See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/259903794

Genetic diversity and structure of an endemic

and critically endangered stream river
salamander (Caudata: Ambystoma leorae) in
Mexico

Article in Conservation Genetics · January 2014

Impact Factor: 2.19 · DOI: 10.1007/s10592-013-0520-9

CITATIONS READS

5 290

4 authors:

Armando Sunny Octavio Monroy-vilchis

Universidad Autónoma del Estado de Méxic… Universidad Autónoma del Estado de Méxic…
9 PUBLICATIONS 7 CITATIONS 64 PUBLICATIONS 393 CITATIONS

SEE PROFILE SEE PROFILE

Victor Fajardo Ulises Aguilera-Reyes

Universidad Autónoma del Estado de Méxic… Universidad Autónoma del Estado de Méxic…
17 PUBLICATIONS 46 CITATIONS 20 PUBLICATIONS 95 CITATIONS

SEE PROFILE SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Octavio Monroy-vilchis
letting you access and read them immediately. Retrieved on: 09 May 2016
Conserv Genet (2014) 15:49–59
DOI 10.1007/s10592-013-0520-9
RESEARCH ARTICLE
Genetic diversity and structure of an endemic and critically
endangered stream river salamander (Caudata: Ambystoma
leorae) in Mexico
Armando Sunny • Octavio Monroy-Vilchis •
Victor Fajardo • Ulises Aguilera-Reyes
Received: 23 February 2013 / Accepted: 24 July 2013 / Published online: 8 August 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Small or isolated populations are highly sus-
ceptible to stochastic events. They are prone and vulnera-
ble to random demographic or environmental fluctuations
that could lead to extinction due to the loss of alleles
through genetic drift and increased inbreeding. We studied
Ambystoma leorae an endemic and critically threatened
species. We analyzed the genetic diversity and structure,
effective population size, presence of bottlenecks and
inbreeding coefficient of 96 individuals based on nine
microsatellite loci. We found high levels of genetic
diversity expressed as heterozygosity (Ho = 0.804,
He = 0.613, He* = 0.626 and HNei = 0.622). The popu-
lation presents few alleles (4–9 per locus) and genotypes
(3–14 per locus) compared with other mole salamanders
species. We identified three genetically differentiated
subpopulations with a significant level of genetic structure
(FST = 0.021, RST = 0.044 y Dest = 0.010, 95 % CI). We
also detected a reduction signal in population size and
evidence of a genetic bottleneck (M = 0.367). The effec-
tive population size is small (Ne = 45.2), but similar to
another mole salamanders with restricted distributions or
with recently fragmented habitat. The inbreeding coeffi-
cient levels detected are low (FIS = -0.619–0.102) as is
gene flow. Despite, high levels of genetic diversity A.
Electronic supplementary material The online version of this
article (doi:10.1007/s10592-013-0520-9) contains supplementary
material, which is available to authorized users.
A. Sunny  O. Monroy-Vilchis (&)  V. Fajardo 
U. Aguilera-Reyes
Estación Biológica Sierra Nanchititla, Facultad de Ciencias,
Universidad Autónoma del Estado de México, Instituto literario
# 100, Colonia Centro, CP 50000 Toluca, Estado de México,
Mexico
e-mail: tavomonroyvilchis@gmail.com; omv@uaemex.mx
leorae is critically endangered because it is a small isolated
population.
Keywords Microsatellites  Endemic species 
Endangered species  Conservation  Mexico
Introduction
Small or isolated populations represent ‘‘islands’’ in terms
of gene flow and genetic diversity (Kim et al. 1998). Such
populations are very susceptible to stochastic events (Gibbs
1998; Hicks and Pearson 2003), resulting in fluctuations of
effective population size and other demographic and
environmental parameters that can lead to extinction
(Gibbs 1998). Small populations also tend to have less
genetic diversity than larger ones due to the loss of alleles
through genetic drift and the increased chance of inbreed-
ing (Van Treuren et al. 1991; Templeton and Read 1994;
Garner et al. 2005; Frankham et al. 2005). Reduced genetic
diversity impairs the population’s ability to cope with
environmental variation and can translate to lower fitness
for the individuals in the population, increasing the
extinction risk (Newman and Tallmon 2001; Johansson
et al. 2006). Establishing the genetic structure of small and
isolated populations may be critically important for man-
agement and conservation decisions (Palsbøll et al. 2007).
Although, genetic variability is often less critical for pop-
ulation persistence than factors like habitat loss or frag-
mentation (Lande 1988), it has a decisive role in a long
term because it enables populations to adapt and persist in a
changing environment.
Ambystoma leorae (Taylor 1943), is a micro-endemic
salamander from the ‘‘Sierra Nevada’’, Central Mexico. The
