CSIRO PUBLISHING Invertebrate Systematics, 2018, 32, 689–702 https://doi.org/10.1071/IS17059 The Pleurobemini (Bivalvia : Unionida) revisited: molecular species delineation using a mitochondrial DNA gene reveals multiple conspecifics and undescribed species Kentaro Inoue A,I, David M. Hayes B, John L. Harris C, Nathan A. Johnson D, Cheryl L. Morrison E, Michael S. Eackles E, Tim L. King E, Jess W. JonesF,G, Eric M. HallermanF, Alan D. Christian C,H and Charles R. Randklev A ANatural Resources Institute, Texas A&M University, Dallas, TX 75252, USA. BDepartment of Biological Sciences, Eastern Kentucky University, Richmond, KY 40475, USA. CDepartment of Biological Sciences, Arkansas State University, Jonesboro, AR 72467, USA. DUS Geological Survey, Wetland and Aquatic Research Center, Gainesville, FL 32653, USA. EUS Geological Survey, Leetown Science Center, Kearneysville, WV 25430, USA. FDepartment of Fish and Wildlife Conservation, Virginia Tech, Blacksburg, VA 24061, USA. GUS Fish and Wildlife Service, Blacksburg, VA 24061, USA. HSchool for the Environment, University of Massachusetts, Boston, MA 02125, USA. ICorresponding author. Email: [email protected] Abstract. The Pleurobemini (Bivalvia: Unionida) represent approximately one-third of freshwater mussel diversity in North America. Species identification within this group is challenging due to morphological convergence and phenotypic plasticity. Accurate species identification, including characterisation of currently unrecognised taxa, is required to develop effective conservation strategies because many species in the group are imperiled. We examined 575 cox1 sequences from 110 currently recognised species (including 13 Fusconaia and 21 Pleurobema species) to understand phylogenetic relationships among pleurobemine species (mainly Fusconaia and Pleurobema) and to delineate species boundaries. The results of phylogenetic analyses showed no geographic structure within widespread species and illustrated a close relationship between Elliptio lanceolata and Parvaspina collina. Constraint tests supported monophyly of the genera Fusconaia and Pleurobema, including the subgenus P. (Sintoxia). Furthermore, results revealed multiple conspecifics, including P. hanleyianum and P. troschelianum, P. chattanoogaense and P. decisum, P. clava and P. oviforme, P. rubrum and P. sintoxia, F. askewi and F. lananensis, and F. cerina and F. flava. Species delimitation analyses identified three currently unrecognised taxa (two in Fusconaia and one in Pleurobema). Further investigation using additional genetic markers and other lines of evidence (e.g. morphology, life history, ecology) are necessary before any taxonomic changes are formalised. Additional keywords: DNA barcode, freshwater mussels, generalised mixed Yule-coalescent, molecular systematics, phylogenetics, species delimitation. Received 1 July 2017, accepted 25 October 2017, published online 1 June 2018 Introduction (Bickford et al. 2006). In relatively immobile organisms, there External morphologies, such as molluscan shells and arthropod is often high morphological variation due to interaction between exoskeletons, are the primary characteristics used by taxonomists genetic factors and ambient environmental conditions. For to describe and identify species. However, external morphology example, in freshwater mussels (Bivalvia: Unionida; hereafter, can be influenced by the environment, biological interactions, freshwater mussels), it is known that shell shape and sculpture can and genetics (i.e. phenotypic plasticity) (Via et al. 1995). vary with stream position and hydrodynamic conditions Thus, a single nominal species may exhibit multiple distinct (Ortmann 1920; Watters 1994). At the same time, evolutionary morphologies. In contrast, cryptic diversity can arise under convergence in shell morphologies is relatively common among those same conditions leading to cases where two or more freshwater mussel species (see Haag 2012 for a summary). Thus, distinct species share similar morphological characteristics, phenotypic plasticity combined with the existence of cryptic making them externally indistinguishable (i.e. cryptic species) species makes morphology-based identification of certain Journal compilation Ó CSIRO 2018 www.publish.csiro.au/journals/is 690 Invertebrate Systematics K. Inoue et al. taxonomic groups of freshwater mussels a challenge and likely We used molecular phylogenetic and species delimitation leads to under- and overestimates of the total species diversity approaches to revisit phylogenetic differentiation among within given taxa. The advent of species delimitation via species within Pleurobemini using additional species/ molecular genetic techniques can address both issues by populations collected for this