17 3 NOTES ON GEOGRAPHIC DISTRIBUTION Check List 17 (3): 783–790 https://doi.org/10.15560/17.3.783 Rediscovery and genetic confirmation of the Threeridge Mussel, Amblema plicata (Say, 1817) (Bivalvia, Unionidae), in the Choctawhatchee River, Florida, USA Lauren N. Patterson1*, Susan R. Geda1, Nathan A. Johnson2 1 Florida Fish and Wildlife Conservation Commission, Milton, Florida, USA • LNP: [email protected] • SRG: [email protected] 2 U.S. Geological Survey, Gainesville, Florida, USA • [email protected] * Corresponding author Abstract Recent freshwater mussel research has resulted in rediscovery of several species presumed extinct. We report the rediscovery of Amblema plicata (Say, 1817) in 2019 from the Choctawhatchee River, Florida, USA. Amblema plicata has not been reported in the Choctawhatchee river basin since 1958, more than 61 years ago. This species was col- lected during the long-term monitoring of freshwater mussels in Florida streams. We provide genetic confirmation of our voucher identification using a DNA barcoding approach and discuss potential risks toA . plicata populations in the Choctawhatchee river basin. Keywords Biodiversity threats, distribution, DNA barcoding, freshwater, long-term monitoring, unionid Academic editor: Igor Christo Miyahira | Received 29 December 2020 | Accepted 5 May 2021 | Published 18 May 2021 Citation: Patterson LN, Geda SR, Johnson NA (2021) Rediscovery and genetic confirmation of the Threeridge Mussel, Amblema plicata (Say, 1817) (Bivalvia, Unionidae), in the Choctawhatchee River, Florida, USA. Check List 17 (3): 783–790. https://doi.org/10.15560/17.3.783 Introduction Freshwater mussels within the family Unionidae are one as bioindicators of the overall health of freshwater eco- of the most imperiled faunal groups in the world, with systems (Carlson et al. 2008; Grabarkiewicz and Davis over 65% of the 297 recognized North American spe- 2008). Recent research and survey efforts have resulted cies being of special concern, threatened, endangered, or in the rediscovery of several freshwater mussels that extinct (Williams et al. 1993; Neves et al. 1997; Haag and were presumed extinct (e.g., Campbell et al. 2008; Rand- Williams 2014). Historically, the southeastern United klev et al. 2012; Holcomb et al. 2015; Johnson et al. 2016; States had more freshwater mussel biodiversity than Pfeiffer et al. 2016) and the discovery of many previously any other geographic region on the continent, but pop- unrecognized species (e.g., Johnson et al. 2018; Smith et ulations have experienced declines since the early 20th al. 2018, 2019; Inoue et al. 2020). century (Neves et al. 1997). The highly imperiled status The genus Amblema (Rafinesque, 1820) is endemic of unionid mussels reflects their sensitivity to changes to North America and contains three currently recog- in their environment but allows biologists to use them nized species: A. elliottii (Lea, 1856), A. neislerii (Lea, © The authors. This 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. 784 Check List 17 (3) 1858), and A. plicata (Say, 1817) (Williams et al. 2017). a minimum of 1-person hour. Sites were selected ran- The Threeridge, A. plicata, was first described as Unio domly using point layers generated in ArcMap v. 10.6 plicata Say, 1817. The type locality for A. plicata is in the (Environmental Systems Research Institute, Inc., Red- Ohio River (Williams et al. 2014). There are at least 15 lands, CA, USA). Any vouchered specimens were placed synonyms of A. plicata (Williams et al. 2008), the most on ice for transit and transferred to 95% non-denatured notable being A. perplicata (Conrad, 1841), which has ethanol. been assigned to populations of Amblema from the lower All surveys conducted by the FMCP in the Choc- Mississippi river basin (Mulvey et al. 1997). However, tawhatchee river basin were compiled to develop a con- Mulvey et al. (1997) reported A. plicata and A. perplicata servation status assessment map following the protocol to be conspecific based on genetic similarities recovered created by the Georgia Department of Natural Resources from the 16s rRNA gene, which was later confirmed by (Georgia DNR 2020). The map was developed within Arc- Elderkin et al. (2007). Amblema elliottii was considered Map v. 10.6 (ESRI 2017). Hydrologic Unit Codes (HUC) a synonym of A. plicata prior to the genetic analysis by 12 are shaded in relation to time, allowing the assessment Mulvey et al. (1997), which demonstrated that A. elliottii of spatial and temporal changes. Surveys by FMCP were is more closely related to A. neislerii. The Coosa Fiver- conducted from 18 June 2014 to 29 August 2019. idge, A. elliottii, is endemic to the Mobile river basin in A DNA barcoding approach (Hebert et al. 2003) was Alabama and Georgia. The Fat Threeridge, A. neislerii, conducted to verify our morphology-based identifica- is federally endangered and endemic to the Apalachicola