mole salamander was historically restricted to six locations
123
50
within the protected area Iztaccihuatl-Popocatepetl National
Park (Fig. 1, IPNP). Recent research identified a new and
relict locality of A. leorae, possibly the last one (Monroy
Vilchis et al. 2012). No data exist on their ecology and basic
biology, and some experts considered this species extinct
(Parra-Olea, pers. comm.). The maximum number of
organisms reported for the only known population studied
was 55 (Lemos-Espinal et al. 1999). The population is next to
the world’s most human populous region. This lack of
knowledge, the limited distribution and the demand for
resources (deforestation, pollution, water) by humans
threaten this species with extinction (SEMARNAT 2010;
Shaffer et al. 2004). Ecological and genetic studies are
important in order to provide information on the status of the
species. In the study area, the threats include the introduction
of cattle, the use of water for human consumption and the
direct collection of specimens for food. Our goals are to
understand the genetic diversity and structure, the effective
population size, the presence of bottlenecks and to determine
the percentage of inbreeding of the last remaining population
of A. leorae in order to develop conservation strategies.
Materials and methods
Study area
Iztaccihuatl-Popocatepetl National Park (IPNP) is located
on the border of the State of Mexico with Puebla. Geo-
graphical coordinates of the study site are 19°210 0900 N,
98°400 1100 W and 19°350 25’’N, 98°660 9700 W, at an altitude of
4,130 masl (Fig. 1). We sampled individuals from a 1 km
stream confined to a small alpine grassland (Muhlenbergia
sp.) surrounded by forest (Pinus hartwegii and Abies
Fig. 1 Map of Mexico
a showing Iztaccı́huatl-
Popocatépetl-Zoquiapan
National Park. b The points
marked from 2 to 7 are historic
populations considered extinct
and the number 1 is the study
population at Volcano Tláloc.
c Volcano Tláloc showing the
sample sites
123
Conserv Genet (2014) 15:49–59
religiosa). The vertebrate community consists of 25 species
of amphibians and reptiles, 46 species of birds, and 38
species of mammals (Monroy Vilchis et al. 2012). Given its
proximity to Mexico City, the area is subject to constant
stress by land use changes mainly by human settlements and
the exploitation of its forest resources.
Population sampling and molecular analyses
We sampled monthly for 1 year. The search for specimens
was intensive and all were caught by hand, between 9:00
am and 5 pm. Sampling was done in two rivers separated
by 0.56 km2. The rivers join after 0.68 km2. The rivers
flow over plains and gentle slopes, with pools approxi-
mately 5 m from each other. There are three basic sub-
strates (stones, sand and mud) and the mole salamanders
where captured every time in the same place. We obtained
96 tissue samples (tail tip) and place immediately in 90 %
ethanol, and then frozen at -20 °C until processed. We
performed DNA extraction with a commercial kit Dneasy
Blood and Tissue Kit (Qiagen), following the manufac-
turer’s instructions. We assessed DNA quantity and quality
with a biophotometer (Eppendorf, Hauppauge, New York)
and in 1.5 % agarose gel stained with 0.5 lg/ml ethidium
bromide and observed with UV light.
Microsatellite typing
Genomic DNA was used as template for amplification of
nine loci following published protocols (Parra-Olea et al.
2007), with an Apollo PCR Touchscreen ATC201. We
multiplexed amplified products on an ABI Prism3730xl
and sized with a Rox-500 standard in GENEMAPPER v.
4.0 (Applied Biosystems, Foster City, CA, USA). Multiple
Conserv Genet (2014) 15:49–59
samples were sized at least twice to assure reproducibility
and correct readings.
Statistical analyses
Identification of identical genotypes for recapture
and potential scoring errors
We performed an analysis using the software Gimlet 1.3.2
Valière (2002) in order to identify recaptured individuals
and reduce error in the interpretation of the results (Kohn
et al. 1999). The presence of null alleles and other typing
errors was determined using MICROCHECKER (Van
Oosterhout et al. 2004). These alleles may affect estima-
tions of population differentiation, by reducing genetic
diversity within populations (Chapuis and Estoup 2007).
Genetic structure
We used the software Geneland 3.2.2 (Guillot et al. 2005),
this software uses a Bayesian algorithm implemented in a
Markov Chain Monte Carlo scheme considering genetic
data and geographical coordinates. We choose the most
suitable combination of models for our data: we assumed a
correlated allelic frequencies model and a true spatial
