Content uploaded by Benjamin Minault Fitzpatrick
Author content
All content in this area was uploaded by Benjamin Minault Fitzpatrick on Oct 24, 2020
Content may be subject to copyright.
Patterns of differential introgression in a salamander
hybrid zone: inferences from genetic data and ecological
niche modelling
M. W. H. CHATFIELD,*§ K. H. KOZAK,† B. M. FITZPATRICK‡ and P. K. TUCKER*
*Department of Ecology and Evolutionary Biology and Museum of Zoology, University of Michigan, Ann Arbor, MI 48109-
1079 USA, †Bell Museum of Natural History and Department of Fisheries, Wildlife and Conservation Biology, University of
Minnesota, St Paul, MN 55108 USA, ‡Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville,
TN 37996-1610 USA
Abstract
Hybrid zones have yielded considerable insight into many evolutionary processes,
including speciation and the maintenance of species boundaries. Presented here are
analyses from a hybrid zone that occurs among three salamanders – Plethodon jordani,
Plethodon metcalfi and Plethodon teyahalee – from the southern Appalachian Mountains.
Using a novel statistical approach for analysis of non-clinal, multispecies hybrid zones,
we examined spatial patterns of variation at four markers: single-nucleotide polymor-
phisms (SNPs) located in the mtDNA ND2 gene and the nuclear DNA ILF3 gene, and the
morphological markers of red cheek pigmentation and white flecks. Concordance of the
ILF3 marker and both morphological markers across four transects is observed. In three
of the four transects, however, the pattern of mtDNA is discordant from all other
markers, with a higher representation of P. metcalfi mtDNA in the northern and lower
elevation localities than is expected given the ILF3 marker and morphology. To explore
whether climate plays a role in the position of the hybrid zone, we created ecological
niche models for P. jordani and P. metcalfi. Modelling results suggest that hybrid zone
position is not determined by steep gradients in climatic suitability for either species.
Instead, the hybrid zone lies in a climatically homogenous region that is broadly suitable
for both P. jordani and P. metcalfi. We discuss various selective (natural selection
associated with climate) and behavioural processes (sex-biased dispersal, asymmetric
reproductive isolation) that might explain the discordance in the extent to which mtDNA
and nuclear DNA and colour-pattern traits have moved across this hybrid zone.
Keywords: climate change, differential introgression, ecological niche modelling, hybrid zone,
Plethodon, salamander
Received 8 October 2009; revision received 14 June 2010; accepted 15 June 2010
Introduction
Hybrid zones, which may form after secondary contact
between two partially reproductively isolated popula-
tions, have long been utilized in studies of speciation
and the maintenance of species boundaries (Barton &
Hewitt 1985). One important aspect of hybrid zone
studies is that different markers, either molecular or
morphological, may exhibit different patterns in fre-
quency change across a hybrid zone. These differences
may indicate important ecological and evolutionary
dynamics in the gene or gene regions under study (Tee-
ter et al. 2008, 2010) or between the interacting species
(e.g. Arntzen & Wallis 1991; Brito 2007).
One pattern that may emerge in hybrid zone analyses
is that of differential introgression. Numerous studies
have documented differential introgression of mtDNA
relative to nuclear DNA and morphology (e.g. Funk &
Omland 2003; Chan & Levin 2005). Many of these are
phylogenetic studies between closely related species
Correspondence: Matthew W. H. Chatfield,
Fax: (504) 862 8706, E-mail: mattchat@tulane.edu
§Present address: Department of Ecology and Evolutionary
Biology, Tulane University, New Orleans, LA 70118 USA.
2010 Blackwell Publishing Ltd
Molecular Ecology (2010) 19, 4265–4282 doi: 10.1111/j.1365-294X.2010.04796.x
that share mtDNA haplotypes, suggesting a pattern of
current or historic mtDNA gene flow (e.g. Weisrock &
Larson 2006; Linnen & Farrell 2007). Many other cases
documenting mtDNA introgression come from studies
of naturally occurring hybrid zones showing shifts in
clines of mtDNA relative to nuclear DNA and morphol-
ogy (e.g. Arntzen & Wallis 1991; Sequeira et al. 2005;
Vo
¨ro
¨set al. 2006; Brito 2007; Hofman & Szymura 2007;
Leache
´& Cole 2007; Kawakami et al. 2008).
A pattern of differential introgression may indicate
hybrid zone movement. Recent empirical work suggests
that hybrid zone movement may be more common than
once thought (Buggs 2007). This has led to inferences
on the ecological and evolutionary dynamics among
participating species (e.g. Arntzen & Wallis 1991; Hair-
ston et al. 1992; Rohwer et al. 2001; Garcı
´a-Parı
´set al.
2003). In a recent review, Buggs (2007) identified 23
studies which documented hybrid zone movement
and another 16 studies which had patterns consistent
with movement. These studies utilize two different
approaches: first, long-term monitoring of molecular
and morphological markers across a hybrid zone is a
reliable method for detecting movement. Second, analy-
sing differential patterns of introgression across a suite
of markers at a single point in time may allow inference
of movement. One cause of hybrid zone movement is
range expansion or contraction as a result of climate
change. Although evidence for this phenomenon is
limited (but see Britch et al. 2001 and Walls 2009), there
is widespread evidence that species’ ranges have shifted
in response to Pleistocene cooling and warming (e.g.
Davis & Shaw 2001; Peterson et al. 2004; Brito 2007).
Furthermore, shifts in hybrid zone position resulting
from climate change are expected to be greatest for
montane species, as geographically proximate loca-
tions may experience considerable climatic differences
(Hewitt 1996; Guralnick 2007; but see Peterson 2003).
Alternatively, a pattern of differential introgression
may indicate differential selection on the markers under
study or regions that are linked to those markers (Barton
1979). For example, such a pattern might be interpreted
as selection acting differentially on different mtDNA
alleles owing to the important metabolic functions of the
mitochondrion (Boutilier 2001; Nouette-Gaulain et al.
2005, Lynn et al. 2007; Tattersall & Ultsch 2008) or to
nuclear DNA-encoded phenotypic traits exhibited by
one, but not the other, parental species. Lastly, noncoin-
cident, biparentally inherited nuclear and maternally
inherited mtDNA clines may be because of mating or
dispersal asymmetries (Dakin 2006).
In this study, we present an analysis of a naturally
occurring hybrid zone among three species of salaman-
ders in the genus Plethodon. The hybrid zone occurs at
the boundary between the Great Smoky and Balsam
Mountains in the southern Appalachians of North Caro-
lina and Tennessee. The high elevation species P. jor-
dani and P. metcalfi are known to hybridize along two
ridgelines, Balsam Mountain and Hyatt Ridge (Hairston
1950; Highton 1970; Hairston et al. 1992). These ridge-
lines are high elevation corridors that connect the Great
Smoky to the Balsam Mountains, encompassing the
range of P. jordani and a portion of the range of P. met-
calfi, respectively. A third species, P. teyahalee, inhabits
lower elevations throughout much of the southern
Appalachians and hybridizes with the former species at
intermediate elevations (Peabody 1978; Manzo 1988;
Reagan 1992). The most complete study of this system
was conducted by Hairston et al. (1992). These authors
sampled salamanders from the same five localities
along the southern portion of Balsam Mountain each
year for an 18-year period and recorded the amount of
red cheek pigmentation, which is present in P. jordani
but absent in P. metcalfi. That study documented exten-
sive hybridization along the ridgeline, and because no
movement was detected, yielded some insight into the
short-term stability of the hybrid zone.
The analyses presented here expand on this study in
four ways: (i) patterns of spatial variation are examined
for mtDNA, nuclear DNA and morphological markers;
(ii) localities with hybrids among P. jordani,P. metcalfi
and P. teyahalee are analysed across four transects, two
along high elevation ridgelines predominately connect-
ing the ranges of P. jordani and P. metcalfi, and two ele-
vational transects between hybrid localities of the former
species with that of P. teyahalee; (iii) sampling in this
study was performed at a spatially fine scale, allowing
for increased resolution in the detection of introgression;
and (iv) ecological niche models are created for P. jordani
and P. metcalfi to explore whether ecological factors play
a role in determining the position of this hybrid zone. It
is only recently that the GIS-based method of ecological
niche modelling (ENM) has been used in hybrid zone
studies (Cicero 2004; Swenson 2006, 2008; Martı
´nez-Fre-
irı
´aet al. 2008; Swenson et al. 2008), although earlier
studies have incorporated habitat-genotype associations
(Bridle et al. 2001). Moreover, as the width of this hybrid
zone is narrow, the fine scale at which ENM is utilized in
this study represents a novel application to the study
of hybrid zones and highlights the utility of ENM, in
concert with genetic and morphological data, for under-
standing hybrid zone dynamics.
Methods
Study species
Salamanders in the genus Plethodon (family Plethodonti-
dae) comprise a monophyletic group that is distributed
4266 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
throughout the eastern and western United States (Petr-
anka 1998). Although under some debate, the estimated
number of species is around 55 (Collins & Taggart 2009).
All members of this group are fully terrestrial and
undergo direct development; therefore, population den-
sity is generally diffuse and uniform throughout the
environment. Plethodon jordani,P. metcalfi, and P. teyaha-
lee are primarily forest inhabitants, and all three species
(especially P. jordani and P. metcalfi) occur at high densi-
ties (Highton 1970; Merchant 1972; Hairston 1980a,b).
