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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 -
glawson@bridgewater.edu.
Manuscript Editor: John Placyk
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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
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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).
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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
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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).
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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.
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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
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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]).
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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).
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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.
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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. Fields at the University of Virginia for their assistance with the molecular
techniques and input on the project. This research was made possible with funding from the
Virginia Foundation of Independent Colleges, Bridgewater College, the Virginia Academy of
Sciences, and the University of South Carolina Salkehatchie.
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