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Phylogeographic Patterns Among the Subspecies of Notophthalmus viridescens (Eastern Newt) in South Carolina
Gavin R. Lawson and Eran S. Kilpatrick

Southeastern Naturalist, Volume 13, Issue 3 (2014): 444–455

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Southeastern Naturalist G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 444 2014 SOUTHEASTERN NATURALIST 13(3):444–455 Phylogeographic Patterns Among the Subspecies of Notophthalmus viridescens (Eastern Newt) in South Carolina Gavin R. Lawson1,* and Eran S. Kilpatrick2 Abstract - Relatively little work has been done on the population genetics and phylogenetic patterns in Notophthalmus viridescens (Eastern Newt). The most recent study by Gabor and Nice (2004) divided the sampled populations into northern and southern groups rather than along taxonomic lines, and patterns of genetic variation indicated the southern populations were isolated and undergoing genetic drift. To re-evaluate these patterns, we collected sequence data on the mitochondrial ND2 and the flanking tRNAMet genes in fifteen South Carolina populations in the piedmont, sandhills, and lower coastal plain where three of the four subspecies were located (we found no Eastern Newts in the upper coastal plain). Haplotypes did not group by taxonomic designation in phylogenetic analyses, suggesting introgressive hybridization has occurred. Statistical parsimony analysis resolved the haplotype groups into two geographic groups, and partitioning of genetic variation between these groups was significant. We suggest these groups represent populations established during the last glacial maximum, a pattern that has been observed in other pond-breeding salamanders. Introduction Notophthalmus viridescens (Rafinesque) (Eastern Newt; Caudata: Salamandridae) is widely distributed in North America and subdivided into four subspecies on the basis of morphological and life-history characters: N. v. viridescens (Rafinesque) (Red-spotted Newt), N. v. louisianensis (Wolterstorff) (Central Newt), N. v. dorsalis (Harlan) (Broken-striped Newt), and N. v. piaropicola (Schwartz and Duellman) (Peninsula Newt) (Petranka 1998). Eastern Newts inhabit ponds and vernal wetlands throughout their range and have a significant impact on community structure (Morin 1983, Smith 2006, Wilbur et al. 1983). Despite their wide geographic distribution, morphological diversity, and ecological significance, relatively little is known about their population genetics and phylogenetic patterns. The most recent and extensive study of population genetics in Eastern Newts was by Gabor and Nice (2004), who analyzed variation in 18 allozymes among the four subspecies over the species’ range. Their results demonstrated a moderate degree of genetic variation overall, but an absence of significant differentiation among the groups; their findings were consistent with earlier studies (Merritt et al. 1984, Reilly 1990, Tabachnick 1977). Additionally, phylogenetic and phenetic analyses divided the sampled populations into northern, southern, and Florida groups rather than along taxonomic lines (Gabor and Nice 2004), with each group containing at least two subspecies (northern = Red-spotted Newt and Central Newt; southern = Red- 1Bridgewater College, Bridgewater, VA 22812. 2Univiersity of South Carolina Salkehatchie, Division of Mathematics and Science, Walterboro, SC 29488. *Corresponding author - Manuscript Editor: John Placyk Southeastern Naturalist 445 G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 spotted Newt, Central Newt, and Broken-striped Newt; Florida = Central Newt and Peninsula Newt) suggesting introgressive hybridization has occurred within each group (Ball 1998, Minton 1972, Takahashi and Parris 2008). Furthermore, patterns of genetic variation differed among these groups. For example, Gabor and Nice (2004) observed a pattern of lower genetic distances and restricted gene flow with isolationby- distance among northern populations. Among the southern populations, however, isolation-by-distance could not entirely explain the population-genetic relationships among the groups, suggesting that there has been very limited gene flow and that the populations are isolated and undergoing genetic drift (Gabor and Nice 2004). The goal of the current study was to re-evaluate the conclusions of Gabor and Nice (2004) among southern populations of Eastern Newts over a smaller geographic scale using mitochondrial DNA (mtDNA) sequence data. Specifically, we focused our sampling in South Carolina because three