Sampling
Salamanders were sampled along transects from dis-
crete localities with each locality having a radius of
<50 m. Two high elevation transects were established
along Balsam Mountain and Hyatt Ridge (Fig. 1 and
Supporting information Table S1). Salamanders were
collected from 24 localities along the 24-km-long Balsam
Mountain, with a minimum of five salamanders
sampled at each locality; and 13 localities along 6 km of
the southernmost portion of Hyatt Ridge, with a mini-
mum of 10 samples per locality. Two elevational tran-
sects were also created that connect high and low
elevation populations (Fig. 1 and Supporting informa-
tion Table S1). Salamanders were collected from eight
localities, with a minimum of five salamanders per
locality, from the Palmer Creek transect beginning at
1390 m in elevation and extending 8 km to 951 m. The
Mt Sterling transect follows Mt Sterling Ridge for
(a)
(b)
Fig. 1 (a) Map of study area showing collection localities of parental (Plethodon jordani,Plethodon metcalfi, and Plethodon teyahalee)
and hybrid samples. Inset depicts location of study within the continental United States. (b) Expanded map of hybrid zones from A
showing collection localities of hybrid samples. In both a and b, darkened areas = high elevation and light areas = low elevation.
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4267
2010 Blackwell Publishing Ltd
1.5 km to Mt Sterling summit (at 1768 m elevation)
before descending for 2 km to 1134 m. Salamanders
were collected from 13 localities, with a minimum of
five salamanders sampled at each locality.
Parental animals from each of the three species were
also sampled at locations distant from known areas of
hybridization (Fig. 1 and Supporting information
Table S2). These included eight localities in the Great
Smoky Mountains (the range of P. jordani), six from the
Balsam Mountains (within the range of P. metcalfi) and
six from low elevations in the Great Smoky Mountains
(within the range of P. teyahalee). It should be noted
that Highton & Peabody (2000) and Weisrock & Larson
(2006) uncovered two genetically divergent lineages of
P. metcalfi corresponding to the Balsam and Blue Ridge
Mountains. As only populations from the Balsam
Mountains are known to hybridize with P. jordani, only
these populations of P. metcalfi were sampled.
Tissue collection and DNA extraction
Samples were collected in the field from 2004 to 2007
during the months of May–July. Salamanders were cap-
tured by hand and 10–20 mm of the tail tip removed
for genetic analysis. In the field, vials containing tissue
samples were immediately placed in an ice-salt mixture
(approximately )20 C) until they could be transferred
to liquid nitrogen 3–48 h later. At the end of each field
season, samples were removed from the liquid nitrogen
and stored at )80 C. All samples have been catalogued
into the tissue collection of the Division of Reptiles and
Amphibians, Museum of Zoology at the University of
Michigan (under accession number 2008–09 no. 3, and
uniquely identified by field number; Supporting infor-
mation Tables S3 and S4).
DNA was extracted from tail tissue using a standard
phenol-chloroform protocol (Museum of Vertebrate
Zoology, University of California, Berkeley, CA, USA).
Briefly, 0.5–20 mg of frozen tissue was washed in 1 mL
of cold STE buffer and then incubated in a mixture of
lysis buffer, proteinase K and RNase A. Samples were
centrifuged and pellets discarded. The resultant super-
natant was subjected to three rounds of purification
with a phenol-chloroform mixture and centrifugation.
DNA was precipitated in approximately 900 mL of cold
95%ethanol, centrifuged and the supernatant dis-
carded. The resultant pellet was washed twice with
70%ethanol, allowed to dry and resuspended in
100 lL of TE buffer.
Morphological markers
Animals were scored in the field for two morphological
markers. The first, red cheek pigmentation, is present in
all the animals captured within the range of P. jordani
and absent in parental populations of P. metcalfi and
P. teyahalee. Animals were scored on a 14-point scale,
with 0 indicating the complete absence of red cheek
pigmentation and 13 indicating bright and pervasive
red cheek pigmentation, extending onto the throat,
shoulders and forelimbs. Assigned scores accounted for
both extent and intensity of red pigmentation, as well
as both the right and left cheeks. The second marker,
white flecking, is present in pure populations of
P. teyahalee, and is absent in populations of P. jordani
and P. metcalfi. The amount and pattern of white fleck-
ing is variable in P. teyahalee, but lateral flecks are gen-
erally abundant and large, while dorsal flecks are
sparse and small. White flecks were scored as being
either present or absent. To achieve consistency in scor-
ing the morphological markers, all animals were scored
by MWHC. Digital photographs of the right and left
sides of the head were taken of each animal. These
images have been catalogued into the digital image col-
lection of the Division of Reptiles and Amphibians,
Museum of Zoology at the University of Michigan
(under accession number 2008–09 no. 3, image numbers
49–960, and uniquely identified by field number;
Supporting information Tables S3 and S4).
mtDNA marker
A series of two single-nucleotide polymorphisms (SNPs)
were identified in the mtDNA gene NADH subunit II
(ND2): the first distinguishes P. jordani from P. metcalfi
and P. teyahalee, and the second distinguishes P. teyaha-
lee from P. jordani and P. metcalfi. Thus, when used in
tandem, the SNPs were diagnostic for each species. The
panel used to identify these single-nucleotide differ-
ences consisted of the following pure parental samples:
18 animals from eight populations within the range of
P. jordani, 16 animals from five populations within the
range of P. metcalfi and 13 animals from six populations
within the range of P. teyahalee (Fig. 1 and Supporting
information Table S3). Approximately 950 base pairs of
the ND2 gene, the entire tRNA
Ala
gene and a portion of
the tRNA
Trp
gene were amplified with the forward pri-
mer MC001 (5¢-TTTCTAACCCAATCTATAGCATCC-3¢)
and the reverse primer MC002 (5¢-GTCTTGCAAGTTC-
GAGTCAGA-3¢), designed using the online software
Primer3 (Fig. 2a). Representative ND2 sequence data
have been deposited in GenBank under accession
numbers HM775317–HM775319. Polymerase chain reac-
tion (PCR) protocols followed Weisrock et al. (2001),
with the inclusion of a 5 -min hot start at 95 C and a
7 -min final extension at 72 C. Sequencing was per-
formed on an ABI 3730 XL automated DNA sequencer
through the University of Michigan DNA Sequencing
4268 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
Core facility. Resulting sequences were aligned using
Sequencher 4.8.
Scoring samples at the mtDNA locus was carried out
using restriction fragment length polymorphism (RFLP)
digests. The PCR product for each sample was divided
into two equal aliquots and digested with BanI and MfeI
restriction enzymes. When used in tandem, RFLP diges-
tions were unambiguous when scoring the ND2 gene.
Digestion with BanI cut the PCR product of P. jordani
into fragments with approximate lengths of 300 and 650
base pairs, while leaving the products of P. metcalfi and
P. teyahalee whole. Similarly, digestion with MfeI cut
the PCR product of P. teyahalee into fragments with
approximate lengths of 280 and 670 base pairs, while
leaving the products of P. jordani and P. metcalfi whole
(Fig. 2a). On rare occasions, P. jordani samples (i.e.
those cut with BanI) shared the P. teyahalee allele and
were cut with MfeI; however, because P. teyahalee was
never cut with BanI, resulting fragments remained diag-
nostic. Banding patterns from the RFLP digestions were
visualized and scored on 2%NuSieve gels.
Nuclear DNA markers
As with the mtDNA marker, a panel was developed to
identify diagnostic nuclear SNPs. The panel consisted
of 12 animals from six localities in the range of P. jor-
dani, 11 animals from five localities in the range of
P. metcalfi and eight animals from five localities in the
range of P. teyahalee (Fig. 1 and Supporting information
Table S3). Two SNPs (separated by four base pairs)
were identified in the nuclear gene interleukin enhancer
binding factor 3 (ILF3) that, when used in tandem,
could distinguish the three parental species. Samples
were scored at the nuclear gene markers by sequencing
each PCR product and scoring the sequences by eye.
Approximately 280 base pairs of the middle exon (and
partial sequences of the surrounding introns) of ILF3
were amplified with the forward primer MC003
(5¢-CCAGGCATTTATGCATCCTT-3¢) and the reverse
primer MC004 (5¢-CGTGCTAGCCTCGGTAACAT-3¢),
designed using Oligo 6.71 (Fig. 2b). PCR was per-
formed using a hot start of 94 C for 3 min, 20 cycles at
94 C for 30 s, 65 C minus 0.5 C⁄cycle for 30 s and
72 C for 1 min, followed by 20 cycles at 94 C for 30 s,
55 C for 30 s and 72 C for 1 min, with a final exten-
sion at 72 C for 8 min (E. Jockusch pers. comm.).
Sequencing and alignments were performed as
described earlier.
Hybrid zone analysis
To address questions about concordance or discordance
between markers, we could not use cline-fitting
methods typically applied to two-species hybrid zones
(e.g. Barton & Baird 1998) or traditional cytonuclear
ND2 tRNA
Ala
tRNA
Trp
G/GAACC C/AATTG
0178
846
11261041
MfeI
MC001 MC002
BanI
472
0
P. j or da ni TA C CTATATG G
P. metcalfi TA C CTATGTG G
P. teyahalee TA C TTATGTG G
Intron IntronExon
278
MC003 MC004
133 137
(a)
(b)
Fig. 2 (a) Map of the mtDNA ND2 gene with adjacent tRNA genes and relative positions of forward (MC001) and reverse (MC002)
primers. Expanded sections of the gene show the six-base-pair restriction enzyme (BanI and MfeI) recognition sites and cut sites (indi-
cated by ‘ ⁄’) that, when used in tandem, are diagnostic for Plethodon jordani,Plethodon metcalfi, and Plethodon teyahalee. (b) Partial map
of nuclear ILF3 gene showing relative positions of the middle exon and two introns and the forward (MC003) and reverse (MC004)
primers. Expanded section depicts diagnostic SNPs (in bold). Base pairs are indicated below arrows in both A and B. Maps are not
drawn to scale.
