Molecular Phylogenetics and Evolution 145 (2020) 106722
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Molecular Phylogenetics and Evolution
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Genetic variation and admixture of red-eared sliders (Trachemys scripta T elegans) in the USA ⁎ James F. Parhama,b, , Theodore J. Papenfussc, Anna B. Sellasb,d, Bryan L. Stuarte, W. Brian Simisonb a Department of Geological Sciences, California State University, Fullerton, CA 92834, USA b Center for Comparative Genomics, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA c Museum of Vertebrate Zoology, 3101 Valley Life Sciences Building, University of California, Berkeley, CA 94720-3160, USA d Sana Biotechnology, 1 Tower Place, STE 500, South San Francisco, CA 94080, USA e North Carolina Museum of Natural Sciences, Raleigh, NC 27601, USA
ARTICLE INFO ABSTRACT
Keywords: The most ubiquitous, abundant, and invasive turtle on Earth, Trachemys scripta elegans (TSE, “red-eared slider”), is Red-eared slider one of four taxa in a clade that is native to the USA and adjacent Mexico (three subspecies of Trachemys scripta plus RES Trachemys gaigeae). The present range-wide study of this clade is based on 173 known-locality mtDNA sequences TSE combined with ddRAD libraries for 43 samples emphasizing the western part of the range of TSE, its contact with Hybrid that of T. gaigeae, and anthropogenic hybrids between TSE and T. s. scripta. The data presented here are the first to Turtle sample the TSE × T. s. scripta intergrade zone or TSE × T. s. scripta crosses from introduced turtles. In the western Invasive Genetic pollution part of its range (New Mexico and Texas), most samples of TSE from the Pecos River have mtDNA haplotypes Trachemys gaigeae matching T. gaigeae. Structure analysis of SNPs from the ddRAD show evidence of genetic admixture between T. Trachemys scripta troostii gaigeae and TSE in all included samples from the Rio Grande and Pecos River. These populations also exhibit T. gaigeae-like head stripes, i.e., a postorbital marking that does not reach the eye. The genetic and morphological data are thereby reconciled, as both suggest that these TSE are intergrades. We recommend that these populations continue to be considered TSE, despite the admixture with T. gaigeae. In the Eastern United States, some samples of the morphologically intermediate subspecies T. s. troostii are not genetically distinct from TSE and some samples share morphological characters and genetic affinities with T. s. scripta. Based on these observations we conclude that the taxon T. s. troostii represents intergrades between TSE and T. s. scripta and should not be considered a valid taxon. Near the already established part of the intergrade zone between TSE and T. s. scripta, TSE mtDNA hap- lotypes have naturally introgressed into typical-looking samples of T. s. scripta in Georgia. Hybrids between in- troduced TSE and T. s. scripta are also confirmed deeper within the natural range of T. s. scripta in South Carolina and Virginia. Given the examples of feral hybrids deep within its range shown here and elsewhere, the threat of genetic pollution of T. s. scripta by feral TSE is established.
1. Introduction for native turtles the threat is potentially compounded by TSE as direct competitors and vectors for disease (Somma et al., 2019). For all of The most ubiquitous and abundant turtle on Earth, Trachemys scripta these reasons, TSE is considered one of the 100 worst invasive species elegans (TSE hereafter), is native to the USA (Fig. 1) where it is com- (GISD, 2019). The taxa most threatened by TSE are other taxa of Tra- monly known as the red-eared slider. TSE are the most frequently kept chemys (some of which are endangered [Tortoise and Freshwater Turtle pet turtle around the world and are farmed at industrial scales (Hughes, Specialist Group, 1996; van Dijk and Flores-Villela, 2007]) through 2000; Shi et al., 2008; Herrel and van der Meijden, 2014; Mali et al., genetic pollution. Previous studies have demonstrated the ability for 2014). Through escapes and intentional release, TSE has spread far related testudinoid turtles to hybridize across lineages that have been beyond its natural borders to every continent except Antarctica separated for tens of millions of years (Parham et al., 2001; Buskirk (Tracking Red-Eared Sliders, 2019; GISD, 2019). Wherever TSE are et al., 2005; Stuart and Parham, 2007). Not surprisingly, there are introduced they impact ecosystems through their omnivorous diet, but documented hybrids between introduced TSE and other taxa of
⁎ Corresponding author. E-mail address: [email protected] (J.F. Parham). https://doi.org/10.1016/j.ympev.2019.106722 Received 11 June 2019; Received in revised form 13 December 2019; Accepted 20 December 2019 Available online 23 December 2019 1055-7903/ © 2020 Elsevier Inc. All rights reserved. J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722
Fig. 1. Map showing major morphological and genetic groupings of Trachemys in the USA. Small squares refer to localities of samples in this study (see Appendix), with color shading of squares corresponding to mtDNA haplotype. Circles refer to samples in the ddRAD analyses. Admixed samples are represented by pie charts (larger circles) corresponding to the Structure analysis of SNPs from the ddRAD libraries. Samples that are mentioned in the text are numbered on the map.
