Molecular Phylogenetics and Evolution 130 (2019) 45–59 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Towards a global phylogeny of freshwater mussels (Bivalvia: Unionida): Species delimitation of Chinese taxa, mitochondrial phylogenomics, and T diversification patterns Xiao-Chen Huanga,b,1, Jin-Hui Sua,1, Jie-Xiu Ouyangc, Shan Ouyanga, Chun-Hua Zhoua, ⁎ Xiao-Ping Wua, a School of Life Sciences, Nanchang University, Nanchang 330031, China b Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, 69120 Heidelberg, Germany c Medical Laboratory Education Center, Nanchang University, Nanchang 330031, China ARTICLE INFO ABSTRACT Keywords: The Yangtze River Basin in China is one of the global hotspots of freshwater mussel (order Unionida) diversity DNA barcoding with 68 nominal species. Few studies have tested the validity of these nominal species. Some taxa from the Unionidae Yangtze unionid fauna have not been adequately examined using molecular data and well-positioned phylo- Yangtze River genetically with respect to the global Unionida. We evaluated species boundaries of Chinese freshwater mussels, DUI and disentangled their phylogenetic relationships within the context of the global freshwater mussels based on BAMM the multi-locus data and complete mitochondrial genomes. Moreover, we produced the time-calibrated phylo- Host-attraction geny of Unionida and explored patterns of diversification. COI barcode data suggested the existence of 41 phylogenetic distinct species from our sampled 40 nominal taxa inhabiting the middle and lower reaches of the Yangtze River. Maximum likelihood and Bayesian inference analyses on three loci (COI, 16S, and 28S) and complete mitochondrial genomes showed that the subfamily Unioninae sensu stricto was paraphyletic, and the subfamily Anodontinae should be subsumed under Unioninae. In addition, we described two new tribes (Aculamprotulini tribe nov. and Lepidodesmini tribe nov.) in the subfamily Unioninae and one new genus (Parvasolenaia gen. nov.) in the subfamily Gonideinae. Molecular dating analysis suggested freshwater mussels diversified at 346.1 Mya (HPD = 286.6–409.9). The global diversification rate for Unionida was estimated to be 0.025 species/Myr. Our study found only a single well-supported rate shift in Unionida diversification, occurring at the base of the subfamily Ambleminae. The evolution of active host-attraction may have triggered the burst of speciation in Ambleminae, and the environment and geography of the Mississippi River Basin likely sustained this radiation. 1. Introduction Eastern North America and East and Southeast Asia (Bogan, 2008; Graf and Cummings, 2007; Zieritz et al., 2017). Rivers and shallow lakes of Bivalves of the order Unionida (freshwater mussels) have a global the Yangtze River Basin, China are inhabited by the richest and most distribution with very high levels of endemism, inhabiting rivers and diverse assemblage of freshwater mussel species found in Asia, with lakes on all continents except Antarctica (Graf and Cummings, 2007). nearly 68 nominal species, or 68.7% percent of the 99 species found in They are divided into six families (i.e. Unionidae, Margaritiferidae, China (Zieritz et al., 2017). For over a century, taxonomic studies of Hyriidae, Etheriidae, Iridinidae, and Mycetopodidae), two of which Unionida species in this region were primarily based on shell mor- (Unionidae and Margaritiferidae) are widespread across the northern phology (He and Zhuang, 2013; Heude, 1875–1885; Lin, 1962; Liu continents (Bogan, 2008; Graf and Cummings, 2007). Freshwater et al., 1979; Tchang and Li, 1965; Tchang et al., 1965). However, the mussels have a distinct life cycle, in which most species are dependent Yangtze unionid fauna was poorly studied using molecular data. Past on host fishes for their parasitic larvae (i.e. glochidia and lasidia). works have focused either on some genera (Zhou et al., 2007)orona Global hotspots of freshwater mussel diversity and endemism are sparse sampling of genera represented by only one or two species using ⁎ Corresponding author. E-mail address: [email protected] (X.-P. Wu). 1 Co-first authors. https://doi.org/10.1016/j.ympev.2018.09.019 Received 4 April 2018; Received in revised form 18 August 2018; Accepted 28 September 2018 Available online 08 October 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved. X.