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4269
2010 Blackwell Publishing Ltd
disequilibrium analyses for the two-species case (As-
mussen et al. 1987). Instead, two different types of anal-
yses were performed to explore differential patterns of
introgression. The first consisted of two chi-square con-
tingency table tests on the entire data set that were used
specifically to test for differential introgression of
mtDNA relative to the ILF3 marker. The first test was
performed on individual samples and tested the null
hypothesis of no difference in relative abundance of
P. jordani,P. metcalfi and P. teyahalee genotypes
between the two markers. The second test compared
sample localities that were classified by their most com-
mon genotype at the nuclear and mtDNA markers and
tested the null hypothesis that the number of sites dom-
inated by each species’ genotype was the same for the
two markers. In both tests, significance was assigned at
the P£0.05 level.
Second, we used generalized log-linear models
(GLMs) to fit specific genetic models to the genotypic
contingency table for the entire data set (Table 1).
Genotypes were grouped according to whether they
were (i) ‘parental’ (homozygous at the nuclear marker
and had homospecific mtDNA), (ii) ‘homozygous
hybrid’ (homozygous at the nuclear marker and had
heterospecific mtDNA), (iii) ‘2-way heterozygote’ (het-
erozygous at the nuclear marker and had mtDNA from
one of the nuclear parents) or (iv) ‘3-way heterozygote’
(heterozygous at the nuclear marker and had mtDNA
from a third parental species). Although these designa-
tions apply only to the two genetic markers assayed
(‘parentals’ might be genetically mixed at other parts of
the genome), they allow us to specify a symmetrical
model in which the genotypic contingency table is pre-
dicted only by the marginal genotype frequencies and
these four hybrid categories. In addition to this sym-
metrical model and the null model (in which each pos-
sible 2-locus genotype is predicted only by the
frequencies of the single-locus genotypes), we tested
nine asymmetrical models constructed by including
interaction terms between single-locus genotypes and
the generic hybrid categories. These asymmetrical mod-
els incorporate variation within hybrid categories
according to the specific ancestry of genotypes. These
analyses test for patterns of association (linkage disequi-
librium) between mtDNA and ILF3 genotypes that
might be caused by spatial structure, mating behaviour
and ⁄or selection. Given the spatial structure of sam-
pling, we expected to reject the statistical null model
therefore what is most interesting is the biological inter-
pretation of symmetrical versus asymmetrical models.
Models were fitted using Poisson GLMs in the stats
package of R 2.6.2 (http://www.r-project.org).
Two methods were used to assess the suitability of
each model. The first is the Akaike information criterion
(AIC). AIC can be used to decide on the best of a given
set of models, or as a multimodel inference tool that
assesses the suitability of the full set of models given
the data (Burnham & Anderson 2004). Models are
ranked according to their AIC values, with lower values
indicating higher suitability. Often, as is done here, AIC
values are given as DAIC, which is the difference of
each AIC value from that of the best model. The second
method uses the residual deviance as a measure of the
goodness-of-fit of a model relative to a saturated model,
which acts as a baseline (Agresti 2007). Higher values
indicate more variation is unaccounted for by the model
and, therefore, lower values indicate models with a bet-
ter fit. Significance levels are determined by comparison
of each model to the saturated model. Therefore, an
insignificant residual deviance (P> 0.05) means the sat-
urated model does not fit significantly better than the
tested model, which we would infer is an adequate
description of the data because the residual variance is
adequately explained as sampling error.
Ecological niche modelling
Hybrid zones that are maintained by a balance between
dispersal of hybrids and selection against those hybrids
are termed tension zones and may move so as to
Table 1 Contingency table depicting genotype groupings
1
used in the generalized log-linear models
mtDNA Genotypes
2
Nuclear Genotypes
3
J⁄JM⁄MT⁄TJ⁄MJ⁄TM⁄T
J P HoH HoH He2 He2 He3
M HoH P HoH He2 He3 He2
T HoH HoH P He3 He2 He2
1
Genotype groupings of parental (P), homozygous hybrids (HoH), 2-way heterozygotes (He2), and 3-way heterozygotes (He3) are as
described in the text.
2
mtDNA genotypes are as follows: J = P. jordani,M=P. metcalfi, and T = P. teyahalee.
3
Each allele in the nuclear genotype is given for homozygotes (J ⁄J, M ⁄M, and T ⁄T) and heterozygotes (J ⁄M, J ⁄T, and M ⁄T).
4270 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
coincide with geographic barriers, population density
troughs (i.e. regions of poor quality habitat for one or
both parental lineages) or ecotonal regions (Barton &
Hewitt 1985). As such, environmental factors (e.g.
climate) might play an important role in maintaining
the spatial locations of hybrid zones. For example,
many hybrid zones are associated with ecotones where
lineages that are adapted to different climatic conditions
come into geographic contact and interbreed (e.g.
Cicero 2004; Swenson 2006). Alternatively, hybrid zones
are also common in environmentally homogeneous
regions where previously isolated lineages that share
similar climatic requirements come into geographic con-
tact. The latter type of hybrid zone may be maintained
primarily by dispersal of parental forms and endoge-
nous selection against hybrids (Barton & Hewitt 1985),
or they may correspond to locations where previously
isolated lineages are in the process of merging (e.g.
Pereira & Wake 2009).
To explore whether climatic factors might influence
the structure of the hybrid zone, we used ecological
niche modelling (ENM) to predict the geographic distri-
bution of climatically suitable habitats for P. jordani and
P. metcalfi, and the extent to which such locations geo-
graphically overlap. We used Maxent version 3.2 to
model the potential geographic distributions of P. jor-
dani and P. metcalfi. Briefly, Maxent predicts the
expected distribution of a species using data on the
environmental conditions where it is known to occur
and randomly selected background locations in the
study area. Maxent is a general approach for character-
izing probability distributions from incomplete informa-
tion and computes a probability distribution that
describes the relative suitability of each grid cell as a
function of the environmental variables at all the known
occurrence locations (Phillips et al. 2006). When the
model is projected into geographic space, it produces a
map of the species’ potential geographic distribution.
To construct the models, we used 19 temperature and
precipitation variables from the WordClim data set with
30-second spatial resolution (Hijmans et al. 2005a) and
georeferenced occurrence locations for P. jordani
(n= 374) and P. metcalfi (n= 289) obtained from the
U.S. National Museum of Natural History. Models for
P. teyahalee are not included as the model resolution is
not fine enough to permit meaningful interpretation in
the narrow area of hybridization between this species
and P. jordani and P. metcalfi. Most of the records used
in the analysis were collected by R. Highton and
assigned to species using morphology and allozymes.
Locality records occurring within the same map pixel
were removed to avoid pseudoreplication. To calibrate
the model, we used quadratic features, and default
parameters for the number of background pixels, regu-
larization, the convergence threshold and the maximum
number of iterations (following Phillips et al. 2006). We
randomly selected 75%of the occurrence locations to
construct the model; the remaining 25%were set aside
to test the model. We calculated the area under the
receiving operator characteristic (AUC) to test whether
the model could discriminate between the test localities
and 10 000 localities randomly selected from across the
study region (defined as the United States east of the
Mississippi River).
Maxent assigns a continuous suitability score to each
grid cell in the study area (referred to as the cumulative
probability). Thus, to map locations where suitable hab-
itats for both species come into geographic contact or
overlap, a threshold value for presence–absence must
be employed. For each species, we recorded the cumu-
lative probability associated with each georeferenced
occurrence location. We then classified as climatically
unsuitable, any grid cell falling in the lower 5th percen-
tile of this empirical distribution of suitability scores.
Finally, given this threshold for suitability, we used the
grid overlay function in DIVA GIS 5.2 (Hijmans et al.
2005b) to map the locations of habitats that were suit-
able for both P. jordani and P. metcalfi. Our threshold
adequately captures patterns of climatic suitability as
it results in very few presence locations (5 of 663)
incorrectly being classified as occurring in unsuitable
habitats.
As predicted by tension zone theory, an association
might exist between allele frequency and climate as a
result of the hybrid zone settling at a geographic bar-
rier, population density trough or ecotonal area (Barton
& Hewitt 1985). If selection associated with climatic fac-
tors influences the position of the hybrid zone, one
might expect an association to exist between climatic
suitability and gene frequencies. For example, if the
nuclear DNA of P. jordani confers greater fitness to the
climatic conditions in the hybrid zone, then one would
expect the P. jordani ILF allele to be present in greater
frequency at sites that have higher suitability for P. jor-
dani than for P. metcalfi. To explore this possibility, we
employed a hybrid index to score the proportion of
P. jordani and P. metcalfi alleles at each site. Our index
ranged from 1 (only P. jordani alleles present) to )1
(only P. mecalfi alleles present). Positive scores indi-
cated a greater frequency of the P. jordani allele; nega-
tive scores indicated a greater frequency of the
P. metcalfi allele. Sites in which P. jordani and P. met-
calfi alleles were present in equal frequencies received a
score of 0. Similarly, we scored whether each site was
more climatically suitable for P. jordani or P. metcalfi by
calculating the difference in climatic suitability (i.e.
cumulative probabilities from Maxent) between the two
species. This climatic suitability index ranged from 100
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4271
2010 Blackwell Publishing Ltd
to )100, with the former score indicating that a site has
the maximum suitability for P. jordani and is com-
pletely unsuitable for P. metcalfi, and the latter score
indicating that a site has the maximum suitability for
P. metcalfi and is completely unsuitable for P. jordani.
Similar to the allele frequency index, scores of zero
indicated that a site is equally suitable for both species.