Trachemys (Forstner et al., 2014; Mitchell, 1994; Palmer and Braswell, hybridization with introduced TSE, and in some cases both types of 1995; Parham et al., 2013; Seidel et al., 1999). admixture (natural and anthropogenic) are occurring in close proxi- Trachemys is a clade of freshwater turtles that is endemic to North mity. Our study tests the following questions: (1) What are the major and South America that includes 25 taxa (species and subspecies; genetic groupings within this widespread clade and how do they cor- Parham et al., 2015; TFTSG, 2017). The four taxa that occur in the USA relate with morphology-based taxa?; (2) Can more modern methods (three subspecies of Trachemys scripta plus T. gaigeae) are considered a reconcile the apparent conflict between morphological and genetic data monophyletic group (Parham et al., 2015). Of these four, TSE had the (i.e., is there genetic evidence for genetic intergradation to match the largest geographic range even before it became an invasive species, widespread intermediate morphologies)?; (3) What is the nature of the naturally occurring in drainages from the Rio Grande to Mississippi lineage boundaries between parapatric taxa that have different levels of (Fig. 1). In the eastern part of its range, TSE has a broad range of genetic variation (e.g., between TSE and T. gaigeae vs. TSE and T. s. morphological intergradation with Trachemys scripta scripta (yellow- scripta)?; (4) Finally, given the purported threat of genetic pollution bellied slider) in and around the Alabama River drainage (Carr, 1952; from introduced TSE, is there any genetic evidence of admixture Mount, 1975). Pure T. s. scripta occur east of the Eastern Continental throughout the range of Trachemys taxa in USA? We address these Divide, although introduced TSE can be found throughout its range questions using a combination of genetic markers (mtDNA and ddRAD (Somma et al., 2019) and hybridization has been reported based on SNPs). observations of intermediate specimens (Mitchell, 1994; Palmer and Braswell, 1995). In the western part of its range, TSE is parapatric with 2. Materials and methods T. gaigeae (Big Bend slider), a vulnerable (van Dijk, 2011) species that is restricted to the upper Rio Grande. Downstream from T. gaigeae, in the 2.1. Sampling lower Rio Grande and Pecos River, TSE are morphologically distinct from other TSE and have occasionally been referred to as intergrades Our study is based on 173 known-locality genetic samples with with T. gaigeae (Hamilton, 1947; Degenhardt and Christiansen, 1974; corresponding museum voucher specimens (Appendix). Four of the Stuart, 2000). The intergrade hypothesis was refuted by a study using sequenced samples are outgroups (Pseudemys gorzugi, Trachemys neb- allozymes (Seidel et al., 1999), but has never been followed up with ulosa, Trachemys venusta [n = 2]). Our ingroup sampling emphasizes other molecular methods. In addition to this unresolved contact zone, the western part of the range of TSE and its contact with that of T. hybridization of T. gaigeae with introduced TSE was hypothesized based gaigeae. We consider the Mexican species Trachemys hartwegi to be a on morphology and allozyme electrophoresis (Seidel et al., 1999) and separate species from T. gaigeae following Parham et al. (2015). The later confirmed with a combination of mitochondrial DNA and micro- 169 samples of the ingroup include 17 T. gaigeae from seven different satellites (Forstner et al., 2014). Aside from these studies, all other localities in New Mexico and Texas, 22 TSE from 10 different localities genetic studies of TSE have been limited to their inclusion in broader in the Pecos River drainage, 16 TSE from nine different localities in the phylogenetic works (e.g., Jackson et al., 2008; Wiens et al., 2010; Fritz Rio Grande, 62 other TSE from other, mostly western, parts of their et al., 2011; Parham et al., 2013, 2015). range in USA (IL, 2; IN, 3; KS, 4; KY, 3; LA, 4; MS, 6; NM, 6; OK, 2; TX, The present study is the first to include range-wide sampling ofthe 28), four samples from introduced populations of TSE (Cayman Islands, clade that includes all four Trachemys in the USA. Understanding the 3; FL, 1), 22 samples from the intergrade zone of TSE and T. s. scripta genetics of invasive species such as TSE is important for determining (AL, 22;), five suspected anthropogenic hybrids between introduced source populations as well as the routes and numbers of invasions TSE and native T. s. scripta (SC, 1; VA, 4), 16 T. s. scripta (FL, 2; GA, 11; (Holland, 2001; Handley et al., 2011). Both T. gaigeae and T. s. scripta NC, 1; SC, 2), and 9 T. s. troostii (NC, 2; TN, 7). This study includes have purported examples of intergradation with native TSE and samples from the type locality of most valid Trachemys taxa native to
2 J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722 the USA inasmuch as they are known (T. gaigeae, Boquillas [Rio Grande 2.4. ddRAD data analysis Village], Brewster Co., TX, samples 12–17; T. s. elegans: Fox River, White Co., IL, near New Harmony, IN, samples 113–114; T. s. scripta: Raw Illumina sequences were de-multiplexed using the Stacks v1.29 Berkeley Co., SC, sample 158). For T. s. troostii, we do not have any (Catchen et al., 2011) ‘process_radtags’ script (resulting in 182 million samples from the type locality (Cumberland River, TN) but do include reads with the number varying between 43 thousand and 4.6 million seven samples from adjacent Cumberland Plateau in TN (samples 69, reads per individual. All ddRAD data sequences were deposited as an 103–107, 145). Taxonomic identification of ingroup samples was based NCBI BioProject (Appendix). Loci were assembled using the Stacks v. on morphological characteristics of the voucher specimens, i.e., color 2.3e (Catchen et al., 2013) denovo_map.pl pipeline and its component and amount of head striping and plastral patterning. For all 173 sam- programs. To eliminate the possibility that mtDNA is driving the RAD ples we sequenced a partial sequence of the control region to represent Structure pattern of asymmetrical introgression, mtDNA was filtered the mitochondrial genome. In order to characterize the genetic varia- out of the raw illumina reads by mapping the raw reads to a Trachemys tion at the contact of T. gaigeae and T. scripta, a subset of 43 samples of scripta mitochondrial genome (Genbank accession NC_011573.1) in CLC Trachemys gaigeae and T. scripta were further analyzed using ddRAD Genomic Workbench 7.0.3 (CLC Bio, Aarhus, Denmark) and running (with an emphasis [n = 29] from the western part of the range). the remaining reads in the Stacks pipeline. Loci were generated by the merging of three or more similar “stacks.” Those “stacks” in which the 2.2. Mitochondrial DNA sequencing and analyses number of reads were more than two standard deviations above the mean were assumed to be repetitive elements and removed from the Genomic DNA was extracted from approximately 25 mg of liver analysis. Loci were identified by aligning the sequence reads from each tissue using the Qiagen DNeasy tissue kit (Qiagen, Valencia, CA) fol- individual to the assembled contigs resulting in 468,175 genotyped loci lowing the manufacturer's instructions. We ran standard PCR reactions with an effective per-sample coverage of 7.3x and a mean number of and sequenced a ~660 bp region of mtDNA control region from the 5′ sites per locus of 95.2. Using the “populations” component in stacks we end using the primers DES-1 (5′-GCATTCATCTATTTTCCGTTAGCA-3′) set the minimum number of populations a locus must be present in to and DES-2 (5′-GGATTTAGGGGTTTGACGAGAAT-3′) (Starkey et al., two (-p 2) and the minimum percentage of individuals in a population 2003). Sequencing was done on an ABI PRISM 3130xl Genetic Analyzer required to process a locus to 80% (-r 0.8). We used the ‘write_sin- (Applied Biosystems). Contigs were quality trimmed and assembled in gle_snp’ option to extract a single SNP from each locus. The resulting Geneious 11.1.4 (https://www.geneious.com), and aligned using the dataset consisted of 23,582 loci with 21,210 variant sites. From this MAFFT 1.3.7 plugin (Katoh and Standley, 2013) for Geneious using the dataset, Structure 2.3.4 (Pritchard et al., 2000) input files were pro- “Auto” settings. Uncorrected pairwise distances (p) were calculated duced using the structure option in “populations.” Using Structure, we using PAUP* 4.0a166 (Swofford, 2003). All mitochondrial sequences evaluated varying numbers of clusters (K = 1–6) and six runs of were deposited in GenBank (Appendix). 100,000 generations for each K value with burn-in values of 10,000. The phylogenetic analyses were performed using RAxML 8.2.10 The six Structure output files for each K value were all submitted to (Stamatakis, 2014). We used the “-f a” option which performs a rapid Structure Harvester (Earl and von Holdt, 2012), which uses the Evanno bootstrap analysis and searches for the best scoring maximum like- method (Evanno et al., 2005) to estimate the K value that best fits the lihood tree. We used the AutoMRE option, which is an a posteriori data. Structure Harvester also outputs input files for CLUMPP bootstopping analysis to determine when enough bootstrap analyses (Jakobsson and Rosenberg, 2007), which helps correct for the organi- have been performed to reach convergence using the majority-rule zational problem of ‘label switching’ (cluster values from each replicate consensus tree criterion, which resulted in 650 bootstrap replicates. are assigned different columns) and corrects for stochastic differences of cluster solutions across replicates of the same K value. We used 2.3. ddRADlibrary prep Distruct 1.1 to create vector graphics of the Structure charts (Fig. 3B).
We also used the populations script in Stacks to calculate FST, expected Samples were extracted using DNeasy Tissue kits (Qiagen) following and observed homozygosity, and mean nucleotide diversity (π). the manufacturer’s protocol for animal tissues, and stored at −20 °C. We generated a maximum likelihood tree (Fig. 3A) from the ddRAD We prepared ddRAD libraries for 43 individuals (T. gaigeae, n = 7; TSE, data using a phylip file generated by the Stacks2 ‘populations’ script. n = 29; invasive TSE × T. s. scripta hybrids, n = 4; T. s. scripta, n = 3) The ‘–phylip’ option produces a phylip formatted alignment file with using the double digest protocol of Peterson et al. (2012). We followed nucleotides that are fixed-within, and variant among populations. To the Peterson et al. (2012) protocol closely with few modifications, in- generate an alignment with all 43 individuals, we created a population cluding a 3-hour digest at 37 °C using the SphI and MluCl restriction input file with each individual assigned to its own population andran endonucleases (New England Biolabs; enzyme pair was chosen based on the ‘populations’ script with the ‘–phylip’ and ‘–write_single_snp’ op- Table 1 of Peterson et al. [2012]). After cleaning using Dynabeads M- tions. This produced an alignment with 43 OTUs and a length of 91,219 270 Streptavidin (Life Technologies), universal P2 adaptors were li- positions. We used the same RAxML parameters used for the mtDNA gated to each DNA fragment. Additionally, a uniquely barcoded P1 control region analysis, which resulted in the autoMRE stopping the adapter was ligated to each of sixteen individuals for three groups of bootstrap analysis at 50 replicates. sixteen individuals. These groups were later pooled and a unique Illu- mina (Illumina) index was added to each group of sixteen and pooled 3. Results together. Prior to pooling, DNA was cleaned using magnetic beads (as above). Pooled DNA was size-selected using a Blue Pippin Prep (Sage 3.1. Mitochondrial DNA results Science) with all fragments between 376 and 450 bp recovered. The unique Illumina indices were incorporated onto the P2 adaptor end of Phylogenetic analyses of partial sequences of the control region DNA fragments using a real-time library amplification kit (Kapa Bio- (Fig. 2) identify three reciprocally monophyletic clades that correspond systems). The concentration of each pool was standardized and com- to the taxa T. gaigeae, TSE, and T. s. scripta. Uncorrected (p) distances of bined for HiSeq 2000 Illumina sequencing. Libraries were quantified control region sequence within T. gaigae ranged from 0 to 0.16%, within using a High Sensitivity DNA Kit on a 2100 Bioanalyzer (Agilent T. s. scriptaranged from 0 to 1.37%, and within TSE ranged from 0 to Technologies) and sequenced at the Harvard Genomics Core facility on 1.98%. TSE shows the highest mtDNA haplotype diversity in the wes- an Illumina HiSeq 2000 (100 bp paired-end reads). Sequencing runs tern part of its range (KS, OK, NM, TX; Fig. 2). One mtDNA haplotype is resulted in ~200 million reads passing initial quality control at the broadly distributed in the southeastern and midwestern USA (samples sequencing facility. 100–142) including most samples of T. s. troostii (samples 103–109).