model (Guillot et al. 2005), with a coordinate uncertainty
value of 100 m. We performed 10 independent runs with
1,000,000 iterations, thinning = 100 and burnin = 100,
using K = 10, K = 5, K = 3, K = 2 and K = 1 in each
trial. Once we got the maximum number of possible pop-
ulations (in this case K = 3), we proceeded with the
assigning of individuals, using 20 independent runs with
1,000,000 iterations (thinning = 100, burnin = 1,000). In
addition, we did an assignment analysis with the software
GeneClass version 2.0 (Piry et al. 2004), in which each
individual’s genotype is analyzed to determine to which
population each individual belongs, based on the genotypic
frequencies present in the populations (Paetkau et al.
1995). In order to estimate their degree of genetic structure,
we used several approaches. Given that microsatellites are
closer to the stepwise mutation model than the infinite
allele model, we calculated RST (Slatkin 1995; Michalakis
and Excoffier 1996). However, because the sample size is
low and few loci, the FST values give a more conservative
assessment (Gaggiotti et al. 1999). We calculated FST
based on Weir and Cockerham (1984), using Arlequin
version 3.5.1.2 (Excoffier and Lischer 2010). We also
calculated Jost’s (2008) D, using Software for the Mea-
surement of Genetic Diversity (SMOGD; Crawford 2010)
with 1,000 replicates in the bootstrapped parameters.
To analyze the distribution of the genetic variance
between and within populations, we used an analysis of
molecular variance (AMOVA) (Excoffier et al. 1992)
51
based on FST and RST. We calculate the significance using
a Wilcoxon test with 30,000 permutations of genotypes
among populations. In order to detect the degree of simi-
larity of the sampling sites based on the species genotypes,
we did a factorial correspondence analysis of the micro-
satellite data using Genetix 4.05 (Belkhir et al. 2004). This
test graphically projects the individuals in the factor space
defined by the similarity of their allelic states.
Genetic diversity
We assessed genetic variability in the population by esti-
mating the observed (no) and effective (ne) number of
alleles, observed (Ho) and expected (He) heterozygosity,
(He*). Expected heterozygosity corrected for small sample
sizes and Nei’s unbiased expected heterozygosity (HNei—
Nei 1973), using the software POPGENE version 1.31
(Yeh et al. 1997) and Genetic studio (Dyer 2009), for each
population-by-locus combination and then averaged over
all loci to produce population estimates.
Linkage disequilibrium between pairs of microsatellite
loci and departures from Hardy–Weinberg equilibrium
(HWE) within each sample and locus were evaluated with
a Markov chain approximation (10,000 dememorizations,
1,000 batches, and 10,000 iterations per batch) of the
Fisher’s exact test performed and calculated the unbiased
P value with a Markov chain algorithm in Genepop 4.0
(Raymond and Rousset 1995).
In order to identify non-neutral loci, we used the soft-
ware LOSITAN (Antao et al. 2008) based on an F-outlier
method and coalescent simulations to produce a null dis-
tribution of FST values based on an infinite island model for
the populations and an infinite allele model or a stepwise
mutation model for polymorphism. Loci with high FST are
under directional selection, and loci with low FST are under
stabilizing selection. We ran 100,000 iterations with a
significance of P B 0.05.
Isolation by distance, historic demography and genetic
bottlenecks
In order to test the correlation between the pairwise genetic
and geographical distance, we performed a Mantel test
(Mantel 1967) in GenAlEx v. 6 (Peakall and Smouse 2006)
with 10,000 randomizations. With the software, Popula-
tions 1.2.32 (Langella 2002) was constructed Neighbour-
Joining tree (NJ) with the Nei0 s standardized genetic dis-
tance (Nei 1972) with 1,000 bootstraps. We calculated the
effective population size (Ne) by a method that estimates
Ne from linkage disequilibrium (LD), using a Jackknife
method with LD values between pairs of loci and a random
mating system, with the software LDNE v.1.31 (Waples
2006; Waples and Do 2008).
123
52
We used the software BOTTLENECK version 5.1.26
(Cournet and Luikart 1996; Piry et al. 1999) to test for a
genetic signature of recent historical reduction in the
effective population size (i.e., a bottleneck), based on the
two-phase model, which is an intermediate model of evo-
lution considered more appropriate for microsatellites
(Cournet and Luikart 1996). Accordingly, we estimated the
observed and expected heterozygosity under the two-phase
model, with settings of 90 % stepwise mutation model,
10 % infinite allele model, and 10 % variance; and used