Positive scores indicated that a site was more suitable
P. jordani; negative scores that a site was more suitable
for P. metcalfi. We then used Spearman’s rank correla-
tion to test for a significant association between the
allele frequency- and habitat-suitability scores, and the
habitat-suitability scores and cheek colouration.
We caution that distribution-based niche modelling
makes the implicit assumption that biotic factors (i.e.
competition) do not prevent species from occupying the
full extent of climatic conditions in which they can sur-
vive and successfully reproduce. If competitive interac-
tions strongly influence a species distribution, then the
geographic extent of climatically suitable habitats could
be drastically underestimated (Pearson & Dawson 2003;
Kozak et al. 2008). However, given that the niche mod-
els of both species predict large areas of suitable habitat
beyond their empirical range limits (and in the hybrid
zone), it does not appear that biotic interactions have
strongly biased our results in such a way (see Results).
Finally, some ENM algorithms have been criticized
for overfitting climatic variables to species’ presence
records and climatic variables (Peterson et al. 2007).
Given that models of P. jordani and P. metcalfi (and
many other species of Plethodon, see Kozak & Wiens
2006) predict large areas of highly suitable habitat out-
side of their empirical distributions, overfitting does not
appear to have strongly influenced the predicted distri-
butions of these species. Nevertheless, if the climatic
niche breadth of either species has been underestimated
either because of biotic interactions or because of over-
fitting, then the overlapping zone of suitable habitats
for P. jordani and P. metcalfi in the hybrid zone would
in reality be even wider. Thus, our conclusion that loca-
tion and dynamics of the hybrid zone are not associated
with steep gradients in climatic suitability for either
species (see Results) is robust to these potential short-
comings of ENM algorithms.
Results
General patterns
Every salamander captured within the range of pure
Plethodon jordani had at least some red cheek pigmenta-
tion (mean = 8.02, range = 1–13, n= 90), while red pig-
mentation was entirely absent in pure P. metcalfi
localities (n= 55) and P. teyahalee (n= 21). Similarly,
the presence of white flecks was found to be diagnostic
for pure P. teyahalee (n= 11) and was completely absent
in pure P. jordani and P. metcalfi localities (Supporting
information Table S3). Within the hybrid zone, the
number of animals collected per transect is as follows:
Balsam Mountain, 5–16 animals per locality, 264 ani-
mals total; Hyatt Ridge, 10–15 per locality, 155 total;
Palmer Creek, 5–16, 86 total; and Mt Sterling, 5–13 per
locality, 140 total (Supporting information Table S4).
Salamanders captured within the hybrid zone show a
wide range of cheek pigmentation scores (range = 0–13,
n= 645), and 14 individuals showed the presence of
both red cheek pigmentation and white flecks.
Hybrid zone analysis
Most combinations of genotypic classes are represented
in the hybrid zone (Table 2). Curiously, no P. met-
calfi ⁄P. teyahalee heterozygotes were found at the nuc-
lear marker. Similarly, no individuals were found that
were homozygous for P. teyahalee at the nuclear marker
while having P. jordani mtDNA. Other genotypic
classes are also uncommon. For example, only a single
individual was sampled that was heterozygous for
P. jordani and P. metcalfi at the nuclear marker but had
P. teyahalee mtDNA. Similarly, only two individuals
were sampled that were homozygous for the P. metcalfi
nuclear allele but had P. teyahalee mtDNA. Most other
hybrid genotype combinations are moderately well
represented. For example, P. jordani ⁄P. metcalfi hetero-
zygotes at the nuclear marker with P. jordani mtDNA
were found in 28 individuals; P. jordani ⁄P. teyahalee
heterozygotes at the nuclear marker with P. metcalfi
mtDNA were found in 15 individuals; and P. jor-
dani ⁄P. teyahalee heterozygotes with P. jordani mtDNA
were found in 8 individuals. Results from the contin-
gency table test on individuals show that P. metcalfi
mtDNA is most common, while the P. jordani ILF3
genotype is most common (v
2
= 193.1835, d.f. = 2,
P< 0.0001; Table 3). Similarly, results from the contin-
gency table test on localities show that P. metcalfi
mtDNA is most common and the P. jordani ILF3 geno-
type is most common at the majority of localities
(v
2
= 9.0195, d.f. = 2, P= 0.0110; Table 4). This suggests
P. metcalfi mtDNA is more widespread than the P. met-
calfi ILF3 genotype, and the P. jordani ILF3 genotype is
more widespread than P. jordani mtDNA.
The general linear models depict general patterns of
association that might be functions of the population
spatial structure, nonrandom mating, natural selection,
or any combination thereof. Based on resulting DAIC
values and residual deviances, the models may be cate-
gorized into three groups (Table 5). The first group
contains those models with large DAIC values and
4272 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
significant residual deviances, meaning these models
are not suitable given the data. This group includes the
null model, the symmetrical model and the following
asymmetrical models: P. metcalfi ⁄P. teyahalee heterozyg-
otes, P. metcalfi homozygotes, P. jordani ⁄P. metcalfi het-
erozygotes and P. jordani ⁄P. teyahalee heterozygotes (all
are ILF3 genotypes). The second group contains those
models with moderate DAIC values and smaller,
although still significant, residual deviances. This group
includes P. jordani homozygotes (ILF3 genotype), and
the models varying P. jordani and P. metcalfi mtDNA.
One interesting trend observed in this group is the
overrepresentation of P. jordani homozygotes at the
ILF3 marker, which suggests P. jordani hybrids are
more likely to backcross with P. jordani than with
P. metcalfi. Another trend is the atypical patterns of
P. jordani and P. metcalfi mtDNA, which reflects an
overrepresentation of P. metcalfi mtDNA and a subse-
quent underrepresentation of P. jordani mtDNA. The
third group contains two models that have low DAIC
values and insignificant residual deviances. These mod-
els – P. teyahalee homozygotes (at the ILF3 marker) and
P. teyahalee mtDNA – are the only two models not
rejected by the goodness-of-fit test. Overall, these results
demonstrate associations among genotypes (rejection of
the null model), with a disproportionate underrepresen-
tation of P. teyahalee genotypes in hybrids (for the sym-
metrical model, the residual deviance for ‘parental’
P. teyahalee genotypes is positive, and the residual devi-
ances of all hybrid genotypes with P. teyahalee mtDNA
or ILF3 alleles are negative). This finding is consistent
with observations that P. teyahalee is the most ecologi-
cally (based on elevation differences in species ranges)
and morphologically (Highton & Peabody 2000) diver-
gent of the three species.
Three patterns emerge from the Balsam Mountain
transect: first, at four localities near the centre of the
transect (LB, LC, PO, and FR), the presence of P. teyaha-
lee was detected both morphologically and genetically
(Fig. 3). This occurs at Pin Oak Gap, a low point in the
otherwise high elevation of the Balsam Mountain ridge-
line. Second, frequencies of the P. jordani nuclear allele
and the incidence of red cheek pigmentation are in
close agreement. That is, populations from localities
along the ridgeline that show a predominance of the
P. jordani nuclear allele also have a high cheek pigmen-
tation score (e.g. localities LG, BH, BG, and SP; Fig. 3).
Third, frequencies of P. jordani mtDNA are largely dis-
cordant with respect to those of the nuclear allele and
red cheek pigmentation. This is most clearly seen in
localities GF, BI, LG, BG, LB, SM, SP, HC, CB, and CA
(Fig. 3). Notably, this represents a shift of P. metcalfi
mtDNA northwards relative to the P. jordani ILF3 allele
and morphology.
The pattern of differential introgression is not seen in
the Hyatt Ridge transect (Fig. 4). Rather, there is coinci-
dence among all markers. Also unlike Balsam Moun-
tain, the P. teyahalee allele does not appear on Hyatt
Ridge, except for one individual that is heterozygous at
the ILF3 marker (with P. jordani mtDNA and no white
flecks) and one apparently pure P. teyahalee from the
southernmost collection locality (which has a lower ele-
vation than most other sites on the ridgeline).
The pattern of differential introgression is strongly
apparent in the Palmer Creek transect (Fig. 5). The
Table 2 Summary of salamander samples from the hybrid
zone that are classified by their nuclear and mtDNA geno-
types. The number of animals with at least some red on their
cheeks and with at least some white flecks is given in paren-
theses, respectively
Nuclear DNA
mtDNA
P. jordani P. metcalfi P. teyahalee
P. jordani ⁄P. jordani 180 (177.1) 168 (142.2) 15 (10.6)
P. metcalfi ⁄P. metcalfi 4 (3.0) 96 (12.3) 2 (0.2)
P. teyahalee ⁄P. teyahalee 0 (0.0) 5 (3.1) 47 (2.42)
P. jordani ⁄P. metcalfi 28 (27.0) 67 (25.3) 1 (0.0)
P. jordan ⁄P. teyahalee 8 (8.1) 15 (10.2) 9 (5.5)
P. metcalfi ⁄P. teyahalee 0 (0.0) 0 (0.0) 0 (0.0)
Table 3 Contingency table showing individual sample data
used to test for differential patterns of introgression
Species
mtDNA
Nuclear
DNA
Total
Obs. Exp. Obs. Exp.