3 J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722
matching T. gaigeae. Of the 21 samples of TSE from the Pecos River, 17 have T. gaigeae mtDNA haplotypes. Fifteen of the 17 TSE from the Pecos share a single mtDNA haplotype. Four samples of TSE from the Pecos River were found to have TSE mtDNA. On the eastern side of the range of T. scripta, mtDNA haplotypes of TSE occur throughout the natural intergrade zone with T. s. scripta south of the Appalachian Mountains. TSE mtDNA haplotypes penetrate east into the range of typical looking T. s. scripta in Georgia. In contrast to the single T. gaigeae mtDNA haplotype recovered in TSE in the Pecos River, there is higher variation among the TSE mtDNA haplotypes in- trogressed into T. s. scripta (n = 8, Fig. 2). Away from the intergrade zone mtDNA haplotypes of TSE also appear deep within the range of T. s. scripta at areas where anthropogenic hybrids from feral populations are identified based on the presence of intermediate-looking individuals (samples 142, 159,162–163). Finally, the taxon T. s. troostii cannot be differentiated from TSE with mtDNA.
3.2. ddRAD DNA results
The Delta K value for K = 2 (Fig. 3B) was 99.36 and all other Delta K values were below 30. The two clusters shown in the Structure ana- lysis of SNPs from the ddRAD libraries correspond to T. gaigeae and T. scripta with FST estimated at 0.201. The subspecies of T. scripta are not distinguished by the structure analysis of SNP data or by the any of the divergence or fixation statistics, likely because of the low number of pure T. s. scripta (n = 3) and T. s. troostii (n = 2) in the ddRAD analysis. Observed and expected heterozygosity for T. gaigaea (0.034 and 0.030) was lower than for T. scripta (0.123 and 0.217), with similar results calculated for mean nucleotide diversity, π, for T. gaigaea (0.037) and T. scripta, (0.240). The ddRAD data show evidence of genetic admixture between T. gaigeae and T. scripta in samples from the Rio Grande and Pecos River (Fig. 3B). Phylogenetic analyses of SNP data (Fig. 3A) place the admixed in- dividuals of T. gaigeae and TSE as a paraphyletic grade between the two species. Two samples of T. s. troostii do not form a clade and are nested within TSE. The T. s. troostii sample from TN formed a clade with nearby midwestern TSE, whereas the T. s. troostii sample from North Carolina (sample 108) formed a clade with samples identified as T. s. scripta or T. s. scripta intergrades. The three samples of T. s. scripta form a clade exclusive of TSE, although like T. s. troostii, nested among TSE. Anthropogenic hybrids from South Carolina and Virginia with either T. s. scripta or TSE mtDNA haplotypes grouped with the pure T. s. scripta.
4. Discussion
Our study set out to ask the following questions: (1) What are the major genetic groupings within this widespread clade and how do they correlate with morphology-based taxa?; (2) Can more modern methods reconcile the apparent conflict between morphological and genetic data (i.e., is there genetic evidence for genetic intergradation to match the widespread intermediate morphologies)?; (3) What is the nature of the Fig. 2. Phylogenetic (ML) tree showing the three main mtDNA haplotype clades lineage boundaries between parapatric taxa that have different levels of of USA Trachemys (T. gaigeae, TSE, and T. s. scripta) with dashed lines. genetic variation (e.g., between TSE and T. gaigeae vs. TSE and T. s. Outgroups are not shown. Scale bar represents substitutions per site. Each sample includes a number (Appendix 1), a taxon assignment based on mor- scripta)?; (4) Finally, given the purported threat of genetic pollution phology (labelled with text, but also with white for T. gaigeae, red for TSE, from introduced TSE, is there any genetic evidence of admixture yellow for T. s. scripta, and orange for morphological intergrades), and a locality throughout the range of Trachemys taxa in USA? Using a combination of (state, and country if not the USA) with drainage listed if it is Pecos or Rio genetic markers (mtDNA and ddRAD SNPs) we answer the questions Grande. Circles are given for samples in the ddRAD analyses, and even larger above by (1) Providing the first range-wide assessment of the USA pie charts are given for admixed samples with white corresponding to T. gaigeae Trachemys clade revealing an uneven application of subspecific taxa; (2) and red corresponding to T. scripta. Bootstrap values are shown for clades with Resolving the question of intergradation of T. gaigeae and TSE by branch lengths ≥ 0.01. (For interpretation of the references to color in this showing that populations in the lower Rio Grande and Pecos River are figure legend, the reader is referred to the web version of this article.) admixed; (3) Providing the first genetic characterization of the inter- grade zone between TSE and T. s. scripta; and finally (4) Providing the The most notable discrepancy between mtDNA and accepted species- first genetic confirmation of hybrids between introduced TSEand T. s. level taxonomy involves samples that are traditionally referred to TSE scripta. from the Pecos River in New Mexico that have mtDNA haplotypes The comparison of rapidly evolving, maternally-inherited, mtDNA
4 J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722