study and data from previous determining putative species boundaries within and among studies (Tables S1 and S2). Our objectives were to: (1) delimit species and allow hidden biodiversity to be uncovered. putative species within Pleurobemini, focusing on the genera Freshwater mussels reach their greatest species diversity Fusconaia and Pleurobema, and (2) examine cryptic diversity in North America, with ~300 currently recognised species within Pleurobemini in the context of phylogenetic patterns (Haag and Williams 2014), although diversity may be higher among other mussel species. than is currently recognised due to imperfect understanding of mussel taxonomy, which is based primarily on shell morphology Methods (Williams et al. 1993; Turgeon et al. 1998; Graf and Cummings 2007). For example, recent molecular phylogenetics and Taxon sampling, sample preparation, and sequencing species delimitation studies have uncovered cryptic diversity We included 575 specimens (110 species, 35 genera/subgenera) among freshwater mussels (Serb et al. 2003; Gangloff et al. (Tables S1 and S2), of which 223 specimens (105 species, 35 2006; Jones et al. 2006; Inoue et al. 2013; Pfeiffer et al. 2016; genera/subgenera) (Table S2) were obtained from GenBank, Perkins et al. 2017), providing empirical support that mussel and we assimilated previously unpublished data for 352 diversity within North America continues to be underestimated. specimens from 42 species representing 11 genera for the The current classification within the family Unionidae current study. A list of taxa, waterbody of collection, unique recognises seven subfamilies, of which three occur in North identifiers, and GenBank accession numbers are provided in America: Ambleminae, Anodontinae, and Gonideinae (Lopes- Table S1. We extracted genomic DNA from mantle tissue via Lima et al. 2017). Ambleminae contains four tribes: Amblemini, a CTAB/chloroform extraction method (Saghai-Maroof et al. Lampsilini, Pleurobemini, and Quadrulini (Lopes-Lima et al. 1984) or using the PureGene DNA extraction kit (Gentra 2017). Many species within these groups have not been examined Systems, Minneapolis, MN) following manufacturer’s using molecular phylogenetics, resulting in their exact taxonomic guidelines. After quantification using fluorescence (Labarca placement being uncertain. Resolving these taxonomic and and Paigen 1980), extracted DNA was diluted to 10 ng mL–1 systematic uncertainties is important for the development of and used as a template in polymerase chain reaction (PCR) for the sound conservation actions, such as listing under the US mtDNA cytochrome c oxidase subunit I (cox1). We used cox1 Endangered Species Act and design of associated recovery plans. primers and the thermal conditions for PCR described in The Pleurobemini was recognised as a distinct subfamily Campbell et al.(2005). Products from PCR were visualised by Hannibal (1912). Campbell and Lydeard (2012a) provided using 1% agarose gel electrophoresis and purified with the first modern review of the systematic and taxonomic QIAquick Gel Extraction Kit (Qiagen) or ExoSAP-IT history of this group using external morphology, anatomy, (Affymetrix). We performed cycle sequencing reactions with and molecular phylogenetics, which resulted in several BIGDYE 3.1 and sequenced bidirectionally using an ABI Genetic nomenclatural revisions. For example, the genera Quincuncina Analyzer (Applied Biosystems). Sequences were visually aligned and Lexingtonia were synonomised within Fusconaia, and in DNADYNAMO (Blue Tractor Software, Ltd) and an open- Pleuronaia was elevated from synonomy within Fusconaia. reading frame was verified. We used MAFFT 7 (Katoh and Several genera within Pleurobemini were removed from the Standley 2013) to perform multiple sequence alignment. tribe, which included Cyclonaias Ortmann & Walker, 1922, Uniomerus Conrad, 1853, and the new genus Reginaia Campbell & Lydeard, 2012. Furthermore, due to phylogenetic Phylogeny estimation distinctiveness, Campbell and Lydeard (2012a) suggested The primary aim of this study was to examine phylogenetic elevation of the subgenera Eurynia and Sintoxia from relationships within two genera (Fusconaia and Pleurobema) synonomy within Elliptio and Pleurobema, respectively, and in Pleurobemini; therefore, we included 62 species from potential placement of Pleurobema collina in a new genus. 10 pleurobemine genera/subgenera (Elliptio, Elliptoideus, Subsequently, Perkins et al.(2017) analysed the molecular Eurynia, Fusconaia, Hemistena, Parvaspina, Plethobasus, systematics of P. collina and described the new genus Pleurobema, Pleurobema (Sintoxia), and Pleuronaia) and 47 Parvaspina, which included the species previously recognised species representing three subfamilies in Unionidae