Plethodon jordani 227 365 868 730 1095
P. metcalfi 369 235 336 470 705
P. teyahalee 82 78 152 156 234
Total 678 678 1356 1356 2034
Table 4 Contingency table showing sample localities classified
by their most common genotype
Species
mtDNA
Nuclear
DNA
Total
Obs. Exp. Obs. Exp.
Plethodon jordani 27 36 45 36 72
P. metcalfi 35 27.5 20 27.5 55
P. teyahalee 12 10.5 9 10.5 21
Total 74 74 74 74 148
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4273
2010 Blackwell Publishing Ltd
upslope (western) end of the transect begins in a region
containing P. jordani and P. metcalfi hybrids (locality
PC); however, the ILF3 and mtDNA markers show a
prevalence of P. metcalfi alleles. The average cheek pig-
mentation score is 0.9, with 2 of 11 animals having
some red pigmentation and a considerable number of
P. jordani ILF3 alleles (8 of 20 alleles). There is, how-
ever, a complete absence of P. jordani mtDNA at this
site. The presence of the P. jordani ILF3 allele extends
downslope to 1000 -m elevation, almost to the valley
floor. The presence of some red cheek pigmentation
extends to the lowest elevation sampled (locality BK at
951 m with an average cheek pigmentation score of
0.125). This pattern is in contrast to the complete
absence of P. jordani mtDNA along the transect. Pat-
terns in white flecks are consistent with those of red
cheek pigmentation and nuclear DNA. Thus, there is a
clear discordance with P. jordani mtDNA being
restricted to the highest elevations sampled despite the
presence of the P. jordani ILF3 allele and morphology
extending much of the way to the valley below.
Patterns of marker frequencies are more complicated
along the Mt Sterling transect (Fig. 6). Along the ridge-
line of the western portion of the transect (localities
ZM-ZI; 1561–1768 m elevation), there is a predominance
of the P. jordani ILF3 allele, mtDNA and morphology,
although P. metcalfi mtDNA is slightly overrepresented.
At localities beginning immediately off the ridge top,
there is a complete absence of P. jordani mtDNA. This
is in sharp contrast to the patterns of the P. jordani ILF3
allele and red cheek pigmentation, both of which pre-
dominate down to an elevation of 1280 m. As in the
Palmer Creek and Balsam Mountain transects, patterns
in P. teyahalee (white flecks) are largely coincident with
those of red cheek pigmentation and nuclear DNA.
Thus, the same pattern of discordance between P. met-
calfi mtDNA and the P. jordani ILF3 allele and morphol-
ogy that is found in the Palmer Creek transect is also
seen in the Mt Sterling transect.
Ecological niche models
The predicted geographic distributions for P. jordani and
P. metcalfi are shown in Fig. 7. The area under the receiv-
ing characteristic (AUC) shows that the ecological niche
models strongly discriminate between randomly selected
locations across the study region and the training
(AUC
P. jordani
= 0.99, AUC
P. metcalfi
= 0.97) and the test
localities (AUC
P. jordani
= 1.0, AUC
P. metcalfi
= 0.99).
The geographic distributions of climatically suitable
habitats for P. jordani and P. metcalfi are not entirely
overlapping. Nevertheless, it seems unlikely that
divergent climatic adaptation or the presence of popula-
tion density troughs influences the position of the
hybrid zone. The climatic conditions are highly suitable
for both species at many of the sampling locations in
the hybrid zone (Fig. 7). Across the hybrid zone, there
is no relationship between allele frequency scores
and climatic suitability scores (mtDNA ·climatic suit-
ability: q=)0.197, P> 0.35; ILF ·climatic suitability:
q=)0.107; P> 0.618). Furthermore, there is also no
relationship between cheek colouration scores and habi-
tat-suitability scores (q=)0.145; P> 0.496). Thus, it
seems unlikely that natural selection associated with cli-
mate and ⁄or gradients in population density influences
the dynamics of the hybrid zone.
Discussion
In this study, we document strong discordance in
the extent to which mtDNA and nuclear DNA and
Table 5 Results of general linear models showing the Akaike
information criterion values (given as DAIC), residual devi-
ances (and associated degrees of freedom) and significance
levels
Model
Residual
DAIC
1
Deviance
2
d.f.
3
P-value
4
Null 387.3449 405.4704 10 6.4467 ·10
)81
Symmetrical 55.6383 69.7638 8 5.4763 ·10
)12
Asymmetrical
5
P. metcalfi ⁄P. teyahalee –– ––
P. metcalfi ⁄P. metcalfi 57.0183 69.1439 7 2.2001 ·10
)12
P. jordani ⁄P. metcalfi 54.0597 66.1852 7 8.6866 ·10
)12
P. jordani ⁄P. teyahalee 54.0597 66.1852 7 8.6866 ·10
)12
P. jordani 15.8975 24.0230 5 2.1491 ·10
)4
P. metcalfi 13.2398 21.3654 5 6.9090 ·10
)4
P. jordani ⁄P. jordani 12.8195 24.9450 7 7.7603 ·10
)4
P. teyahalee ⁄P. teyahalee 1.8222 13.9477 7 0.052120
P. teyahalee 0.0000 8.1255 5 0.14950
1
Akaike information criterion values given as the difference
from the best model.
2
Residual deviance is a measure of the goodness-of-fit of a
model to the data. Higher values indicate that more variation
is unaccounted for by the model and, therefore, lower values
indicate models with a better fit.
3
Degrees of freedom of the residual deviance.
4
Insignificant residual deviance (P> 0.05) means the residual
variance is adequately explained as sampling error and,
therefore, the model is an adequate description of the data.
Insignificant residual P-values are given in bold.
5
Results for all nine possible asymmetrical models are given
(except for P. metcalfi ⁄P. teyahalee heterozygotes because none
were found at the nuclear marker). Each asymmetrical model
was constructed by adding interaction terms involving the
listed marker genotype and all relevant genotypic categories.
Genotypes with ‘ ⁄’ are diploid ILF3 genotypes and all others
are mtDNA genotypes.
4274 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
colour-pattern traits have moved across a salamander
hybrid zone in the Great Smoky Mountains. A variety
of selective (natural selection associated with climate)
and behavioural processes (sex-biased dispersal, asym-
metric reproductive isolation) acting alone or together
might explain this discordance. Here, we discuss the
evidence favouring each of these explanations.
Unlike some hybrid zones (e.g. Cicero 2004; Swenson
2006; Martı
´nez-Freirı
´aet al. 2008; Swenson et al. 2008),
we found no evidence that the location of the hybrid
zone between P. jordani and P. metcalfi is associated
with steep gradients in climate or habitat suitability.
The ecological niche models suggest that the climatic
conditions are suitable for both species across much of
the hybrid zone. Similarly, we found no relationship
between the climatic suitability scores for P. jordani and
P. metcalfi across the transect and the proportion of
samples having alleles that were diagnostic for either
species. Thus, it seems unlikely that exogenous selection
associated with climate could promote the movement of
either P. metcalfi mtDNA (e.g. Boutilier 2001; Nouette-
Gaulain et al. 2005; Lynn et al. 2007; Tattersall & Ultsch
2008) or P. jordani nuclear DNA across the hybrid zone
(e.g. Doiron et al. 2002; Bachtrog et al. 2006). The eco-
logical niche models do suggest that the hybrid zone
lies in a region containing suitable habitat for both
P. jordani and P. metcalfi. Thus, while exogenous, cli-
mate-associated selection may not be acting to maintain
hybrid zone position, the hybrid zone may nonetheless
be constrained by this region of overlapping, suitable
habitats. Furthermore, if this hybrid zone is a tension
zone (i.e. maintained by a balance between dispersal
TB
LU
DB
15
15
15
100
100
100
46.5
46.6
47.1
GF
BI
LG
BH
15
15
5
10
100
100
100
100
92.2
47.0
46.7
95 9
BH
BG
LB
LC
10
10
10
13
100
100
100
100
95.9
92.4
86.1
90.7
PO
PC
FR
SM
10
11
10
10
100
99.8
100
75 8
90.1
87.8
89.0
66 6
SM
SP
BB
CM
10
10
9
9
75.8
100
100
100
66.6
91.2
91.2
100
1km
HC
CB
CA
PG
10
10
10
10
100
75.5
75.8
75.8
46.9
47.2
66.7
100
N1km
ST
WK
HT
11
5
16
75.8
75.8
76.2
100
100
91.5
P. jordani P. metcalfi P. teyahalee
P. me tc al
f
i/ P. te
y
ahalee P.
j
ordani / P. metcal
f
i
Legend:
Fig. 3 Map of Balsam Mountain showing transect (bold line), collection localities, sample sizes, marker scores and habitat-suitability
values. Pie charts are interpreted as follows: mtDNA = proportion of samples at a given locality that are diagnostic for each parental
species, nuclear DNA = proportion of alleles at a given locality that are diagnostic for each parental species, cheek score = the aver-
age scaled cheek pigmentation score (i.e. average score divided by the average for pure parental Plethodon jordani), and white
flecks = the proportion of animals that have at least some white flecks. Habitat-suitability values are extracted from the ecological
niche models presented in Fig. 7 and are given as a percentage from 0 to 100. Note that more than one collection locality may lie
within a single grid cell; therefore, identical values may not be independent from one another.