Fig. 3. ddRAD analyses of USA Trachemys. A. Phylogenetic (ML) tree based on the SNP data. Bootstrap values for all clades are 100 unless otherwise indicated. Scale bar represents substitutions per site. Dashed lines show the different boundaries of T. gaigeae, T. gaigeae mtDNA, and T. gaigeae SNPs. Each sample includes a number (Appendix 1), a taxon assignment based on morphology (labelled with text, but also with white for T. gaigeae, red for TSE, yellow for T. s. scripta, and orange for morphological intergrades), a locality (state, and Country if not the USA) with drainage listed if it is Pecos or Rio Grande, circles for samples in the ddRAD analyses, and even larger pie charts for admixed samples. B. Structure analysis of SNPs from the ddRAD libraries, (K = 2, T. gaigeae in white, T. scripta in red) showing evidence of genetic admixture in the Rio Grande and Pecos River. (For interpretation of the references to color in this figure legend, the reader is referred to the web versionof this article.) with more slowly evolving, biparentally-inherited, nuclear DNA, as well 4.1. Admixture of TSE and T. gaigeae as gross morphology and geographic distribution, provides a range- wide framework for understanding the evolutionary underpinnings of The populations of TSE in the Rio Grande drainage downstream TSE and other Trachemys native to the USA. Although it is not an em- from the range of T. gaigeae (including the Pecos River) exhibit T. gai- phasis of this study, the range-wide assessment of TSE should be useful geae-like head stripes, i.e., a postorbital marking that does not reach the for determining source populations for this globally invasive species. eye (Fig. 1; Stuart, 2000; Stuart and Ward, 2009). Based on allozymes In the following sections we discuss our results by region, first and morphometrics, Seidel et al. (1999) argued against calling the TSE discussing the western part of the range, specifically the admixture of in the Rio Grande or Pecos River intergrades, highlighting the genetic TSE and T. gaigeae (4.1) and the taxonomic status of the intergrades and morphometric distinctiveness of T. gaigeae. Forstner et al. (2014) (4.2). Next, we discuss our genetic results as they pertain to the re- reported some hybridization from the easternmost edge of the range of lationship of TSE to the two eastern taxa such as the possibility of in- T. gaigeae in the Rio Grande, but the scope of their project did not ex- tergradation with and taxonomic status of T. s. troosti (4.3) and the tend to the lower Rio Grande or Pecos River. natural and anthropogenic admixture of TSE with T. s. scripta (4.4). The data presented here show that TSE from the Rio Grande and Pecos River are admixed with T. gaigeae. Seventeen of 21 Pecos River samples assigned to TSE based on morphology show T. gaigeae mtDNA haplotypes (Fig. 2). A subsample of these Pecos River samples (n = 8)
5 J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722 were further analyzed by ddRAD data, revealing a mixture of T. gaigeae 1999; Stuart and Ward, 2009). Therefore, the recognition of an inter- and TSE SNPs. The higher frequency of T. gaigeae mtDNA in these Pecos grade zone here raises questions about the species status of T. gaigeae. River samples is similar to that noted for hybrids between TSE and T. Some other authors employ a strict Biological Species Concept and so gaigeae by Forstner et al. (2014) in the Rio Grande, who suggested that consider all Trachemys to be one species with all named taxa as sub- differential receptiveness to the male courtship behavior of the other species T. scripta (e.g., Legler and Vogt, 2014). Other authors recognize species could explain this asymmetrical introgression. The ddRAD the distinctiveness of other Trachemys species despite intergrade zones analyses also included four samples from the Rio Grande that showed T. (e.g., Parham et al., 2013; TFTSG, 2017). The taxonomic history of gaigeae SNPS, although in lower frequencies than in the Pecos River. In Trachemys was recently reviewed by Seidel and Ernst (2016), who contrast to the Pecos River samples, all samples of TSE from the Rio identified the variable use of species vs. subspecies as the major source Grande have TSE mtDNA haplotypes. A putative hybrid sample (in- of taxonomic instability in the genus. The most widely-used nomen- termediate morphology; sample 42) from within the range of T. gaigeae clature for Trachemys (Seidel, 2002; Parham et al., 2013, 2015; Seidel also differs from the previously reported hybrids (Forstner et al., 2014) and Ernst, 2016; TFTSG, 2017) employs a Phylogenetic Species Concept by having TSE mtDNA haplotype. Sample 42 could be a translocated that applies the species rank for most morphologically and genetically sample from the lower Rio Grande, where admixed samples with TSE distinct lineages, whereas subspecies are unevenly applied to some taxa mtDNA occur. In any case, sample 42 and the intergrades in the lower that show intergradation (e.g., T. scripta sspp.), but not others (see Rio Grande show that hybridization does not always involve T. gaigeae Parham et al., 2013), or have unclear boundaries (e.g., T. decussata females. sspp.), or else are not readily diagnosed (e.g., T. stejnegeri sspp.). The TSE from both the Rio Grande and Pecos River show evidence This ad hoc system is imperfect, but since all current taxonomic of admixture and an intermediate morphology and so should be con- arrangements of Trachemys employ subspecies, any proposed taxo- sidered intergrades. All sampled individuals show predominantly TSE nomic scheme that eliminated this rank would necessitate many no- SNPs. In the Rio Grande, the frequency of T. gaigeae SNPs generally menclatural changes and so should not be undertaken lightly. Because declines as samples get further from the range of T. gaigeae (Fig. 1). the subspecies rank is problematic, as well as the sake of current sta- Similarly, in the Pecos River, the sample (80) with the least amount of bility, we recommend that T. gaigeae continue to be recognized as a T. gaigeae SNPs is furthest from the current range of T. gaigeae. The distinct species. Furthermore, at the current time, the most significant frequency of T. gaigeae SNPs is greater and more consistently dis- genetic break between T. gaigeae and T. scripta still coincides with the tributed in the Pecos River than in the lower Rio Grande. The persis- current species borders (Figs. 2, 3) and they are otherwise morpholo- tence of T. gaigeae SNPs in TSE from the Pecos River compared to the