DNA Barcoding Permits Identification of Potential Fish ...

DNA Barcoding Permits Identification of Potential Fish Hosts of Unionid Freshwater Mussels Author(s): Nathaniel T. Marshall, Joshua A. Banta, Lance R. Williams, Marsha G. Williams and John S. Placyk, Jr Source: American Malacological Bulletin, 36(1):42-56. Published By: American Malacological Society https://doi.org/10.4003/006.036.0114 URL: http://www.bioone.org/doi/full/10.4003/006.036.0114

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Amer. Malac. Bull. 36(1): 42–56 (2018)

DNA barcoding permits identification of potential fish hosts of unionid freshwater mussels Nathaniel T. Marshall1,2, Joshua A. Banta1, Lance R. Williams1, Marsha G. Williams1, and John S. Placyk, Jr1 1

Department of Biology, University of Texas at Tyler, 3900 University Blvd., Tyler, Texas 75799, U.S.A.

2

Current address - Great Lakes Genetics/Genomics Laboratory, the Lake Erie Center and Department of Environmental Sciences, The University of Toledo, 6200 Bayshore Rd., Oregon, Ohio 43616, U.S.A., [email protected]

Abstract: Fish have an ecologically significant role in the life-history of unionid freshwater mussels, as the larvae of most species are obligate ectoparasites (glochidia) on fish hosts. Although this ecological interaction is vital to freshwater mussel conservation, there is a paucity of data on fish-host specificity for many species. A species-specific DNA barcoding dataset utilizing the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene was used to identify 154 glochidia attached to wild fish collected from March through August of 2013 in the Sabine and Neches rivers in Texas, U.S.A. These data include the first report of potential hosts for two state-threatened species, Fusconaia askewi (Marsh, 1896) and Pleurobema riddellii (I. Lea, 1862), as well as potential hosts for Amblema plicata (Say, 1817), Obliquaria reflexa (Rafinesque, 1820), Plectomerus dombeyanus (Valenciennes, 1827), Potamilus purpuratus (Lamarck, 1819), Quadrula mortoni (I. Lea, 1831), Q. verrucosa (Rafinesque, 1820), and Truncilla truncata (Rafinesque, 1820). Cyprinella lutrensis appears to be the primary host for F. askewi, as 50% (54/108) of its glochidia were found on this minnow species alone. Pleurobema riddellii may be a cyprinid specialist, infesting only C. lutrensis and Pimephales vigilax. Alternatively, F. askewi may be a host generalist, as glochidia were found encysted on 17 fish species suggesting that host fish availability may not be an important factor contributing to observed population declines. The findings here will be instrumental in the future conservation of these species, through the translocation to correct habitat and developing successful propagation programs.

Key words: glochidia, ND1, parasite-host, Texas, unionids

The freshwater mussel fauna of North America is the richest of any continent, comprising ~300 of 840 global species (Graf and Cummings 2007). However, this diversity is currently threatened, in part, by habit degradation, dam construction, and introduced species (Williams et al. 1993, Strayer et al. 2004, Haag 2012). As a result of these mostly anthropogenic influences, over 70% of freshwater mussels endemic to North America are currently listed as endangered, threatened, or species of special concern (Haag and Williams 2014). Unfortunately, the management of this group is made difficult due to a lack of life-history information on its members, which is critical for successful conservation management strategies (Hoggarth 1992, Ford and Oliver 2015). An important life-history trait unique to freshwater mussels is their unusual life cycle in which larvae (glochidia) of most species are obligate ectoparasites on fish hosts (Kat 1984), believed to have evolved as a dispersal mechanism (Wächtler et al. 2001). Glochidia only successfully metamorphosize into juveniles on suitable host fish species (Neves et al. 1985). However, host fish identification is lacking for many mussels (Hoggarth 1992). In fact, of the 52 species found in Texas, only 34 have information on potential fish hosts, with data missing for 15 state or federally listed species (Ford and Oliver 2015). Artificial propagation of juvenile mussels is a necessary method for conservation and recovery