default values (70 % stepwise mutation model, 30 % infi-
nite allele model, and 10 % variance). Both settings were
run with 10,000 replicates. Excess heterozygosity was
tested using a Wilcoxon test. We estimated another mea-
sure of population size reduction, the Garza–Williamson
index (M, the ratio of number of alleles to range in allele
size) and the critical value (Mc), with the software Criti-
calM. M-values lower than the critical number are indic-
ative of historical population declines. The latter was done
based on 10,000 simulations and parameters from the
two-phase mutation model, as described in Garza and
Williamson (2001).
Inbreeding and relatedness
Finally, we evaluated the relatedness among individuals
within each population, and between populations in order
to test the levels of endogamy, with the software MLRE-
LATE (Kalinowski et al. 2006), which has the advantages
of being designed for microsatellites, is based on maximum
likelihood tests, and considers null alleles. We also esti-
mated the within-population coefficient of genetic relat-
edness, r (Queller and Goodnigh 1989) in GenAlEx v. 6
(Peakall and Smouse 2006). We bootstrapped allelic data
within populations 999 times to derive 95 % confidence
intervals for r estimates; populations with non-overlapping
bootstrap intervals are statistically distinct. We also per-
muted genotypes from all populations 999 times and
derived upper and lower 95 % confidence intervals (CIs)
for r based on all populations. These intervals represent the
range of r expected if random mating occurs across all
populations. Population r-values that fall above the upper
bound of the 95 % CI indicate that reproductive skew,
inbreeding or drift are increasing relatedness, despite
potential gene flow among localities.
Results
Identification of same genotypes and potential scoring
errors
Do not exist same genotypes and null alleles in any loci.
123
Conserv Genet (2014) 15:49–59
Genetic population structure
Three subpopulations (subpop1 N = 31, subpop2 N = 22
and subpop3 N = 44) were defined with GENELAND
based on 20 runs {[Ln Pr (K = 3) = -3,581.49]} the
individuals were systematically distributed in the same
groups. We performed data analysis considering the sub-
populations together (population) and the three other ones
independently (subpop1, subpop2 and subpop3). The
assignment analyses assigned 56 % of the individuals to
the three subpopulations found with Geneland (Table A1)
correctly. Another result regarding genetic differentiation
showed significant values (FST = 0.021, RST = 0.044 and
Dest = 0.010, 95 % CI. Table 1), in agreement with the
AMOVA results that revealed the majority of genetic
variation resided within populations (98 %; P = 0.01),
followed by that among populations (2 %; P = 0.01, Table
A8). Genetic structuring was low, three subpopulations can
be clearly distinguished (Fig. A3), and the same topology
was obtained with the principal components analysis (Fig.
A4). The NJ tree found that the ancestral subpopulation is
the subpop2 with a bootsrap of 69 %. The Nei0 s value
between the subpop1 and subpop2 was 0.081, between
subpop2 and subpop3 0.045, and subpop1 and subpop3 are
sister groups with a bootstrap of 100 % and a Nei0 s value of
0.041 (Fig. A2).
Genetic diversity
We found 43 alleles across the nine loci, with a range of 4–9
(average 5.4) alleles per locus for the population: subpop 1
had 3–8 (mean = 4.3), subpop2 had 3–6 (mean = 3.9) and
subpop3 had 3–8 (mean = 5) (Table 2 and Table A2).
Atig52.143, Atig52.115 and At52.2 were the most variable
loci (Fig. A6). We found 67 genotypes for the population
(Table A3). The smaller number of genotypes per locus was
three to At52.20, At52.10 and At52.34, however the largest
was 14 (Atig52.143). Atig52.143 had the highest number of
heterozygous genotypes (10), while the lowest was at
At52.10 At52.20 had 2. Loci that had the lowest and highest
number of homozygous genotypes were At52.34, with 0 and
At52.1 with 5. The subpopulations with the highest number
of genotypes was subpop1 and subpop3 with 48, and the
lower was subpop2 with 39. Expected and observed
heterozygosities in the population showed high values
(Ho = 0.804, He = 0.613, He* = 0.626 and HNei = 0.622),
while subpop1 had slightly higher (Ho = 0.849,
He = 0.616, He* = 0.626 and HNei = 0.616), subpop2
(Ho = 0.762, He = 0.627, He* = 0.641 and HNei = 0.627)
and subpop3 (Ho = 0.803, He = 0.596, He* = 0.602 and
HNei = 0.596) (Table 2). False discovery rate correction
tests found departures from HWE in one locus in the popu-
lation and in the subpop2 and subpop3 due to heterozygote
Conserv Genet (2014) 15:49–59 53