gically, genetically, and ecologically distinct (Seidel et al., 1999; Stuart Lower Rio Grande might reflect the fact that the Pecos River is more and Ward, 2009). We also recommend that T. scripta in the lower Rio isolated from other populations of TSE. Grande and Pecos River continue to be considered T. scripta, despite the The admixture of TSE and T. gaigeae suggest an ancient contact admixture with T. gaigeae. This is because they are morphologically and between the two species. Until the Pleistocene (<2.6 Ma), the upper genetically most similar to T. scripta as well as the prediction that more Rio Grande was part of a system of internally draining (endorheic) data will likely confirm a clinal decrease in T. gaigeae influence in the basins terminating in what is now the Chihuahuan Desert (Galloway lower Rio Grande making any other taxonomic boundary somewhat et al., 2011). This ancient endorheic basin could explain the origin of T. arbitrary. Finally, we propose that future studies should include more gaigeae as a distinct lineage. Subsequently, the Rio Grande evolved into detailed analyses of Trachemys contact zones and that the uneven use of its current configuration and began draining to coastal lowlands con- subspecies needs to be further evaluated. nected to the Mississippi River drainage. This Pleistocene drainage evolution facilitated the contact between T. gaigeae and TSE (Legler and 4.3. Taxonomic status of T. s. troostii Vogt, 2014). A Pleistocene contact is recent enough to explain the lack of differentiation between the mtDNA haplotypes of TSE in the Pecos Trachemys scripta troostii is native to the Cumberland and Tennessee River and T. gaigeae, and that timing is consistent with that hypothe- rivers (Carr, 1937, 1952) and intergrades with TSE in Kentucky (Ernst sized other freshwater vertebrates in the region (Echelle and Echelle, and Jett, 1969). Unlike the other three taxa of Trachemys studied here, 1978; Smith and Miller, 1986). the nine samples of T. s. troostii did not form distinct clades in either the Unfortunately, human activities complicate our ability to fully re- phylogenetic analyses of mtDNA or nuclear SNP data. Three different construct the evolutionary history of Trachemys in this region. For ex- mtDNA haplotypes were recovered, with seven of the samples sharing ample, human introductions make it difficult to discern between hy- the broadly distributed TSE mtDNA haplotype (see 3.1). In addition to brids and translocated intergrades (see above). Furthermore, the not being genetically distinct in the markers used here, of the four taxa construction of dams along the Pecos and Rio Grande has altered the of Trachemys native to the USA, T. s. troostii is the least morphologically distribution of freshwater vertebrates by changing the flow regime. For distinct. In fact, Carr (1952) stated that from a morphological per- example, the spread of invasive fishes in dammed drainages in western spective one could consider that “troostii is merely a population of in- USA is well documented, resulting in hybrid swarms (Moyle and Light, tergrades, in that its characters are almost precisely intermediate be- 1996). In some cases, the replacement can be extremely rapid; the Rio tween those of scripta and elegans.” Grande silvery minnow (Hybognathus amarus) was replaced in the Pecos In support of the hypothesis that T. s. troostii are intergrades, pre- in less than 10 years by the plains minnow (Hybognathus placitus), an viously undescribed samples (108–109) from a population of Trachemys introduced congener from the Mississippi drainage (Hoagstrom et al., in North Carolina referred to T. s. troostii by Beane et al. (2008) match 2010). Trachemys gaigeae is adapted to heterogeneous desert rivers, but most characters of T. s. troostii except that some individuals also show drainage impoundments create more lentic habitats favored by TSE vertical postorbital blotches (Fig. 4; similar to that seen in T. s. scripta, (Stuart and Ward, 2009; Forstner et al., 2014). Because the range of T. Fig. 1). Despite having mtDNA matching TSE, in the phylogenetic gaigeae does not currently contact the Pecos River, the hypothesis of analysis of SNP data, a sample (108) of this North Carolina population ancient contact is the favored explanation for the admixed individuals formed a clade with samples identified as T. s. scripta or T. s. scripta in the Pecos River and lower Rio Grande. intergrades. Because of the remote location of these samples, we submit that the intermediate morphological and genetic characteristics arose 4.2. Taxonomic status of T. gaigeae × T. scripta intergrades naturally (i.e., not the result of introduced T. s. scripta). The retention of T. s. scripta characters in the NC samples likely reflect that they are far- Previous studies cited the lack of an intergrade zone between TSE removed from the range of TSE, similar to the pattern seen for the and T. gaigeae to argue for the species status of T. gaigeae (Seidel et al., genetic influence of TSE samples in the upper reaches of the Pecos River
6 J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722
haplotypes. This is the first confirmation of genetic pollution of T. s. scripta with feral TSE. Given the examples of feral hybrids deep within its range shown here and elsewhere, the threat of genetic pollution of T. s. scripta by feral TSE is established.
CRediT authorship contribution statement
James F. Parham: Conceptualization, Methodology, Data curation, Writing - original draft, Visualization, Supervision, Project adminis- tration, Funding acquisition. Theodore J. Papenfuss: Conceptualization, Resources, Writing - review & editing. Anna B. Sellas: Investigation, Data curation. Bryan L. Stuart: Resources, Writing - review & editing. W. Brian Simison: Conceptualization, Methodology, Formal analysis, Resources, Data curation, Writing - re- view & editing, Funding acquisition.
