species of Cristaria collected and described from the Mekong Basin. Simpson (1900, 1914) in his comprehensive Unionidae revision regarded A.bellua as a valid species belonging to the genus Cristaria. This author noted that C.bellua is more rhomboid and inflated than C.plicata (Simpson, 1914). Preston (1912) in his work also held the same view. Later, Haas (1969) synonymized C.bellua with C.plicata. Brandt (1974) and He and Zhuang (2013) also listed C.bellua as a synonym of the latter species. Brandt (1974) suggested there were two morphotypes of Cristariaplicata in the Mekong, such as “a thin-shelled, inflated race from still water pools in the Province of Kon Kaen and a compressed, thicker-shelled race from the Mekong”. This author also noticed that “this species has semi-oval glochidia without hooks” and it “cannot be placed among Anodontinae as Haas (1969), Vokes (1967: 213) a.o. suggested” (Brandt 1974; Sayenko et al. 2005; Goncalves et al. 2022). Sayenko et al. (2005) noted that what Brandt named C.plicata could be another taxon. He and Zhuang (2013) contradicted Simpson’s conclusion about the distinct shell shape of C.bellua (see above) and reasoned that specimens of C.plicata from Hubei Province, China, also have the same shape characteristics and there are no conchological differences between both species. Dang et al. (1980) recognized C.bellua as a valid taxon along with three other Cristaria species for the fauna of Vietnam.
In recent studies on Far East Asian and Russian unionids (Bolotov et al. 2020; Lopes-Lima et al. 2020), the molecular and biogeographic information for Cristaria species from China, South Korea, Russia, Japan, and Vietnam was summarized. However, DNA sequences for Cristaria from the Mekong were not available until now. Interestingly, recent explorations in the Tonle Sap Lake and its tributaries in Cambodia, the type locality of Anodontabellua, did not reveal any Cristaria specimens (Ng et al. 2020).
We collected living specimens of Cristariacf.plicata from the Mekong Basin during fieldwork in Laos and generated new DNA sequences for subsequent analyses. This work aims to (1) clarify the taxonomy and distribution of C.plicata; (2) revise the taxonomic status and estimate the phylogenetic position of the nominal species Anodontabellua Morelet, 1866 from the Mekong Basin; and (3) provide some aspects of the evolutional history of the genus Cristaria using paleogeographical, as well as molecular, morphological, and biogeographical information.
Materials and methodsData sampling
Mussel specimens, preliminarily identified as Cristariacf.plicata, were collected from three localities of the Mekong Basin in Laos (Tables 1, 2). Tissue snips for DNA analyses were then preserved in 96% ethanol. The shell and tissue samples were deposited in the Russian Museum of Biodiversity Hotspots (RMBH thereafter), N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences (Arkhangelsk, Russia).
Information on the COI, 16S rRNA, and 28S rRNA gene sequences of Cristariabellua from the Mekong Basin, Laos.
Specimen Voucher
Locality
NCBI GenBank acc. nos.
COI
16S rRNA
28S rRNA
RMBH biv 813/1
Nam Ngum River
ON704642
ON695881
ON695893
RMBH biv 813/2
Nam Ngum River
ON704643
ON695882
ON695894
RMBH biv 813/3
Nam Ngum River
ON704644
ON695883
ON695895
RMBH biv 853/1
Nam Don River
ON704645
ON695884
ON695896
RMBH biv 853/2
Nam Don River
ON704646
ON695885
ON695897
RMBH biv 891/1
Tributary of Nam Ngiep River
ON704647
ON695886
n/a
RMBH biv 891/2
Tributary of Nam Ngiep River
ON704648
ON695887
ON695898
RMBH biv 891/5
Tributary of Nam Ngiep River
ON704649
ON695888
ON695899
Occurrences of Cristaria species from Mekong Basin, Indochina based on museum records.
Species
Catalog number
Country
Original locality
Coordinates (Latitude, Longitude)
Collection data
Cristariabellua
RMBH biv 813
Laos
Nam Ngum River
18.2123, 102.9998
I. N. Bolotov, I. V. Vikhrev, K. Inkhavilay, E. S. Konopleva, Yu. E. Chapurina and locals leg., 26 February 2020
Cristariabellua
RMBH biv 853
Laos
Nam Don River
17.4781, 104.7630
I. N. Bolotov, I. V. Vikhrev, K. Inkhavilay, E. S. Konopleva, Yu. E. Chapurina and locals leg., 29 February 2020
Cristariabellua
RMBH biv 891
Laos
Tributary of Nam Ngiep River
18.5783, 103.5998
I. N. Bolotov, I. V. Vikhrev, K. Inkhavilay, E. S. Konopleva, Yu. E. Chapurina and locals leg., 5 March 2020
Cristariabellua
MCZ_175610
Cambodia
Lake Touli-Sap, Cambodia, Indo China
13.0, 104.0
A. Morelet leg.
Cristariabellua
BMNH_1965147
Cambodia
lac Toui-Sap [Cambodia]
13.0, 104.0
A. Morelet leg.
Cristariabellua
UMMZ_231091
Thailand
River Lam Chi at Gaeng Nam Ton, south of Kon Kaen, [Thailand]
16.3851, 102.7707
23 April 1965
Cristariabellua
FMNH_296520
Thailand
Thailand, Mun River near Bon Tum
15.3195, 103.6754
R. A. Brandt leg., 5 May 1964
Cristariabellua
ANSP_162038
Cambodia
Grand Lac, Cambodia
13.0, 104.0
F. Baker leg.
Cristariabellua
ANSP_56516
Cambodia
Lake Touli-Sap, Cambodia
13.0, 104.0
n/a
Cristariabellua
MNHN-IM-2022-16006
Thailand
Gaeng Nam Ton, south of Kon Kaen, Thailand
16.385, 102.7707
R. A. Brandt leg., 1974
Cristariabellua
MNHN-IM-2022-16005
Cambodia
Gr. Lacs, Cambodge
13.0, 104.0
M. Kermorgan leg., 1884
Cristariabellua
MNHN-IM-2022-16001
Cambodia
Grand Lacs, Cambodge
13.0, 104.0
M. Kermorgan leg., 1884
Cristariabellua
MNHN-IM-2022-16004
Cambodia
Grand Lak, Cambodia, French Indo-China
13.0, 104.0
F. Baker leg.
Cristariabellua
MNHN-IM-2022-16003
Cambodia
Ban Don Cau, Mekong
12.4833, 106.0167
Bavay leg.
Cristariabellua
MNHN-IM-2020-16002
Cambodia
Cambodge, Somron Seng
12.22083, 104.8012
n/a
Cristariabellua
MCZ_102063
Laos
Houtene, confluence of Nam Hinbourne and Mekong rivers, Laos, French Indo China
17.5851, 104.6098
J. Bequaert leg., 28 February 1934
Cristariabellua
MCZ_266165
Thailand
Thailand: Gaeng Nam Ton, Nam Kaen, S of Son Kaen
16.3851, 102.7707
R. A. Brandt leg., 4 May 1967
Cristariabellua
MCZ_280900
Thailand
Thailand: Gaeng Lawa, 12 km NE of Ban Pai, Khon Kaen Prov.
