Molecular Phylogenetics and Evolution 137 (2019) 114–126 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Unioverse: A phylogenomic resource for reconstructing the evolution of T freshwater mussels (Bivalvia, Unionoida) ⁎ John M. Pfeiffera, , Jesse W. Breinholta,b, Larry M. Pagea a Florida Museum of Natural History, 1659 Museum Road, Gainesville, FL 32611, United States b RAPiD GENOMICS, 747 SW 2nd Ave IMB #314, Gainesville, FL 32601, United States ARTICLE INFO ABSTRACT Keywords: Freshwater mussels (order Unionoida) are a diverse radiation of parasitic bivalves that require temporary larval Anchored hybrid enrichment encystment on vertebrate hosts to complete metamorphosis to free-living juveniles. The freshwater mussel-fish Phylogenetics symbiosis represents a useful relationship for understanding eco-evolutionary dynamics in freshwater ecosys- Bivalvia tems but the practicality of this promising model system is undermined by the absence of a stable freshwater Unionoida mussel phylogeny. Inadequate character sampling is the primary analytical impediment obfuscating a coherent Unionidae phylogeny of freshwater mussels, specifically the lack of nuclear molecular markers appropriate forre- Unionida constructing supraspecific relationships and testing macroevolutionary hypotheses. The objective of this studyis to develop a phylogenomic resource, specifically an anchored hybrid enrichment probe set, capable of capturing hundreds of molecular markers from taxa distributed across the entirety of freshwater mussel biodiversity. Our freshwater mussel specific anchored hybrid enrichment probe set, called Unioverse, successfully captures hun- dreds of nuclear protein-coding loci from all major lineages of the Unionoida and will facilitate more data-rich and taxonomically inclusive reconstructions of freshwater mussel evolution. We demonstrate the utility of this resource at three disparate evolutionary scales by estimating a backbone phylogeny of the Bivalvia with a focus on the Unionoida, reconstructing the subfamily-level relationships of the Unionidae, and recovering the sys- tematic position of the phylogenetically unstable genus Plectomerus. 1. Introduction biology, but also our appreciation of freshwater ecology and evolution more broadly (e.g., comparative biogeography, coevolution, commu- Freshwater mussels (order Unionoida) are an ancient, globally dis- nity assembly). tributed, and species-rich radiation distinguished from all other bi- Beyond the macroevolutionary insights extended from a robust valves by their restriction to freshwater ecosystems and having an ob- phylogeny of the Unionoida are its varied uses to the freshwater mussel ligate parasitic life history (Graf and Cummings, 2006, 2007). These conservation community (e.g., identifying and preserving phylogenetic parasitic bivalves require temporary larval encystment on vertebrate diversity, utilizing synapomorphy to infer ecological interactions and hosts, primarily freshwater fishes, to complete metamorphosis from population-level processes) – an enterprise aimed at stewardship of one parasitic larvae to free-living juveniles. This parasitic relationship is of the world’s most threatened animal groups (Lopes-Lima et al., 2018). thought to be Triassic in origin (> 200MY; Watters, 2001) and ubi- The objective of the present study is to develop an accessible phylo- quitous in permanent freshwater habitats globally, making it a con- genomic resource, specifically an anchored hybrid enrichment probe sequential selective force shaping the evolution and ecology of both set, that facilitates more data-rich and taxonomically inclusive re- symbionts. The freshwater mussel-fish symbiosis represents an inter- constructions of freshwater mussel evolution. esting and useful relationship for better understanding eco-evolutionary The application of molecular phylogenetics has transformed fresh- dynamics of two of the most diverse, ecologically important, and im- water mussel systematics but limited phylogenetic consensus has periled animals in freshwater ecosystems. However, the practicality of emerged in regard to the early evolution of the Unionoida. This is this promising evolutionary and ecological model system is undermined especially true of the relationships of the families and the suprageneric by the absence of a stable freshwater mussel phylogeny. This systematic clades of its most diverse radiation, the Unionidae (Graf, 2013). In- shortcoming not only limits our understanding of freshwater mussel adequate character sampling appears to be the primary analytical ⁎ Corresponding author. E-mail address: [email protected] (J.M. Pfeiffer). https://doi.org/10.1016/j.ympev.2019.02.016 Received 28 November 2018; Received in revised form 25 January 2019; Accepted 18 February 2019 Available online 21 February 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved. J.M. Pfeffer, et al. Molecular Phylogenetfcs and Evolutfon 137 (2019) 114–126 fmpedfment obfuscatfng a coherent phylogeny of the Unfonofda, spe- fncludfng bfvalves ( Gonzalez et al., 2015 ;Lemer et al,. 