Acknowledgments Fig. 4. Sample referred to T. s. troostii with mtDNA matching TSE showing head marking similar to T. s. scripta (Sample 108, NCSM 74447, Madison Co., NC). This project started many years ago in collaboration with Andrew H. Phylogenetic analysis of SNP data placed this sample with those identified as T. Price (Texas Parks and Wildlife Department) and is dedicated to him. s. scripta or T. s. scripta intergrades (Photo by J. Beane). Many people and agencies provided assistance with collections. The late Jim Lee (Hokes Bluff) is thanked for his generosity and assistance and in the lower Rio Grande (see 4.2). with fieldwork in Alabama and the late Charlie Painter is thanked for The overall intermediate morphology of T. s. troostii and the ap- collecting some of the key samples in New Mexico. Kory Heiken assisted parent lack of any genetic distinctiveness of T. s. troostii both argue that with fieldwork in Texas. Jeffrey C. Beane, L. Todd Pusser, Robert A. this taxon is an intergrade of TSE and T. s. scripta. Because of this, we Davis, C. A. d’Orgeix, and E. N. Sismour collected samples deposited at recommend that T. s. troostii should not be considered a valid taxon. NCSM. Jeffrey C. Beane is also thank for his photograph usedin Fig. 4. The range of “T. s. troostii” most likely represents a northern extension For permits we thank Alabama Division of Wildlife and Freshwater of a well-established intergrade zone to the south (see 4.4, Fig. 1) albeit Fisheries, Cayman Islands Department of Environment, Florida Fish and with more genetic influence from TSE in the midwest. Alternatively, the Wildlife Conservation Commission, Georgia Department of Natural intergrades of the western Appalachians may also indicate past contact Resources, Illinois Department of Natural Resources, Indiana Depart- of TSE and T. s. scripta across the Eastern Continetal Divide, which ment of Natural Resources, Louisiana Department of Wildlife and would also explain the persistence of T. s. scripta features in the North Fisheries, Kansas Department of Wildlife, Parks, and Tourism, Kentucky Carolina population of “T. s. troostii.” Department of Fish and Wildlife, Mississippi Museum of Natural Sci- ence, New Mexico Department of Game and Fish, North Carolina Wildlife Resources Commission, Oklahoma Department of Wildlife 4.4. Admixture of TSE and T. s. scripta Conservation, South Carolina Department of Natural Resources, Ten- nessee Wildlife Resources Agency, and the Texas Parks and Wildlife In the eastern USA, intergradation between TSE and T. s. scripta has Department. We also thank the following collections managers in al- been long-recognized based on specimens with intermediate morphol- phabetical order according to the institutional abbreviations given in ogies from Alabama (Carr, 1952; Conant, 1958; Mount, 1975; Conant the appendix: CAS, Lauren Scheinberg and Jens Vindum, Lauren is also and Collins, 1998). The contact between TSE and T. s. scripta is part of a thanked for help with the map figure; MPM RA, Julia Colby (Alan Re- suture zone shared by other southeastern USA taxa that is generally setar of the Field Museum of Natural History, Chicago, IL, also helped hypothesized to result from postglacial range expansions (Swensen and with this specimen); MSB: Tom Giermakowski; MVZ: Carol Spencer; Howard, 2005). The data presented here provide the first genetic as- NCSM, Jeffrey C. Beane; TCWC, Toby Hibbits; TNHC, Travis J. LaDuc. sessment of this natural intergrade zone for Trachemys scripta. All 22 of Funding for this project was provided by a grant from the California our samples from within the range of intergradation had TSE mtDNA State University Program for Education and Research in Biotechnology haplotypes (Figs. 1, 2). TSE mtDNA haplotypes further penetrate east, (CSUPERB; Parham, Simison) as well as a Hagey Award and a Research extending the genetic signature of the intergradation zone well into the Excellence Grant from the California Academy of Sciences, California range of typical-looking T. s. scripta. In some sites, typical-looking T. s. Academy of Sciences (Simison and Parham). scripta with either TSE mtDNA and T. s. scripta mtDNA can be found at the same site (central Georgia, Fig. 1). Unfortunately, our ddRAD Appendix A analysis (k = 2) could not distinguish between TSE and T. s. scripta, probably because of the low number of included T. s. scripta (n = 3; Vouchers for genetic sequences (Mitochondrial DNA sequences, Fig. 2). Additional genomic sampling of T. s. scripta may help elucidate GenBank accessions MN561855-MN562027; ddRAD data, BioProject the genetic structure and age of this contact zone. PRJNA576258) of emydid turtles of the genera Trachemys and Away from this intergrade zone, intermediate morphologies as well Pseudemys used in this study. Museum abbreviations: CAS, California as the presence of TSE mtDNA haplotypes identify examples of hybrids Academy of Sciences Herpetology Collection, San Francisco, California, with feral TSE (Fig. 1). A sample (141) with intermediate morphology USA; MPM RA, Milwaukee Public Museum, Milwaukee, WI, USA; MSB, from South Carolina, well within the range of typical-looking T. s. Museum of Southwestern Biology, University of New Mexico, scripta, has a TSE mtDNA haplotype. The northern part of the range of Albuquerque, NM, USA; MVZ, Museum of Vertebrate Zoology, T. s. scripta intergrades into invasive TSE in northern Virginia and University of California, Berkeley, USA; NCSM, North Carolina Museum Maryland (Mitchell, 1994; Conant and Collins, 1998; Somma et al., of Natural Sciences, Raleigh, NC, USA; TCWC, Texas Cooperative 2019). Four samples (142, 159, 162–163) with intermediate mor- Wildlife Collection, Texas A&M University, College Station, TX, USA; phology from a reported “hybrid swarm” in Virginia (Mitchell, 1994) TNHC, Texas Natural History Collections, University of Texas, Austin, show a mixture of TSE (n = 1) and T. s. scripta (n = 3) mtDNA TX, USA. Vouchers are whole-body preserved specimens. 1. T. gaigeae
7 J.F. Parham, et al. Molecular Phylogenetics and Evolution 145 (2020) 106722
(Brewster Co., TX; MVZ 250723); 2. T. gaigeae (Presidio Co., TX; MVZ (Monroe Co., AL; CAS 255154); 148. T. s. elegans × scripta intergrade 265710); 3–7. T. gaigeae (Socorro Co., NM; MSB 50519-23); 8–11. T. (Tuscaloosa Co., AL; CAS 255159); 149. T. s. scripta (Lowndes Co., GA; gaigeae (Sierra Co., NM; MVZ 250711, 265664-6); 12. T. gaigeae CAS 255138); 150. T. s. elegans × scripta intergrade (Clark Co., AL; CAS (Presidio Co., TX; MVZ 265711); 13–17. T. gaigeae (Brewster Co., TX; 255152); 151. T. s. elegans × scripta intergrade (Monroe Co., AL; CAS TCWC 68545-7, TNHC 50874-5); 18–25. T. s. elegans (Eddy Co., NM; 255155); 152. T. s. scripta (Bleckley Co., GA; CAS 255129); 153. T. s. 250715-6, 265724, 265726-9, 265731); 26–30. T. s. elegans (Eddy Co., scripta (Jefferson Davis Co., GA; CAS 255128); 154. T. s. scripta NM; MVZ 265676, 265678-80, 265725); 31–32. T. s. elegans (De Baca (Bleckley Co., GA; CAS 255130); 155–156. T. s. scripta (Macon Co., GA; Co., NM; MVZ 265736, 265733); 33–34. T. s. elegans (Reeves Co., TX; CAS 255133-4); 157. T. s. scripta (Charleston Co., SC; MVZ 250680); MVZ 265683, 265685); 35–36. T. s. elegans (Rusk Co., TX; TNHC 50876; 158. T. s. scripta (Berkeley Co., SC; MVZ 250681); 159. T. s. ele- TCWC 68555); 37. T. s. elegans (Uvalde Co., TX; MVZ 265719); 38. T. s. gans × scripta hybrid (King and Queen Co., VA; NCSM 74672); 160. T. elegans (Travis Co., TX; TCWC 68637); 39. T. s. elegans (Calhoun Co., s. scripta (McDuffie Co., GA; CAS 255124); 161. T. s. scripta (Dare Co., TNHC 50361); 40. T. s. elegans (Travis Co., TNHC 50865); 41. T. s. NC; MVZ 250678); 162–163. T. s. elegans × scripta hybrids (King and elegans (Bexar Co., TX; TNHC 50872); 42–45. T. s. elegans (Brewster Co., Queen Co., VA; NCSM 74674-5); 164. T. s. scripta (Evans Co., GA; CAS TX; MVZ 250717-9, TCWC 68571); 46. T. s. elegans (Uvalde Co., TX; 255125); 165. T. s. scripta (Leon Co., FL; MVZ 241523); 166. T. s. scripta MVZ 265718); 47. T. s. elegans (Bexar Co., TX; TCWC 68549); 48–49. T. (Jefferson Davis Co., GA; CAS 255127); 167–168. T. s. scripta (Lowndes s. elegans (Hidalgo Co., TX; TCWC 68629, TNHC 50864); 50–54. T. s. Co., GA; CAS 255137, 255139); 169. T. s. scripta (Baker Co., FL; MVZ elegans (Quay Co., NM; MVZ 265669-70, 265673-74, 265737); 55. T. s. 241522). 170. Pseudemys concinna (Jefferson Davis Co., GA; CAS elegans (Hemphill Co., TX; TCWC 68567); 56. T. s. elegans (Kenedy Co., 255126); 171. T. venusta (Costa Rica; MVZ 233245); 172. T. nebulosa TX; TCWC 68567); 57. T. s. elegans (Hemphill Co., TX; TCWC 68631); (Mexico; MVZ 137443); 173. T. venusta (Guatemala; MVZ 264176). 58. T. s. elegans (Brooks Co., TX; TCWC 65331); 59. T. s. elegans (Blanco/Gillespie Co., TX; TCWC 66060); 60. T. s. elegans (Lynn Co., TX; Appendix B. Supplementary material TCWC 68569); 61. T. s. elegans (Dawson Co., TX; TCWC 68553); 62. T. s. elegans (Shackleford Co., TX; TCWC 65334); 63–64. T. s. elegans Supplementary data to this article can be found online at https:// (Runnels Co., TX; MVZ 250689-90); 65. T. s. elegans (Eddy Co., NM; doi.org/10.1016/j.ympev.2019.106722. MVZ 265732); 66–67. T. s. elegans (Menard Co., TX; MVZ 250677, 250692); 68. T. s. elegans (Hardeman Co., TX; TCWC 68564); 69. T. s. Referen ces troostii (Sullivan Co., TN; MVZ 265789); 70. T. s. elegans (Zapata Co., TX; MVZ 265715); 71. T. s. elegans (Webb Co., TX; MVZ 265712); Beane, J.C., Davis, R.A., Pusser, L.T., 2008. Distribution record: Trachemys scripta troostii. 72–74. T. s. elegans (Val Verde Co., TX; MVZ 265723, TCWC 68550, Herp. Rev. 39, 482. Buskirk, J.R., Parham, J.F., Feldman, C.R., 2005. On the hybridisation of two distantly 68561); 75. T. s. elegans (Webb Co., TX; 265713); 76–77. T. s. elegans related Asian turtles (Sacalia x Mauremys). Salamandra 41, 21–26. (Kinney Co., TX; MVZ 265720-1); 78. T. s. elegans (Maverick Co., TX; Carr Jr., A.F., 1937. 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