16.1564, 102.6829
R. A. Brandt leg., 4 May 1967
Cristariabellua
SMF_188910
Thailand
Mekong at Tat Panom
16.9443, 104.7308
R. A. Brandt leg., 19 February 1966
Cristariabellua
SMF_220858
Thailand
Thailand: Mekong River at Ban Kum, 12 km N of Ban Dan
15.3886, 105.4940
R. A. Brandt leg., 8 May 1967
Cristariabellua
SMF_220859
Thailand
Mekong River, Ban Dan
15.3239, 105.4930
R. A. Brandt leg., 8 May 1967
Cristariabellua
SMF_220860
Thailand
Thailand: Gaeng Lawa; Kon Kean, 13 km NE Ban Pai
16.1561, 102.6833
R. A. Brandt leg., 4 May 1967
Cristariabellua
SMF_283462
Thailand
Thailand: Gaeng Namton; S of Kon Kaen
16.3851, 102.7707
R. A. Brandt leg., 4 May 1967
Cristariabellua
SMF_319625
Thailand
Lam Chi S Kon Kaen bei Gaen Nam Ton
16.3851, 102.7707
R. A. Brandt leg., 4 May 1967
Cristariabellua
SMF_319627
Thailand
Songkram River, Wannonivat
17.8655, 103.7741
R. A. Brandt leg., 16 February 1966
Cristariabellua
SMF_319628
Thailand
Mekong, Tat Panom
16.9443, 104.7308
R. A. Brandt leg., 19 February 1966
Cristariabellua
SMF_319629
Cambodia
Cambodia: Sekong at Stung Treng
13.5358, 105.9635
R. A. Brandt leg., 26 March 1969
Cristariabellua
SMF_319630
Laos
Laos: Mekong at Muomng khong
14.1169, 105.8562
R. A. Brandt leg., 26 February 1969
Cristariabellua
INHS_32578
Cambodia
Tonle Sap Lake, near Porsat (=Pursat or Pouthisat), Cambodia
12.6494, 104.1559
M. Davis leg., 23 January 2004
Cristariabellua
UF_225991
Cambodia
Kampuchea (Cambodia), Grand Lac
13.0, 104.0
F. Baker leg.
Cristariabellua
UF_507842
Thailand
Mun River and backwater SSE of Ban Pak Nam
15.2655, 104.9803
J. M. Pfeiffer & L. Page leg., 31 January 2016
Cristariabellua
Department of Biology, Faculty of Science, Mahasarakham University
Thailand
Tham Ban Sanam Bin Stream, a tributary of the Choen River, Mekong Basin
16.6147, 101.9783
Nahok et al. (2017)
The type specimens, non-type museum lots, and images of Cristaria from Laos, Thailand, Cambodia, and East Asia were studied in the Museum of Comparative Zoology (MCZ), Harvard University, Cambridge, USA; Natural History Museum (NHMUK), London, Great Britain; Senckenberg Research Institute and Natural History Museum (SMF), Frankfurt, Germany; Muséum national d’histoire naturelle (MNHN), Paris, France; North Carolina Museum of Natural Sciences (NCSM), Raleigh, USA; Florida Museum of Natural History (UF), Gainesville, USA; Academy of Natural Sciences of Drexel University (ANSP), Philadelphia, USA; Illinois Natural History Survey (INHS), Champaign, USA; Field Museum of Natural History (FMNH), Chicago, USA; and University of Michigan Museum of Zoology (UMMZ), Ann Arbor, USA.
Occurrences of Cristaria spp. from the Mekong Basin were obtained based on museum data and published references (Fig. 1, Table 2; Suppl. material 1). The map was made through ESRI ArcGIS 10 software (https://www.esri.com/arcgis). The topographic base of the map was formed using free open sources such as Natural Earth Free Vector and Raster Map Data (https://www.naturalearthdata.com), Global Self-consistent Hierarchical High-resolution Geography, GSHHG v2.3.7 (https://www.soest.hawaii.edu/wessel/gshhg), and HydroSHEDS (https://www.hydrosheds.org).
Occurrences of Cristariabellua from the Mekong Basin based on newly collected and museum data. (1) type locality of Anodontabellua Morelet, 1866: Lake Tonle Sap, Cambodia; (2) sequenced specimens of Cristariabellua: Mekong Basin, Laos; (3) occurrences of Cristariabellua based on museum records from Thailand, Cambodia, and Laos: Mekong Basin.
Morphological analyses were conducted using parameters of shell shape, umbo position, structure of pseudocardinal and lateral teeth, as well as muscle attachment scars (Konopleva et al. 2017, 2019). All specimens were compared with the original descriptions and images of nominal taxa. We analyzed 59 shell contours of the Cristaria representatives, including 3 shells of the type specimens of Anodontabellua, 15 shells of newly collected topotypes, 12 shells of C.plicata from East Asia, and 29 shells of C.plicata from Sundaland (Suppl. material 2). Images of the shells were processed using GIMP v2.10.3 (www.gimp.org). For morphometric analyses, we used Fourier coefficients calculated through SHAPE v1.3 (Iwata and Ukai 2002) as described in previous studies (Konopleva et al. 2017). The results of the Principal Component Analysis (PCA) of Fourier coefficients were visualized using PAST v4.06 (Hammer et al. 2001).
Molecular analyses
Molecular data of mitochondrial (COI and 16 rRNA) and nuclear (28S rRNA) markers were obtained for the eight newly collected Cristaria specimens (Table 1). Total genomic DNA was extracted from ethanol-preserved tissue samples using the NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co. KG, Germany), following the manufacturer protocol. Primers for amplification are shown in Table 3. For amplification we applied marker-specific PCR programs as follows: (i) COI: 95 °C (4 min), 27 repeats at 95 °C (50 s), 47 °C (50 s), 72 °C (50 s), and 72 °C (5 min); (ii) 28S rRNA: 95 °C (4 min), 35 repeats at 95 °C (50 s), 57 °C (50 s), 72 °C (50 s), and 72 °C (5 min); and (iii) 16S rRNA: 95 °C (4 min), 29 repeats at 95 °C (50 s), 46 °C (50 s), 72 °C (50 s), and 72 °C (5 min). Forward and reverse sequence reactions were executed on an ABI PRISM 3730 DNA analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with the ABI PRISM BigDye Terminator v. 3.1 reagents kit. The resulting sequences were checked visually using a sequence alignment editor BioEdit v. 7.2.5 (Hall 1999). The sequences were aligned through the MUSCLE algorithm in MEGA11 (Tamura et al. 2021). Uncorrected genetic p-distances between species within the genus Cristaria were calculated through MEGA11 (Tamura et al. 2021).
Primer sequences using for PCR amplification and sequencing.
Gene fragment
Primer’s name
Direction
Sequence (5'-3')
Reference
COI
LCO1490
Forward
ggtcaacaaatcataaagatattgg
Folmer et al. (1994)
C1-N-2329
Reverse
actgtaaatatatgatgagctca
Simon et al. (1994)
LoboF1
Forward
kbtchacaaaycayaargayathgg
Lobo et al. (2013)
LoboR1
Reverse
taaacytcwggrtgwccraaraayca
16S rRNA
16Sar
Forward
cgcctgtttatcaaaaacat
Palumbi (1996)
16Sbr
Reverse
ccggtctgaactcagatcacgt
28S rRNA
C1
Forward
acccgctgaatttaagcat
Jovelin and Justine (2001)
D2
Reverse
tccgtgtttcaagacgg
Phylogenetic and phylogeographic analyses
The mitochondrial phylogeny was based on the COI dataset using 130 sequences of Cristaria spp., including eight new samples of Cristariabellua from the Mekong (Table 1; Suppl. material 1). Additional sequences of Cristaria were obtained from the NCBI’s GenBank database (Suppl. material 1). The COI dataset was collapsed to 96 unique haplotypes using an online FASTA sequence toolbox (FaBox v1.61; https://birc.au.dk/~palle/php/fabox/; Villesen 2007). The representatives of the subtribe Cristariina (Beringianaberingiana, Buldowskiasuifunica, and Sinanodontaschrenkii) and the family Margaritiferidae (Margaritiferadahurica and Gibbosulalaosensis) were used as outgroup. Maximum likelihood phylogenetic analysis was performed using the online server for IQ-TREE v1.6.12 (W-IQ-TREE) with automatic identification of the most appropriate evolutionary models (Chernomor et al. 2016) and ultrafast bootstrapping algorithm (UFBoot) with 5000 replicates (Hoang et al. 2017). Models of sequence evolution for each partition were calculated through Model Finder (Kalyaanamoorthy et al. 2017) based on Bayesian Information Criterion (BIC) as follows: 1st codon of COI: F81 + I; 2nd codon of COI: TN + G; and 3rd codon of COI: TN + I. The Bayesian Inference (BI) phylogenetic analysis was performed in MrBayes v3.2.7 (Ronquist et al. 2012) at the San Diego Supercomputer Center through the CIPRES Science Gateway (Miller et al. 2010). The same evolutionary models were implemented in the COI dataset. We used the following parameters: two runs with four Markov chains (three heated and one cold, temperature = 0.2), 15,000,000 generations, and tree sampling every 1000th generation, 15% of trees were discarded as burn-in and the majority rule consensus tree was calculated from the remaining trees. Convergence of the MCMC chains to a stationary distribution was checked visually based on the plotted posterior estimates using an MCMC trace analysis tool (Tracer v1.7; Rambaut et al. 2018).