Table 1 Differentiation indices for the population and for each locus
Locus Population
FIS FIT FST RIS RIT RST GST_est G’ST_est DST D Dest

Atig52.143 -0.251 -0.249 0.003 -0.097 -0.103 -0.005 0 0 1.018 0.026 0

Atig52.115 0.102 0.103 0.002 0.112 0.122 0.011 0.003 0.012 1.029 0.042 0.009
At60.3 -0.606 -0.606 -0.0 -0.253 -0.268 -0.012 0 0 1.005 0.008 0
At52.1 -0.105 -0.092 0.032 -0.107 -0.005 0.091 0.018 0.057 1.044 0.063 0.040
At52.2 0.019 0.036 0.049 -0.106 -0.082 0.022 0.029 0.079 1.048 0.069 0.051
A52.6 -0.187 -0.189 -0.005 0.199 0.194 -0.006 0 0 1.008 0.011 0
At52.20 -0.619 -0.595 0.043 -0.128 0.023 0.134 0.028 0.091 1.062 0.087 0.064
At52.10 -0.619 -0.595 0.043 -0.128 0.023 0.134 0.028 0.091 1.062 0.087 0.064
At52.34 -0.382 -0.371 0.022 0.629 0.640 0.028 0.013 0.067 1.068 0.095 0.054
Average -0.294 -0.284 0.021 0.013 0.060 0.044 0.011 0.038 1.038 0.054 Ñ = 0.010
FIS, FST and FIT fixation indices estimated according to Weir and Cockerham (1984), RIS, RST and RIT fixation indices estimated according to
Slatkin (1995), GST_est genetic differentiation, G’ST_est genetic differentiation according to Hedrick (2005), DS component of the genetic diversity
and effective number of distinct genetic groups according to Jost (2008), D current differentiation by Jost (2008), Dest estimated current genetic
differentiation according to Jost (2008) with a correction for small sample sizes, Ñ harmonic mean

deficiency, but at different loci: At52.1 loci found departures
from HWE in the subpop1 due to heterozygote deficiency
(Table 1). We found linkage disequilibrium between the loci
At52.1-At52.34 in the subpop1, At52.20-At52.10 in the
subpop2 and At52.20-At52.10 in the subpop3. Most loci
showed private alleles in each subpopulation (sub-
pop1 = 32, subpop2 = 35 and subpop3 = 45; Table A2
and Fig. A6). The loci At60.3 and A52.6 under both models
(IAM and SMM) were found under balancing selection
FST = -0.012 and -0.009, p = 0. The remaining seven loci
were under neutral selection (Fig. A1 and Table A6).
Isolation by distance, historic demography and genetic
bottlenecks
The Mantel test for isolation-by-distance found a positive
but not significant relationship between geographic and
population genetic distance, (r2 = 0.011, P = 0.09) (Fig.
A5).
Evidence of a recent genetic bottleneck associated with a
heterozygote excess (BOTTLENECK results) was observed
for the three subpopulations (subpop1, subpop2 and sub-
pop3) when analyzed independently and when combined,
under the IAM model (P = 0.01). They also showed a
significant heterozygote excess with the TPM model
(P = 0.01) in the subpop1 and subpop2 and under the SMM
model none showed a significant heterozygote excess (Table
A10). The results from the Garza–Williamson test showed
empirical M values (population = 0.367, subpop1 =
0.414, subpop2 = 0.433 and subpop3 = 0.377) signifi-
cantly lower than Mc for all the subpopulations (popula-
tion = 0.9444, subpop1 = 0.9444, subpop2 = 0.9407 and
subpop3 = 0.9444), indicative of a historical bottleneck
(Table A11 and A12). The effective population size (Ne)
estimated from linkage disequilibrium was Ne = 45.2
(26.7–45.2, 95 % CI) for the population, Ne = 16.7
(13.6–17.1) for subpop1, Ne = 22.3 (21.3–22.3) for sub-
pop2 and Ne = 27.1 (19.6–32.9) for subpop3 (Table A9).
Inbreeding and relatedness
Finally, the values of the inbreeding coefficient (FIS; from
-0.619 to 0.102) indicates that there is no inbreeding in the
locus Atig52.143, Atig52.115, At52.100, At52.200 and
A52.600; and there is moderate inbreeding at locus At60.300,
At52.100 and At52.200 (Table 1 and Table A4). The propor-
tion of individual relatedness within each population was
similar (Table A13). Most individuals were unrelated (popu-
lation = 68.90 %, subpop1 = 62.36 %, subpop2 = 74.45 %
and subpop3 = 66.66 %), followed by siblings (popula-
tion = 26.42 %, subpop1 = 34.40 %, subpop2 = 23.37 %
and subpop3 = 30.56 %), half-siblings (population = 3.96 %,
subpop1 = 2.58 %, subpop2 = 3.03 % and subpop3 =
2.54 %), and parent/offspring (population = 0.70 %, sub-
pop1 = 0.64 %, subpop2 = 0 % and subpop3 = 0.22 %).
Average pairwise relatedness (rqg) was low for all subpop-
ulations, subpop1 = 0.049, subpop2 = 0.010 and sub-
pop3 = 0.072. (Fig. 2).
Discussion
This is the first study on genetic variability of A. leorae
using nine microsatellite loci from the last known popu-
lation of the species. High genetic diversity was found for
the population and for the three subpopulations obtained by

123
54 Conserv Genet (2014) 15:49–59

Table 2 Genetic diversity values per locus for the population and for each subpopulation
Locus
Atig52.143 Atig52.115 At60.3 At52.1 At52.2 A52.6 At52.20 At52.10 At52.34 Average