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4275
2010 Blackwell Publishing Ltd
and selection), then the zone may have settled into this
region of overlapping habitats without actually being
maintained by climate-associated selection.
Male-biased dispersal is often invoked as a mecha-
nism by which nuclear DNA may move across a hybrid
zone more readily than mtDNA (e.g. Jockusch & Wake
2002; Garcı
´a-Parı
´set al. 2003). However, the available
evidence does not support the idea that greater dis-
persal of male Plethodon underlies the discordant pat-
terns of genetic and morphological variation in the
hybrid zone. The Hyatt Ridge transect is particularly
informative in this regard. Along Hyatt Ridge, dispersal
of pure P. metcalfi from the south is impossible, and no
pattern of asymmetrical introgression is observed. If
greater dispersal of male P. jordani were responsible for
the pattern of differential introgression observed in the
Balsam Mountain, Palmer Creek and Mt Sterling tran-
sects, then differential introgression should also be
observed in the Hyatt Ridge transect. In addition, a
recent study that directly tested for male-biased dis-
persal in a related species with similar behaviour and
ecology to P. jordani and P. metcalfi (Plethodon cinereus)
found that dispersal is equally restricted in both sexes
(Cabe et al. 2007).
An alternative explanation for the pattern of discor-
dance of genetic and morphological variation is that the
hybrid zone is moving. The underrepresentation of
P. jordani mtDNA that is seen in the Balsam Mountain,
Palmer Creek, and Mt Sterling transects may have
resulted from a shift in hybrid zone position southward
towards the range of P. metcalfi and downslope
towards the range of P. teyahalee. Support for this
hypothesis comes from laboratory-staged mating trials
between P. jordani and P. metcalfi as reported by Rea-
gan (1992). Specifically, she found that heterospecific
crosses between female P. metcalfi and male P. jordani
yielded about a 10%mating success rate (11 deposited
spermatophores and eight inseminations of 100 staged
crosses), while the reverse cross yielded only a 1%suc-
cess rate (1 spermatophore and 1 insemination of 100
crosses). Under this scenario, the front of the expanding
P. jordani distribution could not effectively remove the
P. metcalfi mtDNA left in its wake because of mating
asymmetry. Consequently, the extensive occurrence of
P. metcalfi mtDNA may best be viewed as a relict, i.e. a
‘footprint’, of the historic range of P. metcalfi.
Lastly, it is not possible to rule out that positive selec-
tion for P. jordani cheek colouration (along with linked
N1km
13RF
etihWkeehCpoP
code N mtDNA ILF3 Score flecks
13
10
10
RF
MS
BN
10
10
HB
HY
10
15
JC
MH
10
15
HS
QU
12
15
QB
QS
10
15
SK
RR
P. jordani P. metcalfi P. teyahalee
P. m e t ca l
f
i / P. teyahalee P.
j
ordani / P. metcal
f
i
Legend:
Fig. 4 Map of Hyatt Ridge showing transect (bold line), collection localities, sample sizes and marker scores. Interpretation of mar-
ker scores is as given in Fig. 3.
4276 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
nuclear genes) contributes to the discordant patterns
seen across the hybrid zone. Early studies on P. jordani
morphology show that red cheek pigmentation has an
aposematic function, serving as a warning to potential
predators (Huheey 1960; Brodie & Howard 1973; Hensel
& Brodie 1976). While not toxic, all three species are
noxious, and when disturbed, such as during a preda-
tor attack, copious amounts of glandular secretions are
released from the skin (Huheey 1960; Brodie & Howard
1973; Hensel & Brodie 1976; Brodie et al. 1979). Another
plethodontid salamander, Desmognathus imitator,is
likely a Batesian mimic of P. jordani, with about 25%of
the population possessing red cheeks like their noxious
model (Orr 1968; Brodie & Howard 1973). Aposematism
and mimicry of red cheek pigmentation suggest this
trait is under selection, and hybrids possessing at least
some red pigmentation may have a selective advantage
when the red pigmentation and the associated noxious
secretions are common, but a disadvantage when rare.
If this is the case, increased fitness of red-cheeked pop-
ulations may be an explanation for the patterns of intro-
gression observed in this study. Additional work on the
introgression of this morphological trait and possible
differences in defensive chemistry between species is
needed.
The dynamics of hybridization seen in the Palmer
Creek and Mt Sterling transects, may differ somewhat
from those found between P. jordani and P. metcalfi
along Balsam Mountain and Hyatt Ridge. First, there is
likely to be differential adaptation of the parental spe-
cies. Plethodon teyahalee is a large species that is
restricted to low elevations, whereas P. jordani and
P. metcalfi are smaller species that are only found in
cool, moist, high elevation habitats. The latter two spe-
cies may be restricted to high elevations as a result of
smaller body size, which leads to greater rates of evapo-
rative water loss (Spotila & Berman1976). Second, previ-
ous studies (Hairston 1980a,b, 1983; Hairston et al.
N1km
PC TRBC BD BFBE BJ BK
Pop code BK
11 5 16 14 1314 58
N
mtDNA
ILF3
Cheek score
White flecks
P. teyahalee
P. jordani P. metcalfi
P. metcalfi / P. teyahalee P. jordani / P. metcalfi
Legend:
Fig. 5 Map of Palmer Creek showing transect (bold line), collection localities, sample sizes and marker scores. Interpretation of
marker scores is as given in Fig. 3.
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4277
2010 Blackwell Publishing Ltd
N1 km
ZM ZL ZK ZJ ZI ZH ZG ZF ZE ZD ZC ZB ZA
Pop code
8 12 12 12 13 12 10 12 11 11 12 10 5N
mtDNA
ILF3
Cheek score
White flecks
P. jordani P. m etcalfi P. teyahalee
P. metcalfi / P. teyahalee P. jordani / P. metcalfi
Legend:
Fig. 6 Map of Mt Sterling showing transect (bold line), collection localities, sample sizes and marker scores. Interpretation of marker
scores is as given in Fig. 3.
Fig. 7 Ecological niche modelling results showing present-day predicted geographic distributions for Plethodon jordani and Plethodon
metcalfi. For comparison, collection localities are shown for both Plethodon jordani (+) and Plethodon metcalfi (O). The inner box encom-
passes the hybrid zone and is expanded to show sampling localities (D) along the Balsam Mountain transect. Colours indicate habi-
tat-suitability values as assigned by Maxent and are given as percentages (cumulative probability ·100; see text for more detail).
4278 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
1987; Adams 2004) have demonstrated that competitive
interactions may play a role in the distributions of
P. jordani and P. teyahalee. Lastly, P. jordani and P. met-
calfi occur at a greater density than P. teyahalee (High-
ton 1970; Merchant 1972; Hairston 1980a,b). Population
density is one determinant of hybrid zone structure
and, when asymmetrical, may result in the movement
of the hybrid zone towards the less dense parental spe-
cies (Barton & Hewitt 1985).
Hypotheses of hybrid zone movement based on pat-
terns of differential introgression of genes and traits
have been proposed in other systems. For example, in
fire salamanders (Salamandra) on the Iberian Peninsula,
Garcı
´a-Parı
´set al. (2003) found strong discordance
between mtDNA on the one hand and allozymes, mor-
phology and life history on the other, which they attrib-
uted to male-biased dispersal. In another study, a
hybrid zone among lizards in the genus Sceloporus of
the western United States appears to have shifted
1.5 km as a result of anthropogenic changes to the habi-
tat (Leache
´& Cole 2007). Lastly, Rohwer et al. (2001)
examined hybridization between warbler species of the
genus Dendroica from the Pacific Northwest. Patterns of
differential introgression between mtDNA and a suite
of morphological markers led the researchers to con-
clude that the hybrid zone is moving. Independent
observations on behaviour and inferences from histori-
cal data suggest mating asymmetry may be the cause of
hybrid zone movement in that system.
The findings presented here have considerable poten-
tial to explain the patterns observed in other hybrid
zones among Plethodon in the southern Appalachians. A
rapid radiation leading to high species richness (High-
ton 1995; Kozak et al. 2006; Wiens et al. 2006) has
resulted in myriad hybrid zones among Plethodon in the
southeastern United States (Highton & Peabody 2000).
Given the mountainous terrain encompassing the
ranges of many of these species, the seemingly narrow
climatic specificity of many high elevation Plethodon
species (Kozak & Wiens 2006, 2010), and past oscilla-
tions in climate, we may reasonably expect many of
these hybrid zones to be dynamic. One such example
occurs between P. shermani and P. teyahalee.Ina
20-year study, Hairston et al. (1992) documented a shift
in hybrid zone position, which they attributed to
changes in land use early last century. More recent
analyses, however, suggest that modern climate change
may actually be driving hybrid zone movement in that
system (Walls 2009).