of the four subspecies (Redspotted, Central, and Broken-striped newts) occur in the state. Our goal was to test the hypotheses that 1) hybridization/gene flow has occurred among these subspecies, and 2) there is evidence of population isolation and restricted gene flow among the populations sampled. Methods Between May 2009 and May 2013, we collected tissue samples (0.5-cm tail tip) from 125 specimens representing 15 populations (defined as all individuals caught from a single wetland) across the state, with subspecies identified based on morphology and published subspecies ranges (Table 1, Fig. 1). We caught newts in long-hydroperiod wetlands (beaver impoundments) and short-hydroperiod wetlands in the piedmont, but exclusively in short hydroperiod wetlands in the coastal plain. All sampled populations were located in the piedmont, sandhills, and lower coastal Table 1. Locality data for Notophthalmus viridescens (Eastern Newt) collected for this study.WMA = Wildlife Management Area, NWR = National Wildlife Refuge, and NF = National Forest. Sample County Subspecies size Locality 1) Pickens N. v. viridescens 5 Beaver pond, Clemson Experimental Forest 2) Greenville N. v. viridescens 10 Buckhorn Lake, Paris Mountain State Park 3) Greenwood N. v. viridescens 10 Beaver pond, Sumter NF 4) Laurens N. v. viridescens 9 Beaver pond, Sumter NF 5) York N. v. viridescens 4 Depression wetland (Squire Road) 6) Aiken N. v. viridescens 10 Recovery pond, Savannah River Site 7) Fairfield N. v. viridescens 10 Depression wetland (Smalls Chapel Road) 8) Chesterfield N. v. viridescens 10 Depression wetland, Carolina Sandhills NWR 9) Jasper N. v. louisianensis 10 Borrow pit, Tillman Sand Ridge Heritage Preserve 10) Beaufort N. v. louisianensis 9 Borrow Pit, Spring Island 11) Colleton N. v. louisianensis 9 Depression wetland, Donnelley WMA 12) Berkeley N. v. louisianensis 6 Cypress pond, Francis Marion NF 13) Georgetown N. v. dorsalis 4 Cypress pond, Sandy Island 14) Marion N. v. dorsalis 8 Depression Wetland, Woodbury WMA 15) Horry N. v. dorsalis 10 Seepage Pools, Waccamaw NWR Southeastern Naturalist G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 446 plain (Griffith et al. 2002). Despite extensive sampling of 41 wetlands in the upper coastal plain, we found no newts in this region. Following total genomic DNA extractions using DNeasy Tissue Kits (Qiagen, Valencia, CA), we amplified 530 bp of the mtDNA ND2 gene and flanking tRNAMet for 125 individuals using Ready-To-Go PCR Beads (GE Healthcare, Fairfield, CT) and the primers L4437 (5'–AAG CCT TCG GGC CCA TAC C–3') and H4980 (5'– ATT TTT CGT AGT TGG GTT TGA TT–3') (Weisrock et al. 2001). The amplification profile entailed a 3-min initial denaturation at 94 °C, followed by 35 cycles of denaturation at 94 °C for 35 s, annealing at 50 °C for 35 min, elongation at 72 °C for 1 min, and a final elongation step at 72 °C for 10 min on a Techne Genius thermo-cycler (Bio-Rad, Hercules, CA). We purified PCR products using GenElute Agarose Spin Columns (SIGMA, St. Louis, MO) and sequenced them using the PCR primers at the Molecular Biology Research Facility, University of Tennessee, Knoxville, TN. Sequences were aligned using CLUSTALX 2.1 to identify haplotypes (Larkin et al. 2007). Figure 1. Collection locality map. Black lines represent the approximate boundaries of the three subspecies, and numbers correspond to the population list in Table 1 (Petranka 1998). Subspecies key: stippled circle = Notophthalmus viridescens viridescens (Red-spotted Newt), dark gray circle = N. v. louisianensis (Central Newt), light gray circle = N. v. dorsalis (Broken-striped Newt), open circle = upper coastal plain collecting localities (3–6 wetlands sampled per locality; see text for explanation). Southeastern Naturalist 447 G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 We estimated phylogenetic relationships among the haplotypes using maximum parsimony (MP) and maximum likelihood (ML) analyses within the program PAUP* 4.0b10 (Swofford 2002) with Notophthalmus perstriatus (Bishop) (Striped Newt), N. meridionalis (Cope) (Black-spotted Newt) and Taricha granulosa (Skilton) (Rough-skinned Newt) as outgroups (Pryon and Wiens 2011). Both analyses were performed using a heuristic search with 10 random-addition replicates, tree-bisection reconnection (TBR) branch-swapping, and characters unordered and equally weighted. The program jModeltest 2.1 was used to select the model of sequence evolution for the ML analysis (Darriba et al. 2012, Guindon and Gascuel 2003). We employed bootstrap resampling to analyze branch support for