of many unionid species (Neves 2004). Successful rearing of unionids is not possible without the prior knowledge of a suitable host fish. Other conservation management strategies rely on the translocation of unionids from degraded/impacted habitat to one that is more suitable (Cope and Waller 1995, Cope et al. 2003). However, the recruitment, and ultimately growth of a population, will be unsuccessful without the presence of a suitable host at the translocation site. Traditional approaches used for host identification involve the artificial infestation of fish in a laboratory setting (e.g. Watters and O’Dee 1998, Khym and Layzer 2000, van Snik Gray et al. 2002). This approach allows for fish to be exposed to one mussel species at a time, leading to easy identification of the juvenile mussel upon successful metamorphosis. Although laboratory trials provide important insight into mussel-fish relationships, they ignore key differences in spatial overlap and behavioral interactions. For example, on Vancouver Island, Anodonta kennerlyi (I. Lea, 1860) glochidia infested four species of fishes, but strongly associated with hosts (those with greater frequency and intensity) using similar habitat (Martel and Lauzon-Guay 2005). Similarly, in Maine rivers, Lampsilis cariosa (Say, 1817) and Leptodea ochracea (Say, 1817) were found to overwhelmingly infest white perch, Morone americana, in the wild, even though laboratory trials demonstrated higher transformation success 42

POTENTIAL FISH HOSTS FOR THREATENED UNIONIDS

on yellow perch, Perca flavescens (Kneeland and Rhymer 2008). To counter the drawbacks of laboratory-based host identification, molecular genetic keys based on restriction fragment length polymorphisms (RLFPs) and/or DNA barcoding have been used to identify glochidia encysted on fish in the wild (Kneeland and Rhymer 2008, Boyer et al. 2011, Zieritz et al. 2012). Although host effectiveness can be hypothesized via laboratory infestations, capturing already parasitized fish and identifying the encysted glochidia provides more meaningful evidence of true host-mussel interactions (Martel and Lauzon-Guay 2005, Kneeland and Rhymer 2008). Additionally, laboratory infestations are constrained to screening hosts for one or a few mussel species at a time, where identifying glochidia from naturally parasitized fish allows for the potential to yield information on host interactions for the complete mussel and fish community. The purpose of this study was to identify mussel glochidia and host fishes in east Texas rivers supporting 37 mussel species including six state-threatened species (i.e., Fusconaia askewi (Marsh, 1896), F. lananensis (Frierson, 1901), Obovaria arkansasensis (I. Lea, 1862), Lampsilis satura (I. Lea, 1852), Pleurobema riddellii (I. Lea, 1862), and Potamilus amphichaenus (Frierson, 1898)). Glochidia found encysted on fish were identified to the species level using DNA barcoding of the mtDNA NADH dehydrogenase subunit 1 (ND1) gene. This knowledge will benefit future translocation and propagation efforts.

MATERIALS AND METHODS DNA sequencing DNA was extracted from adult foot and adductor muscle tissue using an Illustra tissue and cells genomicPrep mini spin kit (GE Healthcare, Buckinhamshire, UK) and resuspended in 100μL of elution buffer. Genomic DNA was extracted from a single glochidium similarly, except the amounts of buffers and proteinase K was reduced by one half and resuspended in 75 μL of elution buffer (Kneeland and Rhymer 2008). All DNA extractions were stored at -20 °C until genetic analysis. Amplification of the ND1 gene was carried out using the primers Leu-uurF and LoGlyR (Serb et al. 2003), following a 20μL polymerase chain reaction (PCR) consisting of 1 unit Taq polymerase, 200μM dNTPs, 50mM KCL, 2mM MgCl2, 0.5μM of each primer, and ~200ng DNA. A negative control was included with each PCR. Reactions were amplified with an Eppendorf Mastercycler gradient thermal cycler with a temperature-controlled lid. Reaction conditions for double-stranded amplification consisted of an initial denaturation at 94 oC for 5min, followed