Hairston et al. (1992) made the assumption that red
cheek pigmentation is neutrally diffusing across the
P. jordani- P. metcalfi hybrid zone. Based on the most
recent warming period during the Hypsithermal Inter-
val (approximately 5000 years ago; see Pielou 1991),
the authors hypothesized that populations of P. jordani
and P. metcalfi migrated along the Balsam Mountain
ridgeline from their mountain top refuges and met
near the centre of the hybrid zone as inferred by the
authors. However, the expanded and finer-scale sam-
pling performed in this study uncovered extensive
hybridization farther north than that documented by
Hairston et al. (1992), thus nearly doubling the width
of the hybrid zone. Furthermore, this study documents
a nonclinal transition between the parental species,
which suggests a much more complex biogeographic
history than the one outlined by Hairston et al. (1992).
A more likely scenario is that repeated bouts of isola-
tion and secondary contact (perhaps as early as eight
million years ago, i.e. shortly after molecular clock
estimates place the date of divergence; Highton 1995;
Kozak et al. 2006; Wiens et al. 2006) have left a com-
plex, mosaic pattern of hybridization. In addition,
severely limited dispersal abilities (as suggested by
small home range sizes; Madison & Shoop 1970; Mer-
chant 1972; Nishikawa 1990), long generation times
(every other year beginning at 4 years of age for
P. jordani and P. metcalfi; Hairston 1983) and stasis
during the 18-year study period of Hairston et al.
(1992) suggest this hybrid zone, if moving, may be
doing so very slowly. Hairston et al.’s (1992) hypothe-
sis of neutral diffusion gives way to one of differential
introgression, and possibly hybrid zone movement, on
an evolutionary time scale not readily measured by
ecological studies.
Acknowledgements
We especially thank the crew at the Appalachian Highlands
Science Learning Center and R. Highton for help with field
logistics. We also thank M. Vance, V. Chatfield and K. Ha-
med for help with field work, and K. Luzynski and A. Conti
for help with laboratory work. This research was funded
through awards to MWHC from the following institutions:
the Department of Ecology and Evolutionary Biology,
the Museum of Zoology, and the Horace H. Rackham School
of Graduate Studies at the University of Michigan; the
Society for the Study of Amphibians and Reptiles; and the
North Carolina Herpetological Society. This manuscript was
greatly improved through the help of three anonymous
reviewers.
References
Adams DC (2004) Character displacement via aggressive inter-
ference in Appalachian salamanders. Ecology,85, 2664–2670.
Agresti A (2007) An Introduction to Categorical Data Analysis.
John Wiley and Sons Inc., New Jersey.
Arntzen JW, Wallis GP (1991) Restricted gene flow in a
moving hybrid zone of the newts Triturus cristatus and
T. marmoratus in western France. Evolution,4, 805–826.
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4279
2010 Blackwell Publishing Ltd
Asmussen MA, Arnold J, Avise JC (1987) Definition and
properties of disequilibrium statistics for association between
nuclear and cytoplasmic genotypes. Genetics,115, 755–768.
Bachtrog D, Thornton K, Clark A, Andolfatto P (2006)
Extensive introgression of mitochondrial DNA relative to
nuclear genes in the Drosophila yakuba species group.
Evolution,60, 292–302.
Barton NH (1979) Gene flow past a cline. Heredity,43, 333–339.
Barton NH, Baird SJE (1998) Analyse v 1.3. Edinburgh:
http://www.biology.ed.ac.uk/research/institutes/evolution/
software/Mac/Analyse/
Barton NH, Hewitt GM (1985) Analysis of hybrid zones.
Annual Review of Ecology and Systematics,16, 113–148.
Boutilier RG (2001) Mechanisms of metabolic defense against
hypoxia in hibernating frogs. Respiration Physiology,128, 365–
377.
Bridle JR, Baird SJE, Butlin RK (2001) Spatial structure and
habitat variation in a grasshopper hybrid zone. Evolution,55,
1832–1843.
Britch SC, Cain ML, Howard DJ (2001) Spatio-temporal
dynamics of the Allenomobius fasciatus-A. socius mosaic
hybrid zone: a 14-year perspective. Molecular Ecology,10,
627–638.
Brito PH (2007) Contrasting patterns of mitochondrial and
microsatellite genetic structure among Western European
populations of tawny owls (Strix aluco). Molecular Ecology,16,
3423–3437.
Brodie ED Jr, Howard RR (1973) Experimental study of
Batesian mimicry in the salamanders Plethodon jordani and
Desmognathus ochrophaeus.American Midland Naturalist,90,
38–46.
Brodie ED Jr, Nowak RT, Harvey WR (1979) The effectiveness
of antipredator secretions and behavior of selected
salamanders against shrews. Copeia,1979, 270–274.
Buggs RJA (2007) Empirical study of hybrid zone movement.
Heredity,99, 301–312.
Burnham KP, Anderson DR (2004) Multimodel inference:
understanding AIC and BIC in model inference. Sociological
Methods Research,33, 261–304.
Cabe PR, Hanlon TJ, Aldrich ME, Connors L, Marsh DM
(2007) Fine-scale population differentiation and gene flow in
a terrestrial salamander (Plethodon cinereus) living in
continuous habitat. Heredity,98, 1–8.
Chan KMA, Levin SA (2005) Leaky prezygotic isolation and
porous genomes: rapid introgression of maternally inherited
DNA. Evolution,59, 720–729.
Cicero C (2004) Barriers to sympatry between avian sibling
species (Paridae: Baeolophus) in local secondary contact.
Evolution,58, 1573–1587.
Collins JT, Taggart TW (2009) Standard Common and Current
Scientific Names for north American Amphibians, Turtles,
Reptiles, and Crocodilians, 6th edn. Publication of The Center
for North American Herpetology, Lawrence, Kansas.
Dakin E (2006) Cytonuclear disequilibria in a spatially
structured hybrid zone. Theoretical Population Biology,70, 82–
91.
Davis MB, Shaw RG (2001) Range shifts and adaptive
responses to Quaternary climate change. Science,292, 673–
679.
Doiron S, Bernatchez L, Blier PU (2002) A comparative
mitogenomic analysis of the potential adaptive value of
arctic charr mtDNA introgression in brook charr populations
(Salvelinus fontinalis Mitchill). Molecular Biology and Evolution,
19, 1902–1909.
Funk DJ, Omland KE (2003) Species-level paraphyly and
polyphyly: Frequency, causes, and consequences, with
insights from animal mitochondrial DNA. Annual Review of
Ecology and Systematics,34, 397–423.
Garcı
´a-Parı
´s M, Alcobendas M, Buckley D, Wake DB (2003)
Dispersal of viviparity across contact zones in Iberian
populations of fire salamanders (Salamandra) inferred from
discordance of genetic and morphological traits. Evolution,
57, 129–143.
Guralnick R (2007) Differential effects of past climate warming
on mountain and flatland Species distributions: a
multispecies North American mammal assessment. Global
Ecology and Biogeography,16, 14–23.
Hairston NG (1950) Intergradation in Appalachian
salamanders of the genus Plethodon.Copeia,1950, 262–273.
Hairston NG (1980a) The experimental test of an analysis of
field distributions: competition in terrestrial salamanders.
Ecology,61, 817–826.
Hairston NG (1980b) Evolution under interspecific competition:
field experiments on terrestrial salamanders. Evolution,34,
409–420.
Hairston NG (1983) Growth, survival and reproduction of
Plethodon jordani: trade-offs between selective pressures.
Copeia,1983, 1024–1035.
Hairston NG Sr, Nishikawa KC, Stenhouse SL (1987) The
evolution of competing species of terrestrial salamanders:
niche partitioning or interference? Evolutionary Ecology,1,
247–262.
Hairston NG, Wiley RH, Smith CK, Kneidel KA (1992) The
dynamics of two hybrid zones in Appalachian salamanders
of the genus Plethodon.Evolution,46, 930–938.
Hensel JL Jr, Brodie ED Jr (1976) An experimental study of
aposematic coloration in the salamanders Plethodon jordani.
Copeia,1976, 59–65.
Hewitt GM (1996) Some genetic consequences of ice ages, and
their role in divergence and speciation. Biological Journal of
the Linnean Society,58, 247–276.
Highton R (1970) Evolutionary interactions between species of
North American salamanders of the genus Plethodon. Part I.
Genetic and ecological relationships of Plethodon jordani
and P. glutinosus in the southern Appalachian Mountains.
Evolutionary Biology,4, 211–241.
Highton R (1995) Speciation in eastern North American
salamanders of the genus Plethodon.Annual Review of Ecology
and Systematics,26, 579–600.
Highton R, Peabody RB (2000) Geographic protein variation
and speciation in salamanders of the Plethodon jordani and
Plethodon glutinosus complexes in the southern Appalachian
mountains with the description of four new species. In:
The Biology of Plethodontid Salamander (eds Bruce RC, Jaeger
R, Houck LD). pp. 31–94, Kluwer Academic ⁄Plenum,
New York.
Hijmans RJ, Guarino L, Jarvis A et al. (2005a) DIVA-GIS
version 5.2 (http://diva-gis.org).
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A
(2005b) Very high resolution interpolated climate surfaces
for global land areas. International Journal of Climatology,25,
1965–1978.
4280 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
Hofman S, Szymura JM (2007) Limited mitochondrial DNA
introgression in a Bombina hybrid zone. Biological Journal of
the Linnean Society,91, 295–306.
Huheey JE (1960) Mimicry in the color pattern of certain
Appalachian salamanders. Journal of the Elisha Mitchell
Scientific Society,76, 246–251.