For phylogeographic analysis, we trimmed the COI sequences of Cristaria spp. from the initial length of 659 bp to the final length of 615 bp. A median-joining network was constructed through Network v4.6.1.3 software with default settings (Bandelt et al. 1999). Locality for sequences AS13MT01 and AS13MT02 (Zhang et al. 2013), NC_012716 (Jiang et al. 2010), and KM233451 (Wang et al. 2014) was set as Central East China.
Divergence time estimates and ancestral area reconstruction
A time-calibrated multi-locus phylogeny (3 codons of СOI + 16S rRNA + 28S rRNA) was based on 71 haplotypes of the Unionidae (Suppl. material 1). The best-fit evolutionary models according to BIC were as follows: 1st codon of COI: F81+I+G; 2nd codon of COI: K3Pu+G; 3rd codon of COI: HKY+I+G; 28S rRNA: TIM2+G; and 16S rRNA: TIM2+I+G. Instead of using the estimated best-fit models, we used the less complex HKY model with corresponding distributions for each partition to avoid overparameterization (Bolotov et al. 2017). Calculations were performed in BEAST v1.10.4 with a lognormal relaxed clock and Yule speciation process with continuous quantile parametrization as priors (Suchard et al. 2018). We used three fossil calibration points with Lamprotula, Cuneopsis, and Cristaria as MRCA (Bolotov et al. 2017) with an exponential distribution prior: mean (lambda) = 9.3, offset = 34. The MRCA of the Unionidae was set at 152 Ma (Lopes-Lima et al. 2021; Zieritz et al. 2021) with an exponential distribution prior: mean (lambda) = 2.7, offset = 152. Two independent runs of 25,000,000 generations were processed, with sampling every 1000 generations. The resulting tree sets were combined using LogCombiner v1.10.4 with 10% burn-in. The ESS values were checked using Tracer v1.7 (Rambaut et al. 2018) and each value was recorded as >400. A maximum clade credibility tree has been computed with TreeAnnotator v1.10.4.
Ancestral area reconstruction was based on three algorithms, i.e., Statistical Dispersal-Vicariance Analysis (S-DIVA), Dispersal-Extinction Cladogenesis (DEC), and Statistical Dispersal-Extinction Cladogenesis (S-DEC) implemented in RASP v3.2 (Yu et al. 2015) as described in Bolotov et al. (2017). We assigned two possible ancestral areas of the Cristaria species: (A) East Asia and (B) Sundaland. The three primary models were combined into an integrative model using the Combine Results option of RASP v3.2 (Yu et al. 2015).
ResultsResults of comparative morphological studies and morphometric analyses
The general outlines of newly collected shells of Cristaria from Laos are mainly similar to the type specimens of Anodontabellua Morelet, 1866, described from the Mekong Basin (Fig. 2). Comparative morphological analyses did not reveal remarkable differences in the shell shape and teeth structure between representatives of Cristariaplicata, the type specimens of Anodontabellua, and newly collected topotypes. Younger individuals usually differ by a well-developed and high post-dorsal wing, which can be smoothed with aging. Old specimens have large, solid, more ovate-elongated shells with more or less developed wings, sharp and long teeth, and well-marked muscle attachment scars.
Type specimens and newly collected shells of Cristariabellua (Morelet, 1866) from the Mekong Basin: (A) paralectotype MCZ 175610, Lake Tonlé Sap, Cambodia; (B) lectotype MCZ 175610, Lake Tonlé Sap, Cambodia; (C) paralectotype NHMUK 1965147, Lake Tonlé Sap, Cambodia; (D) specimen RMBH biv 813/3, Nam Ngum River, Laos; (E) specimen RMBH biv 853/1, Nam Don River, Laos; (F) specimen RMBH biv 813/1, Nam Ngum River, Laos; (G) specimen RMBH biv 891/1, tributary of Nam Ngiep River, Laos; (H) specimen RMBH biv 891/4, the same locality. Scale bar: 2 cm. Photos: Adam J. Baldinger, MCZ [A, B]; Kevin Webb, NHMUK [C]; and Ekaterina S. Konopleva [D–H].
https://binary.pensoft.net/fig/792248
PCA based on Fourier coefficients revealed six principal components (PCs) (Fig. 3), among which PC1 and PC2 explained the maximum of the total shell shape variance (69.8% and 12.7%, respectively). However, a Kruskal-Wallis test revealed only one significant component PC2 (P < 0.05; Suppl. material 3). This component reflects the position of the wing and the equilaterality of shells. Three 95% confidence ellipses corresponding to the topotypes of Anodontabellua, the shells from Mekong Basin, and the shells from East Asia are almost completely overlapped. The lectotype and paralectotypes of Anodontabellua also lie within the 95% confidence ellipses and mainly correspond to the coordinates of topotypes of Anodontabellua and specimens from the Mekong Basin.
Principal component analysis (PCA) for the first two PC axes obtained using Fourier coefficients of the Cristaria shell shapes. The ‘extreme’ shapes are illustrated by the four synthetic shell outlines. The filled regions show 95% confidence ellipses.
https://binary.pensoft.net/fig/792249Results of phylogenetic and phylogeographic analyses
The phylogenetic analysis revealed five species-level clades of Cristaria, i.e. Cristariaplicata, C.clessini, C.truncata, C.bellua, and one undescribed lineage Cristaria sp. (Fig. 4). Cristariabellua represents a separate phylogenetic lineage, which is sister to C.clessini with an uncorrected COI p-distance of 8.0 ± 1.0%. Genetic divergences (mean uncorrected COI p-distances, %) calculated between C.bellua and other related species of the genus are shown in Table 4. The topologies of the mitochondrial COI trees were identical for both the Bayesian and ML analyses. Cristariabellua was recorded as a highly-supported monophyletic clade (1.00/99). Other clades were also well-supported, except for the node leading to Cristariabellua and C.clessini, which was moderately supported in the ML analysis (BS = 60).
Phylogenetic reconstruction of the genus Cristaria. A. Bayesian phylogeny of the mitochondrial data set (three codons of COI) on Cristaria taxa. Scale bar indicates the branch lengths. Black numbers near nodes are Bayesian posterior probabilities (BPP) / ML ultrafast bootstrap support values (BS). The outgroup is not shown. B. A fragment of fossil-calibrated Unionidae tree, including the genus Cristaria, based on the complete data set of mitochondrial and nuclear gene sequences (five partitions: three codons of COI + 16S rRNA + 28S rRNA) (Suppl. material 4). Black numbers under nodes are Bayesian posterior probabilities (BPP) of BEAST v. 1.10.4; red numbers above nodes are the mean node ages, Ma. Node bars represent the 95% HPDintervals. Stratigraphic chart according to the International Commission on Stratigraphy, 2021 (https://stratigraphy.org/chart).
https://binary.pensoft.net/fig/792250
Genetic divergences (mean uncorrected p-distances ± standard error estimate, %) between Cristariabellua from Mekong and other related species of the genus Cristaria from East Asia based on the mitochondrial COI gene sequences.