2019 ), gastro- cffcally the lack of nuclear molecular markers approprfate for estf- pods ( Zapata et al., 2014 ), cephalopods ( Lfndgren and Anderson, 2018 ), matfng supraspecffc relatfonshfps and testfng macroevolutfonary hy- and the phylum as a whole ( Smfth et al., 2011; Kocot et al., 2011 ). potheses ( Graf and Cummfngs, 2006, 2010; Hoeh et al., 2009 ). The Although sequence capture has been used to reconstruct the evolutfon fnadequacfes of the avaflable molecular markers fn descrfbfng the of many specfes-rfch and phylogenetfcally dfverse clades (e.g., hfgher-level relatfonshfps of freshwater mussels fs apparent fn the nu- vertebrates– Lemon et al., 2012 ; brfttle stars– Hugall et al., 2015 ; mfn- merous conlfctfng hypotheses generated usfng the conventfonal two- or nows and relatfves– Stout et al., 2016 ; butterlfes and moths– Brefnholt three-locus reconstructfons ( Ffg. 1 ). Fortunately, approaches for gen- et al., 2018 ; maylfes– Mfller et al., 2018 ), ft has only begun to re- eratfng genome-scale datasets desfgned to estfmate the evolutfonary volutfonfze molluscan phylogenomfcs (Gastropoda, Confdae: relatfonshfps of phylogenetfcally dfvergent groups fs becomfng much Abdelkrfm et al., 2018; Phuong and Mahardfka, 2018 ). One logfstfcal more accessfble for non-model organfsms ( McCormack et al., 2013 ). advantage of AHE over phylotranscrfptomfcs fs that ft has less de- Two fncreasfngly common methods of generatfng phylogenomfc mandfng sample requfrements. Rather than relyfng on fresh tfssue or datasets afmed at estfmatfng hfgher-level evolutfonary relatfonshfps are properly preserved RNA, as fs the case wfth phylotranscrfptomfcs, AHE transcrfptome sequencfng (a.k.a. RNA-seq) and sequence capture (e.g., can be used to sequence the genomfc DNA contafned fn a wfde varfety ultraconserved elements and anchored hybrfd enrfchment). of hfstorfcal samples, fncludfng alcohol-preserved specfmens, drfed Transcrfptomfc approaches to reconstructfng phylogeny typfcally fn- tfssues, and shells ( Yeates et al., 2016; Sproul and Maddfson, 2017; Der volve extractfng hfgh qualfty RNA from fresh or properly preserved Sarkfssfan, 2017 ). The capacfty to more completely leverage the tfssues, next-generatfon sequencfng of cDNA lfbrarfes, orthology de- genomfc resources of hfstorfcal specfmens would be an fmportant de- tectfon, and the assembly of hundreds of multfple sequence alfgnments. velopment fn freshwater mussel systematfcs, especfally gfven the hfgh Sequence capture, and specffcally anchored hybrfd enrfchment (AHE: proportfon of extfnct and rare taxa. Lemmon et al., 2012 ), uses genetfc resources (genomes and tran- The objectfve of thfs study fs to develop an AHE probe set desfgned scrfptomes) to fdentffy target regfons to whfch DNA probes are desfgned to capture hundreds of molecular markers from taxa dfstrfbuted across to hybrfdfze wfth and selectfvely enrfch to facflftate capture durfng the entfrety of freshwater mussel bfodfversfty and demonstrate the next-generatfon sequencfng, resultfng fn hundreds of preselected mo- phylogenetfc utflfty of these locf at multfple evolutfonary scales. We lecular markers sequenced from taxa dfstrfbuted across the desfred desfgned our taxon samplfng to 1) estfmate a backbone phylogeny for phylogenetfc scope. the Bfvalvfa wfth a focus on the Unfonofda, 2) re-evaluate the sub- To date, systematfc malacology has, wfth great success, relfed lar- famfly-level relatfonshfps of the most specfes-rfch unfonofd famfly, the gely on transcrfptomfc approaches to generate phylogenomfc datasets Unfonfdae, and 3) determfne the trfbe-level posftfon of the phylogen- and estfmate the evolutfonary hfstory of dfverse molluscan groups, etfcally unstable North Amerfcan genus Plectomerus . Ffg. 1. Summary of recent multf-locus phylogenetfc hypotheses fncludfng all the famflfes of the Unfonofda, all subfamflfes of the Unfonfdae, and the posftfon of Plectomerus among the subfamfly Amblemfnae. 115 J.M. Pfeiffer, et al. Molecular Phylogenetics and Evolution 137 (2019) 114–126 Table 1 Taxa, vouchers, and source information utilized in phylogenomic analysis of the Bivalvia. Taxon Voucher Accession Source Type PROTOBRANCHIA Nuculidae Ennucula tenuis (Montagu, 1808) NA SRR331123 Smith et al. (2011) Transcriptome Solemyidae Solemya velum Say 1822 NA SRR330465 Smith et al. (2011) Transcriptome PTERIOMORPHA Mytillidae Bathymodiolus platifrons