the MP and ML trees using 1000 and 500 bootstrap pseudoreplicates, respectively (Felsenstein 1985) and then constructed a haplotype network with the program TCS 1.21 to estimate the genealogical relationships among the haplotypes (Clement et al. 2000). Additionally, we conducted Tajima’s D, and Fu and Li’s D* and F* tests with DnaSP v 5.10.1 to determine whether natural selection significantly influenced the data (Fu and Li 1993, Librado and Rozas 2009, Tajima 1989). We employed AMOVA to estimate the structuring of genetic variation among the three subspecies and among the haplotype groups defined in the statistical parsimony analysis using ARLEQUIN 3.5 (Excoffier and Lischer 2010) and tested these estimates for significance with 1000 random permutations. Lastly, we conducted spatial analyses of molecular variation (SAMOVA) to investigate the geographical structure of genetic variation using SAMOVA 1.0 (Dupanloup et al. 2002). SAMOVA identifies geographically homogeneous groups of populations that maximize among population variation (FCT) for a specified number of groups (K). We ran 100 simulated annealing processes for each group number (K = 2 through K = 12). Results Our analyses detected 34 unique haplotypes, and of the 146 variable characters identified, 64 were parsimoniously informative and produced a best-fit model of sequence evolution of TPM1uf+G. Neither Tajima’s D nor Fu and Li’s D* and F* tests were significant which indicated that the data were not meaningfully influenced by natural selection but instead largely reflect background mutation rates (Tajima’s D: -0.54, P > 0.10; Fu and Li’s D*: -0.71, P > 0.10; Fu and Li’s F*: -0.77, P > 0.10). The consensus trees from both the MP and ML analyses were not identical yet recovered several of the same clades supported by bootstrap values >70% including (NvA-NvC, NvQ-NvBB), (D, I, L, O, M, P), (CC, EE, GG), and (E, F, J) (Fig. 2). Although there was little phylogenetic resolution, the haplotypes did not group by taxonomic designation. Furthermore, clade (NvA-NvC, NvQ-NvBB) included both Red-spotted Newt and Central Newt haplotypes and haplotype NvK was found in one specimen of Red-spotted Newt and one of Centra l Newt. The statistical parsimony analysis divided the haplotypes into two groups at the 95% connection limit, group 1 included individuals from all three subspecies and group 2 included Red-spotted Newt and Central Newt (Fig. 3). These groupings were identical to those recovered in the parsimony phylogenetic analysis (Fig. 2). Haplotypes H and DD were recovered at much higher frequency than Southeastern Naturalist G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 448 Figure 2. Consensus trees of the haplotypes generated by maximum parsimony (left) and maximum likelihood (right) with bootstrap values above the clade branches (NvA = Notophthalmus viridescens haplotype A). Subspecies key: stippled circle = Notophthalmus viridescens viridescens (Red-spotted Newt), dark gray circle = N. v. louisianensis (Central Newt), light gray circle = N. v. dorsalis (Broken-striped Newt). Southeastern Naturalist 449 G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 others and were widely distributed geographically (Fig. 3, Table 2). There was significant partitioning of genetic variation at all levels between groups 1 and 2, and variation among the two accounted for nearly 60% of the total. However, there was also significant partitioning when the haplotypes were grouped by subspecies, albeit at a much lower level (Table 3). When the haplotypes were superimposed on the locality map, the two haplotype groups displayed a distinct geographic pattern with group 2 located largely in the Pee Dee watershed and group 1 located primarily in the remainder of the state (Fig. 4). Four haplotypes (H, I, L, M) were found in multiple piedmont populations and four (T, U, CC, DD) were found in multiple lower coastal plain populations, providing evidence of genetic exchange among the populations in the groups. A significant proportion of total variation was partitioned among groups at all values of K, and FCT increased with increasing values of K, reaching a plateau at K ≈ 4 (Fig. 5). For all values of K ≥ 4, some groups consisted of only a single population Figure 3. Haplotype network with two subgroups calculated using statistical parsimony (95% connection limit). Haplotype size is proportional to the haplotype frequeny among the individuals sequenced. Subspecies key: stippled circle = Notophthalmus viridescens viridescens (Redspotted Newt), dark gray circle = N. v. louisianensis (Central