Species
Cristariabellua
Cristariaplicata
Cristariatruncata
Cristariaclessini
Cristariaplicata
8.7±1.0
Cristariatruncata
8.5±1.1
4.4±0.7
Cristariaclessini
8.0±1.0
7.9±1.0
7.6±1.0
Cristaria sp.
11.8±1.2
10.0±1.1
11.2±1.1
11.4±1.2
According to the median-joining network of the COI sequences, C.bellua also represents a divergent lineage and shares five unique haplotypes (Fig. 5). Two haplotypes from the Nam Ngum River are more divergent from others and differ by five nucleotide substitutions. The COI haplotypes of C.bellua are more closely related to C.clessini from Japan, but do not connect to other Cristaria species. The largest COI haplotype diversity is observed in Cristariaplicata, especially for individuals from China. Three species, Cristariatruncata, Cristaria sp. and C.clessini connect to Cristariaplicata.
Median-joining network of the COI gene sequences of Cristaria species (N = 130). The red numbers near branches indicate the numbers of nucleotide substitutions between haplotypes. Size of circles corresponds to the number of available sequences for each haplotype (smallest circle = one sequence). Red small circles indicate hypothetic haplotypes.
https://binary.pensoft.net/fig/792251
Fossil-calibrated biogeographic modeling (Fig. 4; Suppl. material 4) suggests that the Cristaria MRCA originated within East Asia and Sundaland in the Eocene (mean age = 36.5 Ma, 95% HPD = 34.0–41.2 Ma, BEAST BPP = 1.00) with the probability for an East Asian origin of 76.7% and an East Asia + Sundaland origin of 23.3%. The speciation processes mainly occurred during the Miocene, starting from 20.4 Ma, when the ancestors of two clades (C.plicata + C.truncata and C.bellua + C.clessini) diverged.
The split between Cristariabellua and C.clessini is placed in the mid-Miocene (mean age 15.8 Ma, 95% HPD = 6.5 – 29.1 Ma), but with a low support value (BEAST BPP = 0.51). This clade most likely originated in East Asia and Sundaland via a vicariance event (probability of 98.4%)
AnimaliaMalvalesMalvaceae1CF72949-C2C3-5967-A2DE-306B414C9BF8Cristariabellua(Morelet, 1866)=Anodontabellua Morelet, 1866: 167. =Margaron (Dipsas) plicatus – Lea (1870): 74. =Dipsasbellua – Morelet (1875): 331, Fischer (1891): 222. =Cristaria(s.s.)bellua – Simpson (1900): 584. =Cristaria(s.s.)bellua – Preston (1912): 281. =Cristaria(s.s.)bellua – Simpson (1914): 226. =Dipsasplicatus – Dautzenberg and Fischer (1905): 198. =Cristaria(s.s.)plicataplicata – Haas (1969): 387. =Cristariaplicata – Brandt (1974): 278, He and Zhuang (2013): 39, Do et al. (2018): 5; Lopes-Lima et al. (2020): 9. =Cristariabellua – Dang et al. (1980): 537. Type and type locality.
Lectotype MCZ 175610 (in lacu Touli-Sap, Cambogensi) [Cambodia: Lake Tonlé Sap]; designated by Johnson (1956): p. 107, pl. 1, fig. 1 (Fig. 2B).
Type material.
Paralectotype MCZ 175610 (in lacu Touli-Sap, Cambogensi) [Cambodia: Lake Tonlé Sap] (Fig. 2A); Paralectotype NHMUK 1965147 (lac Touli-Sap, Cambodia) [Cambodia: Lake Tonlé Sap] (Johnson 1971: p. 80; Breure et al. 2018) (Fig. 2C).
Material examined.
Laos: Nam Ngum River, 18.2123°N, 102.9998°E, Mekong Basin, 26.ii.2020, 3 specimens [RMBH biv 813, all sequenced], Bolotov, Vikhrev, Inkhavilay, Konopleva, Chapurina, and locals leg. (Fig. 2D, F); Nam Don River, 17.4781°N, 104.7630°E, Mekong Basin, 29.ii.2020, 2 specimens [RMBH biv 853, all sequenced], Bolotov, Vikhrev, Inkhavilay, Konopleva, Chapurina, and locals leg. (Fig. 2E); tributary of Nam Ngiep River, 18.5783°N, 103.5998°E, Mekong Basin, 05.iii.2020, 9 specimens [RMBH biv 891, including biv 891/1, biv 891/2 and biv 891/5 sequenced], Bolotov, Vikhrev, Inkhavilay, Konopleva, Chapurina, and locals leg. (Fig. 2G, H). Museum material examined: listed in Table 2.
Re-description.
Shell rhomboid, rather large, moderately thin and inflated, sub-solid; high posteriorly, young specimens usually with clearly developed wing or somewhat crest smoothing with aging; ventral margin straight or slightly curved; posterior ridge folded. Umbo small, slightly elevated above hinge line. Periostracum of various coloration, from olive-green to brown and blackish; younger individuals usually with dark-green radial rays running from the umbones to the ventral margin, visible on either side of valve; wider stripes usually located on posterior slope. Nacre whitish, may be tinted with yellow; specimens from the tributary of the Nam Ngiep River differ by a pinkish color, mainly for younger shells. Umbo cavity shallow with a few deep pits. Lateral teeth short, more or less developed on each valve. Pseudocardinal teeth extremely thin or underdeveloped. Anterior muscle scars irregular and usually well-developed, posterior muscle scars more shallow, somewhat crescent-shaped.
Habitat and ecology.
The species was recorded from the main channel of the Nam Ngum and Nam Don rivers and a tributary of the Nam Ngiep River, mainly on clay-sandy substrates and at a depth of >1.5–2.0 m. It was found together with representatives of different genera such as Lens, Physunio, Hyriopsis, Pilsbryoconcha, Bineurus, Monodontina, Scabies, and Nyeinchanconcha. Numerous parasitic mites (Acari: Unionicolidae) and their eggs were discovered on the mantle and gills of C.bellua, especially from specimens collected in a tributary of the Nam Ngiep River.
Distribution.
Mekong Basin in Laos, Cambodia, and Thailand.
Comments.
Morelet (1866) did not state how many specimens were collected and studied for the description of Anodontabellua. In the original description, there was only one set of measurements: shell length = 187 mm; shell height = 123 mm; and shell width = 71 mm (Morelet, 1866). In MCZ, there are two specimens under catalog number 175610. One of them was designated by Johnson (1956) as the lectotype of A.bellua and illustrated in his work (Johnson 1956: pl. 1, fig. 1). This author wrote that “this the first time that this species has been figured”. Second (larger) specimen was not discussed. Specimen NHMUK 1965147 was considered a paralectotype (Johnson 1971).
DiscussionMorphological and molecular characteristics of Cristariabellua
In the present study, Cristariabellua was distinguished as a distinct species based on molecular and phylogenetic analyses. The COI haplotypes of Cristariabellua are relatively close to its congener, C.clessini, endemic to Japan (Lopes-Lima et al. 2020). Populations of Cristariabellua, collected from three localities of the Mekong Basin during the field surveys are rather distant, with each sample having its COI haplotype (Figs 4, 5), which may reflect their isolated existence in these water bodies. Nevertheless, comparative morphological and morphometric studies did not reveal any significant differences from its other congener, Cristariaplicata. Historically, Cristariabellua has been synonymized with C.plicata by many authors (Haas 1969; Brandt 1974; He and Zhuang 2013; Do et al. 2018; Lopes-Lima et al. 2020), mainly, due to the high level of conchological similarity of both species. The two species are morphologically very similar and may be distinguished using molecular data and biogeographic patterns only.