Population
Ho 0.816 0.605 0.992 0.642 0.515 0.713 0.981 0.981 1 0.804
He 0.651 0.662 0.610 0.583 0.520 0.605 0.586 0.586 0.716 0.613
He* 0.662 0.673 0.619 0.592 0.552 0.615 0.595 0.595 0.728 0.626
HNEI 0.655 0.681 0.614 0.600 0.518 0.602 0.603 0.603 0.721 0.622
na 9 6 4 6 6 4 4 4 6 5.44
ne 2.890 3.130 2.590 2.500 2.070 2.510 2.520 2.520 3.580 2.700
A 7.177 3.999 3.898 3.919 4.683 3 2.919 2.919 3.997 4.057
Subpop1
Ho 0.838 0.709 1 0.612 0.741 0.806 0.967 0.967 1 0.849
He 0.704 0.697 0.573 0.654 0.595 0.623 0.530 0.530 0.635 0.616
He* 0.715 0.708 0.583 0.664 0.605 0.634 0.539 0.539 0.645 0.626
HNEI 0.704 0.697 0.573 0.654 0.595 0.623 0.530 0.530 0.635 0.616
na 8 4 4 4 5 3 3 3 4 4.220
ne 3.370 3.300 2.340 2.890 2.470 2.650 2.130 2.130 2.740 2.670
A 3 4 4 3 5 4 3 3 6 3.889
Subpop2
Ho 0.772 0.500 1 0.545 0.409 0.636 1 1 1 0.762
He 0.631 0.598 0.615 0.542 0.584 0.615 0.624 0.624 0.807 0.627
He* 0.645 0.612 0.630 0.555 0.598 0.630 0.638 0.638 0.826 0.641
HNEI 0.631 0.598 0.615 0.542 0.584 0.615 0.624 0.624 0.807 0.627
na 3 4 4 3 5 4 3 3 6 3.880
ne 2.710 2.480 2.600 2.180 2.400 2.600 2.650 2.650 5.200 2.830
A 5.809 5.501 3.947 4.265 4.040 3 3.763 3.763 5.885 4.441
Subpop3
Ho 0.837 0.604 0.976 0.767 0.395 0.697 0.976 0.976 1 0.803
He 0.617 0.691 0.639 0.551 0.379 0.574 0.602 0.602 0.705 0.596
He* 0.625 0.699 0.646 0.557 0.383 0.580 0.609 0.609 0.713 0.602
HNEI 0.617 0.691 0.639 0.551 0.379 0.574 0.602 0.602 0.705 0.596
na 8 6 4 5 5 3 4 4 6 5
ne 2.610 3.240 2.770 2.220 1.610 2.340 2.510 2.510 3.390 2.580
A 5.643 5.050 3.874 4.043 4.790 3.407 3.647 3.647 5.870 4.441
Ho observed heterozygosity, He expected heterozygosity, He* heterozygosity expected with a correction for small samples (Dyer 2009), HNEI
expected heterozygosity, na observed number of alleles, ne effective number of alleles, A allelic richness (estimated for 22 individuals, the
minimum number of samples in a population)

Geneland, in contrast with what would be expected for
small (\200 individuals) and isolated populations (Kilpa-
trick 1981; Frankham 1998).
Genetic diversity
There is significant deviation of the Hardy–Weinberg
(HW) proportions due to a heterozygote deficiency. This is
a common result for threatened species with fragmented
populations (Degne et al. 2007; Spear and Storfer 2010;
Castañeda-Rico et al. 2011; Vázquez-Domı́nguez et al.
2012). The explanation for the observed Hardy–Weinberg
disequilibrium could be genetic drift (Hedrick 2005). In
addition, other ecological or behavioral factors such as
social structure, could be affecting movement or repro-
duction. The genetic diversity found was higher than
expected, the values obtained were similar to species that
have been demographically stable (Allendorf and Luikart
2007; Dlugosh and Parker 2008; Ho & 0.85). These levels
of genetic diversity are similar (Goprenko et al. 2007,
Greenwald et al. 2009) or slightly higher than those
reported in other studies with mole salamanders and