Newt), and light gray circle = N. v. dorsalis (Brokenstriped Newt), open circles = ancestral haplotype. Southeastern Naturalist G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 450 indicating that group structure was being lost at this point (Heuertz et al. 2004). For K = 2, FCT = 0.48, with one group including all Santee, Savannah, and ACE Basin populations, and the second including all Pee Dee drainage populations (Fig. 4). For K = 3, FCT = 0.57, with one group including all Pee Dee drainage populations, one including populations 6 and 9–12, and one including populations 1–5 and 7 (Table 1, Fig. 6). Discussion The results of this study are generally consistent with those of Gabor and Nice (2004) and support our hypotheses. The phylogenetic analyses did not separate the haplotypes in accordance with current taxonomy but recovered one clade (NvA-NvC, NvQ-NvBB) in both the MP and ML analyses and a second clade (E, F, J, G DD, FF) in the MP analysis, including haplotypes from multiple subspecies. This effect, in addition to the presence of one haplotype (K) in both Red-spotted Table 3. Results of the AMOVA of Notophthalmus viridescens (Eastern Newt) subspecies in this study with haplotypes grouped in accordance with the results of the statistical parsimony analysis and by subspecies designation. Variance % of total Source of variation df SSD component variance P-value Haplotype groups Among groups 1 82.7 5.72 58.7 <0.001 Among populations/within groups 17 113.3 1.70 17.4 <0.001 Within populations 30 69.6 2.31 23.8 <0.001 Subspecies Among groups 2 81.5 2.37 36.7 <0.001 Among populations/ within groups 12 64.0 0.56 8.7 0.07 Within populations 34 120.2 3.54 54.7 <0.001 Table 2. Distribution of Notophthalmus viridescens (Eastern Newt) haplotypes by locality and by haplotype group. County Group 1 haplotypes Group 2 haplotypes 1) Pickens I, O, P - 2) Greenville H, I, L, M - 3) Greenwood H, M - 4) Laurens H, L, M, N - 5) York H - 6) Aiken H, L, M, N - 7) Fairfield H, I, K J 8) Chesterfield - E, F, G 9) Jasper T, Y, A, AA, BB - 10) Beaufort T, V, W, X - 11) Colleton S, T, U - 12) Berkeley K, Q, R, U - 13) Georgetown CC DD 14) Marion GG DD 15) Horry CC, EE DD, FF Southeastern Naturalist 451 G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 Newt and one of Central Newt, supports our hypothesis of gene flow and introgressive hybridization. However, these patterns may instead reflect incomplete lineage sorting; additional data are needed to evaluate this possibility. These results also suggest that the current subspecies classifications are incorrec t. Statistical parsimony analysis divided the haplotypes into two geographic groups: one in the Pee Dee drainage and the other distributed throughout the remainder of the state. Furthermore, each includes a high-frequency and geographically widespread haplotype, indicating the groups are well-established (Posada and Crandall 2001). This finding suggests gene flow has been historically limited between these population groups despite evidence of haplotype mixing in some populations. In contrast to Gabor and Nice’s results, however, partitioning the haplotypes by subspecies in the AMOVA also accounts for a significant, albeit much lower, proportion of genetic variation. Thus, while hybridization has occurred, it has not been extensive enough to completely obscure taxonomic distinctions. Although group 1 includes haplotypes from all three subspecies, the haplotypes are Figure 4. Distribution of haplotype groups by locality. Black lines represent the boundaries of the three subspecies and dashed lines represent the boundaries of the four main drainages in the state (Petranka 1998). Key: stippled circle = group 1 haplotypes [Notophthalmus viridescens viridescens (Red-spotted Newt), N. v. louisianensis (Central Newt)]; dark gray circle = group 2 haplotypes (N. v. viridescens [Red-spotted Newt], N. v. dorsalis [Brokenstriped Newt]). Southeastern Naturalist G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 452 not randomly distributed in the network but instead group together. The only exceptions are haplotypes A, B, and C, which branch from a Central Newt haplotype rather from another Red-spotted Newt haplotype. This is also the case for group 2 with the exception of haplotype G. Results of the SAMOVA are consistent with the statistical parsimony analysis because the same geographic groups are recovered when K = 2. However, a greater proportion of among-group variation is accounted for when K = 3, suggesting greater geographic partitioning of variation and the subdivision of the Santee-Savannah- ACE