Evolutionary biogeography of Cristaria
For a long time, it was supposed that the range of Cristariaplicata covers the major basins of East and Southeast Asia, including the Mekong River catchment area (Brandt 1974; Lopes-Lima et al. 2020; Graf and Cummings 2021). Such a broad distribution was surprising because this species crosses the drainage divides between separate large freshwater systems such as the Mekong and the East Asian rivers. The crossing of separate drainages was described in previous studies of Southeast Asia, for example, the presence of disjunct mussel species populations in the Mekong and Chao Phraya (Konopleva et al. 2021; Pfeiffer et al. 2021). Earlier, Bolotov et al. (2020) noted that “records from the Mekong Basin may represent a historical human-mediated or natural dispersal event”. It is well-known that C.plicata has historically been used for food, pearl, nacre, and jewelry production (Landmann et al. 2001; Fiske and Shepherd 2007; Nagai 2013) and was dispersed by man (Schneider et al. 2013). Thereby Schneider et al. (2013) doubted the reliability of the genus Cristaria for paleogeographic reconstructions. In contrast, our molecular analyses showed very high COI genetic divergence between Cristariabellua and all its congeners (uncorrected p-distance > 8.0%), and point to long-term isolation in the Mekong Basin with a subsequent speciation event.
Multiple fossils of Cristaria were found among the Paleogene Na Duong, Cao Bang, and Rhin Chua mollusk assemblages (Böhme et al. 2013; Schneider et al. 2013). These records were used in our fossil-calibrated phylogenetic modeling as one of the calibration points. According to our model, the diversification of Cristaria mainly occurred in the Miocene. The MRCA (Middle Miocene) of Cristariabellua from the Mekong Basin and C.clessini from Japan suggests a probable ancient stream capture between the Mekong and the East Asian rivers and the start of the separate evolution of these lineages since this event. The moderate to high level of phylogenetic support for a node of the sister species Cristariabellua and C.clessini as well as a high genetic distance between these lineages may indicate the presence of putative additional taxa, which have not yet been sampled, for example from remote and understudied areas of northern Laos, China, Vietnam or the Korean Peninsula.
Several geological studies support the hypothesis of an ancient stream capture between the paleo-Mekong and paleo-Red rivers. According to the geomorphological reconstruction of Clark et al. (2004), in the past, the Upper and Middle Yangtze, Upper Mekong, Upper Salween, and the Tsangpo (Upper Brahmaputra) rivers drained together to the South China Sea through the paleo-Red River. In this work, the Mekong River is considered a major tributary of the paleo-Red River system before the elevation of southeastern Tibet (Clark et al. 2004). Conversely, analyses by Hoang et al. (2009) showed that the Mekong headwaters (as well as the Ayeyarwady and the Salween) have not been connected to the Red River since the Late Miocene. Furthermore, the drainage of the paleo-Red River included the middle part of the present Yangtze and the headwaters of the modern Pearl River (Clift et al. 2008; Hoang et al. 2009).
There are examples of East Asian fish genera, which cross the Mekong drainage divide, with one or several species being endemic to the Upper Mekong. For instance, representatives of Pareuchiloglanis species inhabit the Upper and Middle Yangtze, Red, and Pearl River basins and at the same time the Mekong drainage (Li et al. 2020). The same distributional patterns are typical for another fish genus, Vanmanenia, which occurs in the Upper Yangtze, Pearl, and Red River drainages and the Upper Mekong (Lancangjiang) (Li et al. 2019).
Recently, the representative of the typical East Asian fish genus Carassius Nilsson, 1832 was described from the Upper Nam Ngum River in the Mekong Basin in central Laos, i.e., C.praecipuusKottelat 2017 (Kottelat 2017). As Kottelat (2017) claimed, the Nam Ngum River together with the Nam Ngiep and Nam Neun have headwaters on the Plain of Jars. One of these rivers, i.e. the Nam Neun, is adjacent to the Ma River Basin and flows through Vietnam into the South China Sea (the Gulf of Tonkin). The fact that the three rivers are separated only by very low divides may suggest a faunal exchange in the past, which has already been shown for a few species (Kottelat 2017). Consequently, Cristariabellua may have been isolated in the Mekong Basin, for example, together with some East Asian fishes.
Further molecular studies and field sampling is necessary to better understand the evolutionary diversification and the biogeography of Cristaria.
Distribution and ecology of Cristariabellua
Currently, Cristariabellua is the sole representative of the genus Cristaria and the only native member of the subfamily Unioninae in the Mekong River drainage. Live specimens of Cristariabellua were recorded in three water bodies of the Mekong basin in Laos, i.e., the Nam Ngum, Nam Don, and Nam Ngiep rivers. The majority of available museum lots of Cristaria from Laos, Cambodia, and Thailand were collected as late as the second part of the 20th century or even earlier (see Table 2). Among the most recent museum records of Cristaria (January 2016), only shells from one locality of the Mun River, Thailand were registered (UF 507842). Additionally, one Cristaria shell was collected from the Choen River basin in Thailand in 2015 (Nahok et al. 2017). The fact that the present living specimens from Laos were collected from a considerable depth (>1.5–2.0) also revealed that mussels of Cristariabellua are not easily accessible for sampling.
As mentioned in the taxonomic account, specimens of Cristariabellua were infested by parasitic mites (Acari: Unionicolidae). However, this is not surprising, because there are multiple observations of mussel-associated mites and their eggs in the congeneric species Cristariaplicata from China (Wen et al. 2006; Wu et al. 2008; Zhang et al. 2018). Probably, Cristariabellua may serve as a nutrition source and shelter for these parasites (Vidrine 1986; Wen et al. 2006).
The Mekong Basin harbors one of the richest fauna of the Unionidae globally with many endemic taxa (Pfeiffer et al. 2018, 2021; Zieritz et al. 2018; Jeratthitikul et al. 2021; Konopleva et al. 2021). The fauna in Laos is threatened by many anthropogenic pressures such as habitat degradation, water pollution, dam construction, and overharvesting of aquatic animals (Bolotov et al. 2014). Hence, Cristariabellua requires conservation attention including accurate monitoring efforts, especially because viable populations in other parts of the Mekong, for instance in the type locality, have not been recorded during recent decades (Ng et al. 2020).
Conclusion
In this study, we revised the taxonomic status and estimate the phylogenetic position of the nominal species Anodontabellua Morelet, 1866, which was earlier considered a synonym of the widespread species Cristariaplicata (Leach, 1814). The molecular and phylogenetic analyses support Cristariabellua as a distinct species, which is relatively close to its congener, C.clessini (Kobelt, 1879). At the same time, C.bellua can hardly be distinguished conchologically from its congener, Cristariaplicata.
All known representatives of the genus Cristaria are distributed in East Asia, except for Cristariabellua, which inhabits the Mekong River drainage. Phylogeographic modelling revealed that the diversification in the genus Cristaria probably occurred in the Miocene. The speciation events in this genus could be linked to the rearrangements of paleo-river basins throughout East and Southeast Asia. We also propose that Cristariabellua may have been isolated in the Mekong Basin together with several East Asian freshwater fish species. Our results highlight that Cristariabellua is a unique representative of the subfamily Unioninae in the Mekong drainage and that this species is of high priority and concern to future conservation efforts.