123
Conserv Genet (2014) 15:49–59
Fig. 2 Mean within-population
pairwise relatedness values (rqg)
between subpopulations of
A. leorae. The red bars are the
above confidence intervals and
the green bars are the lower
confidence intervals with 95 %
confidence with a null
distribution generated with 999
permutations, the blue bars are
the observed kinship mean
conducted with 999 bootstraps
amphibian species (Myers and Zamudio 2004; Steinfartz
et al. 2006; Marsh et al. 2007; Zamudio and Wieczorek
2007; Rhoads 2011). Moreover, the He average is 0.519
with a range from 0.140 to 0.937 regardless of the size of the
population (Curtis and Taylor 2003; Myers and Zamudio
2004; Adams et al. 2005; Jehle et al. 2005); similar results
to those obtained in the present study (He = 0.520–0.716
Table 2). However, when the average number of alleles is
considered, the population and the subpopulations of
A. leorae have fewer alleles than in most other mole sala-
mander and amphibian studies (na = 5.44; Table 2). This is
important to consider because the genetic diversity of
A. leorae could be starting to decline because due to habitat
fragmentation, anthropogenic activities and isolation. The
high levels of heterozygosity in this population could be
caused by high founder size, high effective population size
in the past, multiple paternity or, maybe, overlapping
generations.
Genetic structure and isolation by distance
We have 3 subpopulations (LnPr (k = 3) = -3,581.49)
which coincides with the three substrates present in the
sampled rivers (stones, sand and mud). However, only
56 % of the individuals was correctly assigning to their
original population with Bayesian assignment method
(Table A1). We also found weak population structure
(FST = 0.021, RST = 0.044 and Dest = 0.010; Table 1),
small percentage of variation (2 %) generated by the
AMOVA method (Table A8), and low genetic differenti-
ation with a correlation factor analysis and principal
components analysis (Figs. A3 and A4). With these results,
we conclude that the structure observed is weak; a common
trend found in amphibian populations in small geographi-
cal scales with limited distributions (Rowe et al. 2000;
Newman and Squire 2001; Palo et al. 2003; Funk et al.
2005; Jehle et al. 2005; Spear et al. 2005; Johansson et al.
2006; Giordano et al. 2007; Noël et al. 2007; Zamudio and
55
Wieczorek 2007; Purrenhage et al. 2009). These results
suggest that our genetic groups behave as a metapopula-
tion, rather than isolated populations (Jehle et al. 2005;
Kinkead et al. 2006). No positive correlation exists
between genetic and geographic distances; this is consis-
tent with field data because the individuals are collected
and recaptured in the same place. This pattern generally
occurs in amphibians with highly philopatric tendencies
(Funk et al. 2005; Spear et al. 2005; Savage and Zamudio
2005; Vences and Wake 2007; Gamble et al. 2007; Cal-
houn and deMaynadier 2008; Semlitsch 2008; Wang et al.
2009; Wang and Summers 2010) showing that there is little
gene flow between substrate types. This may also be due to
poor dispersal ability (Trenham and Shaffer 2005; Gamble
et al. 2006; Gamble et al. 2007; Searcy and Shaffer 2008;
Summitt 2009). It is also known that the main environ-
mental factors that influence the locomotion capacity are
low temperatures (Johnson et al. 2010) and habitats with
low vegetation cover (Naughton et al. 2000; Wang et al.
2009). In the Tláloc volcano, the weather is very cold;
water temperature ranges from 6 to 10 °C and air tem-
perature ranges from 4 to 8 °C. Outside the area beside the
rivers there is no continuous forest allowing mole sala-
mander dispersion (Wang et al. 2009). The results suggest
that the limited dispersive capacity and the highly philop-
atric tendencies of this species mimics the pattern of iso-
lation-by-distance (Fig. A5) making the populations
subdivided or little structured (Jehle et al. 2005; Kinkead
et al. 2006). Moreover, no evidence of isolation by distance
pattern implies a balance between drift and gene flow, due
to an ancestral allelic variation. This means the population
could be a relict of a larger ancestral population; showing
that this allelic variation is not due gene flow.
Effective population size
The low values of effective population size (Table A9) are
similar to other mole salamanders (Funk et al. 1999; Jehle
123
56
and Arntzen 2002; Davis and Verrell 2005; Savage et al.
2010), with low effective population sizes Ne = 3.2–37.8
and no more than 100 individuals per population. These
estimates are low but some studies have shown that
effective population sizes are lower than thought (Frank-
ham 2009). Small effective population sizes can occur for a
variety of factors including bottlenecks, genetic isolation,
asymmetry in the proportions of males and females and
difference in reproductive success between individuals
(Tennessen and Zamudio 2003; Myers and Zamudio 2004;
Wang 2009).
Historical demography
There are no recent bottlenecks (Table A10), but we detect
ancestral bottlenecks (Tables A11 and A12) which can be
associated with two factors (1) the founder effect suffered
when this population was separated from a larger ancestral
population and (2) because local people ate mole sala-