Basin group into a piedmont and sandhills plus southern lower coastal plain group. Perhaps this result is not unexpected given the number of ancestral haplotypes separating the haplotypes associated with these two population groups (Table 1, Fig. 3). These results partition the populations more in accordance with current subspecies definitions than do the phylogenetic and statistical parsimony analyses because the Central Newt populations are separated from the Redspotted Newt populations in the piedmont. However, because this group includes one Red-spotted Newt population (Aiken County), it supports our hypothesis of hybridization between subspecies, as does the inclusion of a Red-spotted Newt population (Chesterfield County) with the Broken-striped Newt populations in the Pee Dee group. Gabor and Nice (2004) argued that the population genetic patterns observed in their northern and southern population groups were a product of North America’s recent glacial history. They contended that the southern populations represent refugial populations established during the last glacial maximum, whereas the northern populations represent ones that recolonized the previously glaciated areas of the species’ range following retreat of the ice sheet. Being older, one would predict that the southern populations had more time to accumulate genetic differences Figure 5. FCT values obtained with the program SAMOVA (Dupanloup et al. 2002) as a function of the number of groups of populations (K). Southeastern Naturalist 453 G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 while the younger northern populations would still retain greater genetic connectivity (Gabor and Nice 2004). North America’s glacial history has also been used to explain phylogeographic patterns in other pond-breeding salamanders such as Ambystoma maculatum (Shaw) (Spotted Salamander) (Zamudio and Savage 2003) and Ambystoma tigrinum (Green) (Tiger Salamander) (Church et al. 2003). Each study identified one refugial group occupying the southern coastal plain of the Carolinas and linked to populations in the northeast, and a second refugium associated with the Gulf Coast. Thus, we hypothesize that the haplotype groups identified in this study reflect refugial groups established during the last glacial maximum with the haplotype exchange at their boundaries representing secondary contact following glacial retreat. Whether there are two or three refugia as suggested by the statistical parsimony and SAMOVA analyses, respectively, is uncertain, and additional sampling is needed to fully understand the phylogeographic and population genetic patterns in this species. One of the most unexpected findings of this study was the absence of newts in any upper coastal plain locality during the sampling years, despite the presence of seemingly favorable newt habitats. For example, many of the sampled wetlands Figure 6. Distribution of groups identified by the SAMOVA. Black lines represent the boundaries of the three subspecies and dashed lines represent the boundaries of the four main drainages in the state. Stippled circle = piedmont, dark gray circle = sandhills and southern lower coastal plain, and light gray circle = Pee Dee drainage. Southeastern Naturalist G.R. Lawson and E.S. Kilpatrick 2014 Vol. 13, No. 3 454 supported Lithobates spp. (bullfrog) and Ambystoma opacum (Gravenhorst) (Marbled Salamander) larvae (total dip-net sweeps per site routinely captured >50 of the former and >100 of the latter), which commonly co-occur with newts in both the piedmont and the lower coastal plain. At present, we cannot assess whether this gap is a recent phenomenon related to anthropogenic disturbance (e.g., agriculture and silviculture) in this part of the state or some unobserved natural process. Additional collection efforts in the upper coastal plain in Georgia and North Carolina may provide insight as to whether this absence is unique to South Carolina or part of a larger regional pattern. If newts truly do not occur in the upper coastal plain, it could represent a significant geographic barrier impacting migration/gene flow among populations. Acknowledgments We would like to thank M. Brown, K. Browning, J. Camper, J. Castleberry, M. Danaher, M.L. Edwards, J. Jones, J. Luken, B. Metts, T. Mills, J. Palis, R. Reed, P. Thomas, and J. Waldron for their help locating newt populations, field work, and providing newt tissue samples for this study. We are also grateful to S. Baron, E. Lickey, and R. Puffenbarger at Bridgewater College and P. 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