-C. Huang et al. Molecular Phylogenetics and Evolution 130 (2019) 45–59 one gene (Huang et al., 2002). Although substantial progress has been understanding the evolutionary history of the freshwater mussels, and recently made in identifying the major clades of Unionida and the re- discussed whether active host-attraction clade (i.e. Ambleminae) had lationships among them (e.g. Bolotov et al., 2017a, 2017b; Lopes-Lima an increased diversification rate relative to the remainder of Unionida. et al., 2017b), some genera from the Yangtze fauna have not been adequately sampled and well-positioned phylogenetically with respect 2. Materials and methods to the global Unionida. In addition, little is known about the validity of nominal species in 2.1. Taxon sampling and taxonomic identification the Yangtze River Basin. Species concepts were based mainly on sub- jective morphological characters, and species delimitation of Chinese A total of 302 specimens were collected during 2013–2015 from taxa has been left untouched in previous phylogenetic studies. Our representative sites in the middle and lower reaches of the Yangtze present understanding of species-level freshwater mussel diversity in River. Voucher specimens have been deposited in Nanchang University. the Yangtze River Basin was relied heavily on century-old work of The detailed information of taxa, collection data, vouchers and acces- Pierre Marie Heude (1875–1885) who described about 140 Chinese sion numbers was provided in Supplementary Table S1. Preliminary taxa by recognizing subtle shell variants as distinct species. Though this identification of species was conducted morphologically by referring to approach resulted in an inflated number of species because of pheno- the body of literature (Heude, 1875–1885; Liu et al., 1979; Tchang and typic plasticity, synonymization without rigorous molecular analysis Li, 1965; Tchang et al., 1965). Disputed species and subspecies were from subsequent revisions (e.g. Haas, 1969; He and Zhuang, 2013; Liu provisionally defined as species pending for further molecular analyses. et al., 1979; Simpson, 1900, 1914) lack of recognition of phylogeneti- In the present study, original data sampling comprised 39 (ca. 60%) cally distinctive taxa. For the estimate of North American unionid di- species in 16 (72.7%) genera of the superfamily Unionoidea (i.e. versity, phylogenetic studies have already yielded surprising departures Unionidae + Margaritiferidae) in the Yangtze River Basin, among from traditional classifications (Haag, 2012). Therefore, an estimate of which six Chinese species from five genera have not been included in Chinese unionid diversity awaits rigorous phylogenetic studies based on earlier phylogenetic studies. Additionally, sequences of Unionida spe- molecular data. We herein implemented COI barcoding to fill the gap cies from recent studies on Southeast Asian freshwater mussel assem- where morphometric analyses of congeneric species and comprehensive blage (Bolotov et al., 2017a, 2017b) as well as the revision of the Un- multi-locus data from both mtDNA and nDNA are lacking. ionidae classification (Lopes-Lima et al., 2017b) were included, making While it is increasingly compelling that a small number of genes this study the most taxonomically comprehensive to date for Unionida cannot resolve deep nodes, mitogenome data are known to be highly phylogenetic analyses. effective in disentangling both shallow and deep relationships in freshwater mussels (e.g. Huang et al., 2018; Lopes-Lima et al., 2017a; 2.2. DNA extraction and PCR amplification Zhou et al., 2017). Sequencing of Chinese unionid mitogenomes in re- cent years provided an increasing number of molecular data for re- Total genomic DNA was extracted on the dissected somatic tissues vealing freshwater mussel relationships. However, no attempt has been using TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, made yet to check the source organism of these mitogenomes because China) following to the manufacturer's protocol. To investigate species of the lack of comprehensive molecular data to aid species identifica- delimitation of Chinese freshwater mussels, two loci (F-type COI- tion. mtDNA as the barcode marker and nuclear 28S ribosomal DNA) were Unionida diversity is not equally distributed across the order. generated. For all individuals, an ∼680 bp fragment of COI (LCO22me2 Unionidae is the most species rich and most widespread family in the 5′-GGTCAACAAAYCATAARGATATTGG-3′ and HCO700dy2 5′-TCAGG- order Unionida, the greatest number of which belong to the subfamily GTGACCAAAAAAYCA-3′)(Walker et al., 2007, 2006) and an ∼420 bp Ambleminae, distributed in Nearctic and Mesoamerica; about 342 fragment of 28S (D23F 5′-GAGAGTTCAAGAGTACGTG-3′ and D4RB species have been described in Ambleminae, taking up over half of the 5′-TGTTAGACTCCTTGGTCCGTGT-3′)(Park and Foighil, 2000) were species-level diversity in the family (Graf and Cummings, 2007; amplified. Williams et al., 2017). Therefore, macroevolutionary patterns across PCR conditions and processes were as follows: 94 °C for 5 min; 35 extant