Jockusch EL, Wake DB (2002) Falling apart and merging:
diversification of slender salamanders (Plethodontidae:
Batrachoseps) in the American West. Biological Journal of the
Linnean Society,76, 361–391.
Kawakami T, Butlin RK, Adams M, Paull DJ, Cooper SJB
(2008) Genetic analysis of a chromosomal hybrid zone in the
Australian morabine grasshoppers (Vandiemenella viatica
species group). Evolution,63, 139–152.
Kozak KH, Wiens JJ (2006) Does niche conservatism promote
speciation? A case study in North American salamanders.
Evolution,60, 2604–2621.
Kozak KH, Wiens JJ (2010) Niche conservatism drives
elevational diversity patterns in Appalachian salamanders.
American Naturalist,176, 40–54.
Kozak KH, Weisrock DW, Larson A (2006) Rapid lineage
accumulation in a non-adaptive radiation: phylogenetic
analysis of diversification rates in eastern North American
woodland salamanders (Plethodontidae: Plethodon).
Proceedings of the Royal Society Royal Society B-Biological
Sciences,273, 539–546.
Kozak KH, Graham CH, Wiens JJ (2008) Integrating GIS-based
environmental data into evolutionary biology. Trends in
Ecology and Evolution,23, 141–148.
Leache
´AD, Cole CJ (2007) Hybridization between multiple
fence lizard lineages in an ecotone: locally discordant
variation in mitochondrial DNA, chromosomes, and
morphology. Molecular Ecology,16, 1035–1054.
Linnen CR, Farrell BD (2007) Mitonuclear discordance is
caused by rampant mitochondrial introgression in
Neodiprion (Hymenoptera: Diprionidae) sawflies. Evolution,
61, 1417–1438.
Lynn EG, Zhongping L, Minerbi D, Sack MN (2007) The
regulation, control, and consequences of mitochondrial
oxygen utilization and disposition in the heart and skeletal
muscle during hypoxia. Antioxidants and Redox Signaling,9,
1353–1361.
Madison DM, Shoop CR (1970) Homing behavior, orientation,
and home range of salamanders tagged with tantalum-182.
Science,168, 1484–1487.
Manzo PA (1988) A Morphometric Analysis of Plethodon jordani
Blatchley and its Hybrids. PhD thesis, University of Maryland,
College Park, Maryland.
Martı
´nez-Freirı
´a F, Sillero N, Lizana M, Brito JC (2008) GIS-
based niche models identify environmental correlates
sustaining a contact zone between three species of European
vipers. Diversity and Distributions,14, 452–461.
Merchant H (1972) Estimated population size and home range
of the salamanders Plethodon jordani and Plethodon glutinosus.
Journal of the Washington Academy of Sciences,62, 248–257.
Nishikawa KC (1990) Intraspecific spatial relationships of two
species of terrestrial salamanders. Copeia,1990, 418–426.
Nouette-Gaulain K, Malgat M, Rocher C et al. (2005) Time
course of differential mitochondrial energy metabolism
adaptation to chronic hypoxia in right and left ventricles.
Cardiovascular Research,66, 132–140.
Orr LP (1968) The relative abundance of mimics and models in
a supposed mimetic complex in salamanders. Journal of the
Elisha Mitchell Scientific Society,84, 303–304.
Peabody RB (1978) Electrophoretic Analysis of Geographic
Variation and Hybridization of Two Appalachian Salamanders,
Plethodon jordani and Plethodon glutinosus. PhD thesis,
University of Maryland, College Park, Maryland.
Pearson RG, Dawson TP (2003) Predicting the impact of
climate change on the distribution of species: are bioclimate
envelope models useful? Global Ecology and Biogeography,12,
361–371.
Pereira RJ, Wake DB (2009) Genetic leakage after adaptive and
nonadaptive divergence in the Ensatina eschscholtzii ring
species. Evolution,63, 2288–2301.
Peterson AT (2003) Projected climate change effects on Rocky
Mountain and Great Plains birds: generalities of biodiversity
consequences. Global Change Biology,9, 647–655.
Peterson AT, Martı
´nez-Meyer E, Gonza
´lez-Salazar C (2004)
Reconstructing the Pleistocene geography of the Aphelocoma
jays (Corvidae). Diversity and Distributions,10, 237–246.
Peterson AT, Papes M, Eaton M (2007) Transferability and
model evaluation in ecological niche modeling: a comparison
of GARP and Maxent. Ecography,30, 550–560.
Petranka JW (1998) Salamanders of the United States and Canada.
Smithsonian Institution, Washington DC.
Phillips SJ, Anderson RP, Schapire RE (2006) Maximum
entropy modeling of species geographic distributions.
Ecological Modelling,190, 231–259.
Pielou EC (1991) After the Ice Age: The Return of Life to Glaciated
North America. The University of Chicago Press, Illinois.
Reagan NL (1992) Evolution of Sexual Isolation in Salamanders in
the Genus Plethodon, PhD thesis. The University of Chicago,
Chicago, Illinois.
Rohwer S, Bermingham E, Wood C (2001) Plumage and
mitochondrial DNA haplotype variation across a moving
hybrid zone. Evolution,55, 405–422.
Sequeira F, Alexandrino F, Rocha S, Arntzen JW, Ferrand N
(2005) Genetic exchange across a hybrid zone within the
Iberian endemic golden-striped salamander. Chioglossa
lusitanica Molecular Ecology,14, 245–254.
Spotila JR, Berman EN (1976) Determination of skin resistance
and the role of the skin in controlling water loss in
amphibians and reptiles. Comparative Biochemistry and
Physiology,55, 407–411.
Swenson NG (2006) Gis-based niche models reveal unifying
climatic mechanisms that maintain the location of avian
hybrid zones in a North American suture zone. Evolutionary
Biology,19, 717–725.
Swenson NG (2008) The past and future influence of geo-
graphic information systems on hybrid zone, phylogeo-
graphic and speciation research. Evolutionary Biology,21,
421–434.
Swenson NG, Fair JM, Heikoop J (2008) Water stress and
hybridization between Quercus gambelii and Quercus grisea.
Western North American Naturalist,68, 498–507.
Tattersall GJ, Ultsch GR (2008) Physiological ecology of
aquatic overwintering in ranid frogs. Biological Review,83,
119–140.
Teeter KC, Payseur BA, Harris LW et al. (2008) Genome-wide
patterns of gene flow across a house mouse hybrid zone.
Genome Research,18, 67–76.
DIFFERENTIAL INTROGRESSION IN A SALAMANDER HYBRID ZONE 4281
2010 Blackwell Publishing Ltd
Teeter KC, Thibodeau LM, Gompert Z, Buerkle AC, Nachman
MW, Tucker PK (2010) The variable genomic architecture of
isolation between hybridizing species of house mice.
Evolution,64, 472–485.
Vo
¨ro
¨s J, Alcobendas M, Martı
´nez-Solano I, Garcı
´a-Parı
´sM
(2006) Evolution of Bombina bombina and Bombina variegata
(Anura: Discoglossidae) in the Carpathian Basin: a history of
repeated mt-DNA introgression across species. Molecular
Phylogenetics and Evolution,38, 705–718.
Walls S (2009) The role of climate in the dynamics of a hybrid
zone in Appalachian salamanders. Global Change Biology,15,
1903–1910.
Weisrock DW, Larson A (2006) Testing hypotheses of
speciation in the Plethodon jordani species complex with
allozymes and mitochondrial DNA sequences. Biological
Journal of the Linnean Society,89, 25–51.
Weisrock DW, Macey JR, Ugurtas IH, Larson A, Panenfuss TJ
(2001) Molecular phylogenetics and historical biogeography
among salamandrids of the ‘‘true’’ salamander clade: rapid
branching of numerous highly divergent lineages in
Mertensiella luschani associated with the rise of Anatolia.
Molecular Phylogenetics and Evolution,18, 434–448.
Wiens JJ, Engstrom TN, Chippindale PT (2006) Rapid
diversification, incomplete isolation, and the ‘‘speciation
clock’’ in North American salamanders (genus Plethodon):
testing the hybrid swarm hypothesis of rapid radiation.
Evolution,60, 2585–2603.
This work forms part of M.W.H.C.’s Ph.D. thesis on the evolu-
tionary dynamics of salamanders in the Plethodon glutinosus
group. He is currently studying interactions between the
amphibian chytrid fungus and frogs of the eastern United
States. K.H.K. is interested in the evolutionary processes of
southern Appalachian salamanders, especially as revealed by
ecological niche modelling and physiological limits. B.M.F. stu-
dies conservation, population genetics, and patterns of hybridi-
zation across a variety of salamander taxa. P.K.T.’s research
focuses on speciation genetics, as informed by an extensive
house mouse hybrid zone in Eastern Europe.
Supporting information
Additional supporting information may be found in the online
version of this article.
Table S1 Transects, sites codes, elevation, and latitude and lon-
gitude for animals captured in the hybrid zone
Table S2 Sites codes, elevation, and latitude and longitude for
parental taxa
Table S3 Pure parental individuals (Plethodon jordani,P. met-
calfi, and P. teyahalee) and marker scores used in panel for mar-
ker development
Table S4 Samples (arranged by transect) and marker scores for
all samples used in analyses
Please note: Wiley-Blackwell are not responsible for the content
or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
4282 M. W. H. CHATFIELD ET AL.
2010 Blackwell Publishing Ltd