Acknowledgements
This study was partly supported by the Russian Ministry of Science and Higher Education (project No. FUUW-2022-0056) and the Russian Science Foundation (grant No. 21-17-00126 to I.N.B. and E.S.K.). The field research in Laos was performed within the framework of scientific cooperation between the National University of Laos (Vientiane, Lao PDR) and N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences (Arkhangelsk, Russia). The Portuguese Foundation for Science and Technology (FCT) funded M.L.L. (2020.03608.CEECIND) under the Stimulus of Scientific Employment CEEC Individual 2020 and the project ConBiomics: the missing approach for the Conservation of Freshwater Bivalves Project No. NORTE-01-0145-FEDER-030286. We thank the curators of mussel collections, Dr. Adam J. Baldinger from the Museum of Comparative Zoology, Cambridge, USA and Dr. Jon Ablett from Natural History Museum, UK for discussion and providing high-resolution images of the type specimens of Anodontabellua. We thank Dr. Virginie Heros from Museum national d'Histoire naturelle, France for the assistance with mussel collection. We are also grateful to Dr. Björn Stelbrink and Dr. Frank Köhler for their valuable comments which helped to improve earlier version of this paper.
ReferencesBandeltHJForsterPRöhlA (1999) Median-joining networks for inferring intra-specific phylogenies.16(1): 37–48. https://doi.org/10.1093/oxfordjournals.molbev.a026036BöhmeMAiglstorferMAntoineP-OAppelEHavlikPMétaisGThe PhucLSchneiderSSetzerFTappertRNgoc TranDUhlDPrietoJ (2013) Na Duong (northern Vietnam) – an exceptional window into Eocene ecosystems from Southeast Asia.53: 121–167.BolotovIVikhrevIBespalayaYGofarovMKolosovaJKondakovAFrolovALyubasARomanisTTitovaKMakhrovAArtamonovaVTumpeesuwanS (2014) Ecology and conservation of the endangered Indochinese freshwater pearl mussel, Margaritiferalaosensis (Lea, 1863) in the Nam Pe and Nam Long rivers, Northern Laos.7(4): 706–719. https://doi.org/10.1177/194008291400700409BolotovINKondakovAVVikhrevIVAksenovaOVBespalayaYVGofarovMYKolosovaYSKonoplevaESSpitsynVMTanmuangpakKTumpeesuwanS (2017) Ancient river inference explains exceptional oriental freshwater mussel radiations.7(2135): 1–14. https://doi.org/10.1038/s41598-017-02312-zBolotovINPfeifferJMKonoplevaESVikhrevIVKondakovAVAksenovaOVGofarovMYTumpeesuwanSWinT (2018) A new genus and tribe of freshwater mussel (Unionidae) from Southeast Asia.8(10030): 1–12. https://doi.org/10.1038/s41598-018-28385-yBolotovINKondakovAVKonoplevaESVikhrevIVAksenovaOVAksenovASBespalayaYVBorovskoyAVDanilovPPDvoryankinGAGofarovMYKabakovMBKlishkoOKKolosovaYSLyubasAANovoselovAPPalatovDMSavvinovGNSolomonovNMSpitsynVMSokolovaSETomilovaAAFroufeEBoganAELopes-LimaMMakhrovAAVinarskiMV (2020) Integrative taxonomy, biogeography and conservation of freshwater mussels (Unionidae) in Russia.10(3072): 1–20. https://doi.org/10.1038/s41598-020-59867-7BrandtRAM (1974) The non-marine aquatic Mollusca of Thailand.105: 1–423.BreureASAudibertCAblettJD (2018) Nederlandse Malacologische Vereniging, Leiden, 544 pp.ChernomorOvon HaeselerAMinhBQ (2016) Terrace aware data structure for phylogenomic inference from supermatrices.65(6): 997–1008. https://doi.org/10.1093/sysbio/syw037ClarkMKSchoenbohmLMRoydenLHWhippleKXBurchfielBCZhangXTangWWangEChenL (2004) Surface uplift, tectonics, and erosion of eastern Tibet from large-scale drainage patterns.23(1): 1–20. https://doi.org/10.1029/2002TC001402CliftPDLongHVHintonREllamRMHanniganRTanMTBlusztajnJDucNA (2008) Evolving east Asian river systems reconstructed by trace element and Pb and Nd isotope variations in modern and ancient Red River-Song Hong sediments. Geochemistry, Geophysics, Geosystems 9(4): Q04039. https://doi.org/10.1029/2007GC001867DangNTHoTH (2017) Sciences and Technology Publishing Co, Ha Noi, 363 pp.DangNTThaiTBPhamVM (1980) Sciences and Technology Publishing Co, Ha Noi, 573 pp.DautzenbergPFischerH (1905) Liste des mollusques récoltés par M. le Capitaine de Frégate Blaise au Tonkin, et description d’espèces nouvelles.53: 85–234. https://doi.org/10.5962/bhl.title.13158DoVTTuanLQBoganAE (2018) Freshwater mussels (Bivalvia: Unionida) of Vietnam: Diversity, distribution, and conservation status.21(1): 1–18. https://doi.org/10.31931/fmbc.v21i1.2018.1-18FiskeDShepherdJ (2007) Continuity and Change in Chinese Freshwater Pearl Culture.43(2): 138–145. https://doi.org/10.5741/GEMS.43.2.138FischerP (1891) Catalogue et distribution géographique des mollusques terrestres, fluviatiles & marins d’une partie de l Indo-chine (Siam, Laos, Cambodge, Cochinchine, Annam, Tonkin).4: 87–276. https://doi.org/10.5962/bhl.title.14809FolmerOBlackMHoehWLutzRVrijenhoekR (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.3(5): 294–299.GoncalvesAZieritzALopes-LimaMDeeinGPfeifferJ (2022) Taxonomic revision and conservation assessment of the Southeast Asian freshwater mussel genus Chamberlainia Simpson, 1900. Journal of Molluscan Studies 88(2): eyac008. https://doi.org/10.1093/mollus/eyac008GrafDLCummingsKS (2021) A ‘big data’ approach to global freshwater mussel diversity (Bivalvia: Unionoida), with an updated checklist of genera and species. Journal of Molluscan Studies 87(1): eyaa034. https://doi.org/10.1093/mollus/eyaa034HaasF (1969) Superfamilia Unionacea.88: 1–663.HallTA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.41: 95–98.HammerØHarperDATRyanPD (2001) PAST: Paleontological statistics software package for education and data analysis.4(1): 1–9.HeJZhuangZ (2013) ConchBooks, Harxheim, 198 pp.HoangLWuF-YCliftPDWysockaASwierczewskaA (2009) Evaluating the evolution of the Red River system based on in situ U-Pb dating and Hf isotope analysis of zircons.10(11): 1–20. https://doi.org/10.1029/2009GC002819HoangDTChernomorOvon HaeselerAMinhBQVinhLS (2017) UFBoot2: Improving the ultrafast bootstrap approximation.35(2): 518–522. https://doi.org/10.1093/molbev/msx281IwataHUkaiY (2002) SHAPE: A computer program package for quantitative evaluation of biological shapes based on elliptic Fourier descriptors.93(5): 384–385. https://doi.org/10.1093/jhered/93.5.384JeratthitikulESutcharitCNgorPBPrasankokP (2021) Molecular phylogeny reveals a new genus of freshwater mussels from the Mekong River Basin (Bivalvia: Unionidae).775(1): 119–142. https://doi.org/10.5852/ejt.2021.775.1553JiangWPLiJLZhengRLWangGL (2010) Analysis of complete mitochondrial genome of Cristariaplicata.32(2): 153–162. https://doi.org/10.3724/sp.j.1005.2010.00153 [In Chinese with an English