tion. Genomic DNA was extracted from a small (≤5.0 and Flint river basins in Florida and Georgia. mm2) piece of mantle tissue using the Qiagen PureGene Amblema plicata is currently recognized as stable DNA extraction kit following the standard extraction throughout most of its range, but its conservation status protocol (Qiagen, Hilden, Germany). The protein-coding in some states is undetermined (Williams et al. 2014). mitochondrial DNA gene cytochrome c oxidase subunit Amblema plicata has a widespread historical distribu- I (COI) was amplified using previously published PCR tion in North America. In its northern distribution, A. primers (Campbell et al. 2005) and thermal cycling con- plicata is present from the St. Lawrence River drain- ditions (Johnson et al. 2018) in a 25 µL mixture contain- age in New York throughout the entire Mississippi river ing the following: MyTaqTM Red Mix (12.5 µL; Meridian basin to Louisiana (Mulvey et al. 1997; Williams et al. Bioscience, Memphis, TN, USA), primers (1.0 µL each 2008). On the Gulf Coast, it occurs from the San Anto- at 10 mM), DNA template (1.0 µL at 100 ng µL−1), and nio and Guadalupe rivers in Texas (Howells et al. 1996) 9.5 µL molecular grade H O. PCR product was purified east to the Choctawhatchee River in Florida (Blalock- 2 with Exonuclease I and Shrimp Alkaline Phosphatase Herod et al. 2005). The Choctawhatchee River continues (Promege Corp.) and bi-directionally sequenced on an into Alabama, but A. plicata is not known to occur in ABI 3730 at Molecular Cloning Laboratories (McLAB, this reach (Williams et al. 2008, 2014). Within the state South San Francisco, CA, USA). Geneious v. 10.2.3 of Florida, A. plicata is known from the Escambia and (Kearse et al. 2012) was used to assemble forward and Choctawhatchee River drainages. The population in the reverse contigs before evaluating chromatograms and Escambia River is currently considered stable (Williams forming the consensus sequence, which was then trans- et al. 2014), while the population in the Choctawhatchee lated into amino acid sequences to ensure the absence River was thought to be extirpated (Blalock-Herod et al. of gaps and stop codons using Mesquite v. 3.61 (Mad- 2005) prior to locating a live individual in 2019 during one of our recent surveys. Here, we report the collec- dison and Maddison 2019). The consensus sequence tion of A. plicata in the Choctawhatchee River in 2019, was compared to published sequences on GenBank to the first occurrence in the basin since 1958 (Butler 1989; determine species-level identification using the NCBI Blalock-Herod et al. 2005), and provide the first genetic standard nucleotide BLASTN tool (Zhang et al. 2000) confirmation of the Choctawhatchee River population. with default megablast and nt database options on 20 April 2021. A maximum likelihood analysis (ML) was performed to visualize the relationships of the newly Methods generated sequence with the top 52 matches from the The Florida Fish and Wildlife Conservation Commission BLASTN search. The following nucleotide substitu- Freshwater Mussel Conservation Program (FMCP) rou- tion models were determined for each codon using tinely monitors freshwater mussel populations in Flori- ModelFinder (Kalyaanamoorthy et al. 2017): codon da’s freshwater streams and rivers to provide ecologically 1 – TN+F+I; codon 2 – F81+F; and codon 3 – HKY+F. significant findings to conservation managers. Both ML analyses were performed using IQ-TREE v. 2.0.6 quantitative and qualitative sampling procedures were (Nguyen et al. 2015) with 10 independent runs of ini- performed during these long-term monitoring efforts. In tial tree search and 1000 ultrafast bootstrap replicates to brief, quantitative sampling procedures included excava- assess nodal support (Hoang et al. 2018). The resulting tion of five 0.5-m2 quadrats along three 10-m transects phylogeny was presented as a 50% majority-rule consen- placed perpendicular to flow. Qualitative sampling pro- sus tree with Villosa villosa (B.H. Wright, 1898) (Gen- cedures included visual and tactile survey methods for Bank: MK044978.1) designated as the outgroup. Patterson et al. | Rediscovery of Amblema plicata in the Choctawhatchee River 785 Repositories. UF: University of Florida Museum of Nat- ural History, Invertebrate Zoology Collection. NCSM: North Carolina State Museum of Natural Sciences, Mol- lusks Collection. Results New records. UNITED STATES OF AMERICA – Flor- ida • Walton County, Choctawhatchee River; 30.4887, −85.8653; 5.VI.2019; L.N. Patterson, S.R. Geda, S.N. Casebolt leg.; 1 spec., 73 mm, 95% non-denatured etha- nol, UF 439505. From 2014 to 2019, the FMCP surveyed 110 sites in the Choctawhatchee river basin in Florida, totaling 117 person hours. A total of 16468 individual mussels repre- senting 22 species were collected (Supplemental Table S1). Only one individual of A. plicata was found dur- ing these efforts (Fig.