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by 30 cycles of 94 oC for 45 s, 54 oC for 60 s, and 72 oC for 60 s, and a final extension of 72 oC for 5 min. PCR products were purified using an E.Z.N.A. cycle pure kit (Omega biotek, Norcross, GA) and resuspended in 60 μL of sterile water. Purified DNA was concentrated to the level recommended by Eurofins MWG Operon (20–40 ng/μL) and shipped for sequencing using BigDye Terminator v 3.1 Cycle Sequencing kits (Applied Biosystems). Consensus contig sequences were assembled and edited using BioEdit v7.2.6.1. Development of molecular dataset Fifty-seven sequences (GenBank accession numbers MG030348-MG030375 and MG020448-MG020478) from mussel tissue collected in east Texas (Fig. 1, Table 1) was combined with 117 sequences available on the U.S. National Institutes of Health GenBank database (http://blast.ncbi.nlm. nih.gov), for a total of 174 sequences with representatives across all 37 mussel species. These 174 sequences were aligned using ClustalX, and then analyzed for uncorrected sequence pairwise distances (p-distance) using Mega v7.2 (Tamura et al. 2007). Fish and glochidia sampling Fish sampling was conducted at one site in the Sabine River and one site in the Neches River (Fig. 1). The Sabine River site was sampled every two weeks from March through August and the Neches River site was sampled in June and August of 2013. Fish were captured near a large diverse mussel bed using two sampling methods: beach seining and electrofishing. Sites were composed of different habitats, including riffles and pools, to increase the diversity of fish and mussel species present. Standardized sampling conditions were implemented to have a similar catch per unit effort (CPUE) on each sampling date. The conditions consisted of a three-person sampling team, in a 150 m reach, for one hour. Collections used a 7.5 m bag seine and a Halltech Aquatic Research INC. HT-2000 backpack electrofisher. Fish species were identified in the field, sacrificed with a lethal dose of tricaine methanesulfonate (MS-222, 200 mg/L), and preserved in 95% ethanol. Gills and fins were excised from fish and examined for glochidia under a compound light microscope with the addition of a 2% KOH solution. Individual glochidium were removed with dissecting probes, preserved in 95% ethanol, and stored at -20 oC prior to DNA sequencing. A subset of glochidia was selected for genetic analysis with representatives from every fish species found to be encysted and across the temporal sampling period. More glochidia were selected for analysis from the Sabine River (n = 153) than the Neches River (n = 37), as sampling was more intense in the Sabine. Sequenced glochidia were identified to

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causing problems in separating the two species. Low levels of interspecific variation could reflect very recent divergence, or, as suggested by Burlakova et al. (2012), the incorrect splitting of a single species. Fusconaia askewi is the only one of these two species present in the Sabine River (Ford et al. 2014), and therefore any glochidia identified as this species complex in this river, were labeled as F. askewi. However, both species are found in the Neches River (Ford et al. 2014), and thus glochidia could not be identified precisely. Therefore, any glochidia identified to this species complex from the Neches River were labeled as F. askewi/lananensis. Prevalence and abundance of glochidia A total of 1566 fishes (43 species) were captured and examined for the presence of glochidia from the Sabine River and 142 fishes (13 species) were collected and examined from the Neches River (Table 2). Fishes from the Sabine River were infested with a total Figure 1. Collection sites of adult mussel tissue for the creation of a DNA barcoding dataset. of 6721 glochidia and fishes from the Species sampled from each location are listed in Table 1. Fish sampling for glochidia identifi- Neches River were infested with 824 cation occurred at sites 4 in the Sabine River and 11 in the Neches River. glochidia. All glochidia detected were encysted on gills. Peaks in glochidia abundance were observed throughout the species level by analyzing the genetic divergence with the the sampling season at the Sabine River on early May, early created ND1 dataset. June, and late June (Fig. 2). In the Sabine River, 578 fishes (37%) representing 23 species were infested with one or more glochidia, whereas in the Neches River, 87 fishes RESULTS (61%) representing 7 species were infested with one or more glochidia (Table 2, species captured but not encysted with Assessment of molecular dataset glochidia are found in Supplementary Table 2). All 7 fish A 750 base pair (bp) region of the ND1 gene was used for species infested from the Neches River were also infested interspecific comparisons in order to distinguish unionid in the Sabine River. Among fishes from the Sabine River species. Seven of the east Texas mussel species were sequenced that were captured most often (20 fish examined), for the ND1 gene for the first time, Anodonta suborbiculata Cyprinella venusta was most frequently parasitized (72%) (Say, 1831), Lampsilis hydiana (I. Lea, 1831), L. satura, L. teres and often heavily parasitized (18% with 20 encysted (Rafinesque, 1820), Potamilus amphichaenus, P. purpuratus glochidia, Fig. 3A, B). Cyprinella lutrensis was similarly (Lamarck, 1819), and Toxolasma texasensis (I. Lea, 1860). infested (64%), but was more heavily parasitized (22% Interspecific variation was high between most taxa (well over with 20 encysted glochidia). Similar infestation rates were 3.0% for most pairwise comparisons) allowing for unmistakseen in C. lutrensis and C. venusta from the Neches River able species distinction (i.e., Serb et al. 2003, Boyer et al. 2011) (81% and 75% respectively). Glochidia primarily encysted (Supplementary Table 1). However, the variation between on the family Cyprinidae, with C. lutrensis, C. venusta, and Fusconaia askewi and F. lananensis was low (