abstract]JohnsonRI (1956) The types of Naiades (Mollusca: Unionidae) in the Museum of Comparative Zoology.115: 101–142.JohnsonRI (1971) The types and figured specimens of Unionacea (Mollusca: Bivalvia) in the British Museum (Natural History).20: 73–108. https://doi.org/10.5962/p.314189JovelinRJustineJL (2001) Phylogenetic relationships within the polyopisthocotylean monogeneans (Platyhelminthes) inferred from partial 28S rDNA sequences.31(4): 393–401. https://doi.org/10.1016/S0020-7519(01)00114-XKalyaanamoorthySMinhBQWongTKFvon HaeselerAJermiinLS (2017) Model finder: Fast model selection for accurate phylogenetic estimates.14(6): 587–589. https://doi.org/10.1038/nmeth.4285KlishkoOKLopes-LimaMFroufeEBoganAE (2014) Are Cristariaherculea (Middendorff, 1847) and Cristariaplicata (Leach, 1815) (Bivalvia, Unionidae) separate species? ZooKeys 438: 1–15. https://doi.org/10.3897/zookeys.438.7493KlishkoOKLopes-LimaMFroufeEBoganAEAbakumovaVY (2016) Systematics and distribution of Cristariaplicata (Bivalvia, Unionidae) from the Russian Far East.580: 13–27. https://doi.org/10.3897/zookeys.580.7588KonoplevaESBolotovINVikhrevIVGofarovMYKondakovAV (2017) An integrative approach underscores the taxonomic status of Lamellidensexolescens, a freshwater mussel from the Oriental tropics (Bivalvia: Unionidae).15(3): 204–217. https://doi.org/10.1080/14772000.2016.1249530KonoplevaESPfeifferJMVikhrevIVKondakovAVGofarovMYAksenovaOVLunnZChanNBolotovIN (2019) A new genus and two new species of freshwater mussels (Unionidae) from western Indochina.9(4106): 1–14. https://doi.org/10.1038/s41598-019-39365-1KonoplevaESBolotovINPfeifferJMVikhrevIVKondakovAVGofarovMYTomilovaAATanmuangpakKTumpeesuwanS (2021) New freshwater mussels from two Southeast Asian genera Bineurus and Thaiconcha (Pseudodontini, Gonideinae, Unionidae).11(8244): 1–17. https://doi.org/10.1038/s41598-021-87633-wKottelatM (2017) Carassiuspraecipuus, a dwarf new species of goldfish from the Mekong drainage in central Laos (Teleostei: Cyprinidae).124(3): 323–329.LandmannNHMikkelsenPMBielerRBronsonB (2001) Abrams, New York, 232 pp.LeaI (1870) Lea, Philadelphia, 184 pp. https://doi.org/10.5962/bhl.title.13200LiXZhouWCheXJ (2019) Loaches of Vanmanenia (Cypriniformes: Gastromyzontidae) from Yunnan, China with description of a new species.4603(1): 125–144. https://doi.org/10.11646/zootaxa.4603.1.6LiXDaoWZhouW (2020) Type locality and species identity of Pareuchiloglanissinensis (Hora & Silas) with a description of a new species of the genus from the upper Yangtze River basin in southern China.97(3): 827–844. https://doi.org/10.1111/jfb.14438LoboJCostaPMTeixeiraMAFerreiraMSGCostaMHCostaFO (2013) Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans.13(34): 1–8. https://doi.org/10.1186/1472-6785-13-34Lopes-LimaMHattoriAKondoTLeeJHKimSKShiraiAHayashiHUsuiTSakumaKToriyaTSunamuraYIshikawaHHoshinoNKusanoYKumakiHUtsugiYYabeSYoshinariYHirumaHTanakaASaoKUedaTSanoIMiyazakiJ-IGonçalvesDVKlishkoOKKonoplevaESVikhrevIVKondakovAVGofarovMYBolotovINSayenkoEMSorokaMZieritzABoganAEFroufeE (2020) Freshwater mussels (Bivalvia: Unionidae) from the rising sun (Far East Asia): phylogeny, systematics, and distribution.146: 1–27. https://doi.org/10.1016/j.ympev.2020.106755Lopes-LimaMGürlekMEKebapçıÜŞereflişanHYanıkTMirzajaniANeubertEPriéVTeixeiraAGomes-dos-SantosABarros-GarcíaDBolotovINKondakovAVVikhrevIVTomilovaAAÖzcanTAltunAGonçalvesDVBoganAEFroufeE (2021) Diversity, biogeography, evolutionary relationships, and conservation of Eastern Mediterranean freshwater mussels (Bivalvia: Unionidae).163: 1–23. https://doi.org/10.1016/j.ympev.2021.107261MillerMAPfeifferWSchwartzT (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010. New Orleans, LA, 1–8. https://doi.org/10.1109/GCE.2010.5676129MoreletA (1866) Description d’espèces appartenant à la faune malacologique de l’Indo-Chine.14: 62–64.MoreletA (1875) Séries conchyliologiques comprenant l’énumération de mollusques terrestres et fuviatiles recueillies pendent le cours de diférents voyages, ainsi que la description de plusieurs espèces nouvelles 4. Savy, Paris, 227–377.NagaiK (2013) A History of the Cultured Pearl Industry.30(10): 783–793. https://doi.org/10.2108/zsj.30.783NahokBTumpeesuwanCSrifaATumpeesuwanS (2017) Freshwater molluscan assemblages in upper part of Choen River Basin. Northeastern Thailand.17(1): 11–24. https://li01.tci-thaijo.org/index.php/tnh/article/view/103061NgTHJeratthitikulESutcharitCChhuoySPinKPholyothaASiriwutWSrisonchaiRHoganZSNgorPB (2020) Annotated checklist of freshwater molluscs from the largest freshwater lake in Southeast Asia.958: 107–141. https://doi.org/10.3897/zookeys.958.53865PalumbiSR (1996) Nucleic Acids II: Polymerase Chain Reaction. In: HillisDMoritzCMableB (Eds) Molecular Systematic, 2nd Edn., 205–247.PatnaikBBWangTHKangSWHwangHJParkSYParkEBChungJMSongDKKimCKimSLeeJSHanYSParkHSLeeYS (2016) Sequencing, De Novo Assembly, and Annotation of the Transcriptome of the Endangered Freshwater Pearl Bivalve, Cristariaplicata, Provides Novel Insights into Functional Genes and Marker Discovery.11(2): 1–28. https://doi.org/10.1371/journal.pone.0148622PfeifferJMGrafDLCummingsKSPageLM (2018) Molecular phylogeny and taxonomic revision of two enigmatic freshwater mussel genera (Bivalvia: Unionidae incertae sedis: Harmandia and Unionetta) reveals a diverse clade of Southeast Asian Parreysiinae.84(4): 404–416. https://doi.org/10.1093/mollus/eyy028PfeifferJMGrafDLCummingsKSPageLM (2021) Taxonomic revision of a radiation of Southeast Asian freshwater mussels (Unionidae: Gonideinae: Contradentini+Rectidentini).35(4): 394–470. https://doi.org/10.1071/IS20044PrestonHB (1912) A catalogue of the Asiatic naiades in the collection of the Indian Museum, Calcutta, with descriptions of new species.7: 279–308. https://doi.org/10.26515/rzsi/v7/i3/1912/163128ProzorovaLABogatovVVSayenkoEM (2004) New data on the freshwater mollusk fauna of Sakhalin Island. In: Flora and fauna of Sakhalin Island: Mater. Int. Sakhalin Is. Proj 1. Dalnauka, 138–144.RambautADrummondAJXieDBaeleGSuchardMA (2018) Posterior summarisation in Bayesian phylogenetics using Tracer 1.7.67(5): 901–904. https://doi.org/10.1093/sysbio/syy032RonquistFTeslenkoMvan der MarkPAyresDLDarlingAHöhnaSLargetBLiuLSuchardMAHuelsenbeckJP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space.61(3): 539–542. https://doi.org/10.1093/sysbio/sys029SayenkoEMPearceTASheaEK (2005) Glochidial morphology of selected species of the genera Cristaria Schumacher, 1817 and Sinanodonta Modell, 1945 (Bivalvia: Unionidae) from Far Eastern Russia.20(1–2): 11–21.SchneiderSBöhmeMPrietoJ (2013) Unionidae (Bivalvia; Palaeoheterodonta) from the Palaeogene of northern Vietnam: Exploring the origins of the modern East Asian freshwater bivalve fauna.11(3): 337–357. https://doi.org/10.1080/14772019.2012.665085SimonCFratiFBeckenbachACrespiBLiuHFlookP (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers.87(6): 651–701. https://doi.org/10.1093/aesa/87.6.651SimpsonCT (1900) New and unfigured Unionidae. Proceedings.52: 74–86.SimpsonCT (1914) Bryant Walker, Detroit, 1540 pp. https://doi.org/10.5962/bhl.title.10910SuchardMALemeyPBaeleGAyresDLDrummondAJRambautA (2018) Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10.4(1): 1–5. https://doi.org/10.1093/ve/vey016TamuraKStecherGKumarS (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11.38(7): 3022–3027. https://doi.org/10.1093/molbev/msab120VidrineMF (1986) Revision of the Unionicolinae (Acari: Unionicolidae).12(4): 233–243. https://doi.org/10.1080/01647958608683470VillesenP (2007) FaBox: An online toolbox for fasta sequences.7(6): 965–968. https://doi.org/10.1111/j.1471-8286.2007.01821.xVokesHS (1967) Genera of the Bivalvia: A systematic and bibli-ographic catalogue.51: 103–392.WangHHeLYangXYangSLiCWangX (2014) Determination of the complete mitochondrial genome sequence of mussel Cristariaplicata (Leach). Mitochondrial DNA.27(2): 1478–1479. https://doi.org/10.3109/19401736.2014.953100WenCNiePZhuZ (2006) Population dynamics of the water mite Unionicolaarcuata (Unionicolidae) in the freshwater bivalve Cristariaplicata (Unionidae) in Poyang Lake, eastern China.70(1–2): 123–127. https://doi.org/10.3354/dao070123WuHXieYWenCFengT (2008) Histopathological effect of Unionicolaarcuata eggs on different tissues in Cristariaplicata.26: 544–547. [In Chinese with an English abstract]WuRWLiuYTWangSLiuXJZanattaDTRoeKJSongXLAnCTWuXP (2018) Testing the utility of DNA barcodes and a preliminary phylogenetic framework for Chinese freshwater mussels (Bivalvia: Unionidae) from the middle and lower Yangtze River.13(8): 1–19. https://doi.org/10.1371/journal.pone.0200956YuYHarrisAJBlairCHeX (2015) RASP (Reconstruct Ancestral State in Phylogenies): A tool for historical biogeography.87: 46–49. https://doi.org/10.1016/j.ympev.2015.03.008ZhangHYaoHCuiLDuHLinZGaoXLangXSongJLuoKShiLChenS (2013) Application of COI-based DNA Barcoding for Identifying Animal Medical Materials in the Chinese Pharmacopoeia.15(3): 371–380. [In Chinese with an English abstract]ZhangFChengJWenCHuBYangGJianS (2018) Seasonal variation of a population Unionicolapenicillatus (Unionicolidae) from the freshwater bivalve Cristariaplicata (Unionidae) in Poyang Lake, eastern China.23(2): 288–295. https://doi.org/10.11158/saa.23.2.5ZieritzABoganAEFroufeEKlishkoOKondoTKovitvadhiUKovitvadhiSLeeJHLopes-LimaMPfeifferJMSousaRDoTVVikhrevIZanattaDT (2018) Diversity, biogeography and conservation of freshwater mussels (Bivalvia: Unionida) in East and Southeast Asia.810(1): 29–44. https://doi.org/10.1007/s10750-017-3104-8ZieritzAFroufeEBolotovIGonçalvesDVAldridgeDCBoganAEGanHMGomes-Dos-SantosASousaRTeixeiraAVarandasSZanattaDLopes-LimaM (2021) Mitogenomic phylogeny and fossil-calibrated mutation rates for all F- and M-type mtDNA genes of the largest freshwater mussel family, the Unionidae (Bivalvia).193(3): 1088–1107. https://doi.org/10.1093/zoolinnean/zlaa153Supplementary materials10.3897/zse.99.90784.suppl175134695B5B3391-3C2D-5A6C-A154-79677B75F55F
List of sequences used in this study, including species names, localities, voucher numbers, and GenBank accession numbers
Sequence dataset (Excel file)
List of sequences used in this study, including species names, localities, voucher numbers, and GenBank accession numbers.
https://binary.pensoft.net/file/792252This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Ekaterina S. Konopleva, Ivan N. Bolotov, Ilya V. Vikhrev, Khamla Inkhavilay, Mikhail Yu. Gofarov, Alexander V. Kondakov, Alena A. Tomilova, Yulia E. Chapurina, Tu Van Do, John M. Pfeiffer, Manuel Lopes-Lima, Arthur E. Bogan10.3897/zse.99.90784.suppl275134757E512795-C984-5317-A8DB-1EB00C035E03
Freshwater mussel specimens used in geometric morphometric analyses and results of PCA
Morphometric dataset (Excel file)
Freshwater mussel specimens used in geometric morphometric analyses and results of PCA
https://binary.pensoft.net/file/792253This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Ekaterina S. Konopleva, Ivan N. Bolotov, Ilya V. Vikhrev, Khamla Inkhavilay, Mikhail Yu. Gofarov, Alexander V. Kondakov, Alena A. Tomilova, Yulia E. Chapurina, Tu Van Do, John M. Pfeiffer, Manuel Lopes-Lima, Arthur E. Bogan10.3897/zse.99.90784.suppl37513481E3FB0744-F167-53DF-8FBF-1D2272A07F19
Results of Kruskal-Wallis test, including Chi-square and P-values for each principal component
Morphometric results (Excel file)
Results of Kruskal-Wallis test, including Chi-square and P-values for each principal component.
https://binary.pensoft.net/file/792254This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Ekaterina S. Konopleva, Ivan N. Bolotov, Ilya V. Vikhrev, Khamla Inkhavilay, Mikhail Yu. Gofarov, Alexander V. Kondakov, Alena A. Tomilova, Yulia E. Chapurina, Tu Van Do, John M. Pfeiffer, Manuel Lopes-Lima, Arthur E. Bogan10.3897/zse.99.90784.suppl47513485E37084EE-CFF1-5181-A114-BA8F5355B0F1
Figure S1. Fossil-calibrated Unionidae tree, including the genus Cristaria, based on the complete data set of mitochondrial and nuclear gene sequences (five partitions: three codons of COI + 16S rRNA + 28S rRNA)
Biogeographic analysis results (Tif file)
Figure S1. Fossil-calibrated Unionidae tree, including the genus Cristaria, based on the complete data set of mitochondrial and nuclear gene sequences (five partitions: three codons of COI + 16S rRNA + 28S rRNA). Black numbers under nodes are Bayesian posterior probabilities (BPP) of BEAST v. 1.10.4; red numbers above nodes are the mean node ages, Ma. Node bars represent 95% HPD intervals. Calibration points are marked by red stars. Stratigraphic chart according to the International Commission on Stratigraphy, 2021 (https://stratigraphy.org/chart).
https://binary.pensoft.net/file/792255This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Ekaterina S. Konopleva, Ivan N. Bolotov, Ilya V. Vikhrev, Khamla Inkhavilay, Mikhail Yu. Gofarov, Alexander V. Kondakov, Alena A. Tomilova, Yulia E. Chapurina, Tu Van Do, John M. Pfeiffer, Manuel Lopes-Lima, Arthur E. Bogan