manders, and stopped because the mole salamander pop-
ulations drastically decreased.
Inbreeding and relatedness
Despite the small sample size and the characteristics of this
species (endemic, restricted, isolated) no inbreeding was
detected in the population and in the subpopulations
(Fig. 2, Table A13) coinciding with other mole salaman-
ders and amphibians studies located in small areas (Noël
and Lapointe 2010). We found many siblings, possibly
because we collected most of the individuals from March
to June (19, 38 and 20, Table 3 and Table A13), and most
of them were gilled larva. A. leorae populations may have
some mechanism to avoid inbreeding like P. cinereus,
through chemical recognition (Waldman, 1988; Walls and
Roudebush 1991; Pfennig et al. 1994; Masters and Forester
1995; Cabe et al. 2007).
Conservation implications
Although A. leorae is critically endangered in the world
(Shaffer et al. 2004) and endangered in Mexico
(SEMARNAT 2010), and all historical populations of this
Table 3 Number of samples per month collected at Volcano Tláloc
Number of samples per month
January 12
February 7
March 19
May 38
June 20
123
Conserv Genet (2014) 15:49–59
species have become extinct by pollution and over
exploitation of water resources, no conservation action is
currently under consideration because of limited informa-
tion on population size and current distribution. This last
population probably is an evolutionarily significant unit
(ESU) in terms of conservation (Moritz 1994; Hedrick
et al. 2001). The purpose of defining this unit is to ensure
that the evolutionary heritage will be recognized and pro-
tected (Moritz 1994, 1995). We consider that the best
conservation strategy is the creation of a protected natural
area to preserve the habitat of A. leorae and the creation of
an ecotourism park in order to allow people to be made
aware of the importance of this species.
In conclusion, A. leorae is a micro-endemic and
endangered species with high levels of genetic diversity.
However, it has few alleles and genotypes compared with
other mole salamanders species. The population and the
specie is critically endangered because it is an isolated
small population. Therefore, it is urgent to undertake
conservation strategies to avoid extinction, especially due
to the deterioration and loss of habitat.
Acknowledgments We deeply thank to Dr. Carlos Aguilar Ortigoza
for borrowed a thermalcycler. We thank Brenda Cole and Carl
Mitchell for valuable comments and English editing. We thank all the
students who helped in field. We thank two anonymous reviewers for
their comments that helped improve the manuscript. AS is grateful to
the graduate program Maestrı́a en Ciencias Agropecuarias y Recursos
Naturales to Universidad Autónoma del Estado de México for the
scholarship granted and also the scholarships received from CONA-
CYT and COMECYT.
References
Adams EM, Jones AG, Arnold SJ (2005) Multiple paternity in a
natural population of a salamander with long-term sperm
storage. Mol Ecol 14:1803–1810
Allendorf FW, Luikart G (2007) Conservation and the genetics of
populations. Blackwell, Hoboken
Antao T, Lopes A, Lopes R, Beja-Pereira A, Luikart G (2008)
LOSITAN: a workbench to detect molecular adaptation based on
a Fst-outlier method. BMC Bioinformatics 9:323
Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F (2004)
GENETIX 405 Logiciel sous Windows TM pour la Génétique
des Populations
Cabe PR, Page RB, 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 98:53–60
Calhoun AJK, deMaynadier PG (2008) Science and conservation of
vernal pools in northeastern North America. CRC Press, Boca
Raton
Castañeda-Rico S, León-Paniagua L, Ruedas LA, Vázquez-Domı́n-
guez E (2011) High genetic diversity and extreme differentiation
in the two remaining populations of Habromys simulatus.
J Mammal 92:963–973
Chapuis M, Estoup PA (2007) Microsatellite null alleles and estimation
of population differentiation. Mol Biol Evol 24:621–631
Conserv Genet (2014) 15:49–59
Cournet JM, Luikart G (1996) Description and power analysis of two
tests for detecting recent populations bottleneck from allele
frequency data. Genetics 144:2001–2014
Crawford NG (2010) SMOGD: software for the measurement of
genetic diversity. Mol Ecol Resour 10:556–557
Curtis JMR, Taylor EB (2003) The genetic structure of coastal giant
salamanders (Dicamptodon tenebrosus) in a managed forest.
Biol Conserv 115:45–54
Davis AB, Verrell PA (2005) Demography and reproductive ecology
of the Columbia spotted frog (Rana luteiventris) across the
Palouse. Can J Zool 83:702–711
Degne JF, Stout IJ, Roth JD, Parkinson CL (2007) Population genetics
and conservation of the threatened southeastern beach mouse
(Peromyscus polionotus niveiventris): subspecies and evolution-
ary units. Conserv Genet 8:1441–1452
Dlugosh KM, Parker M (2008) Founding events in species invasions:
genetic variation adaptive evolution and the role of multiple
introductions. Mol Ecol 17:431–449
Dyer RJ (2009) GeneticStudio: a suite of programs for the spatial
analysis of genetic marker data. Mol Ecol Resour 9:110–113
Excoffier L, Lischer HEL (2010) Arlequin suite ver 35: a new series
of programs to perform population genetics analyses under
Linux and Windows. Mol Ecol Resour 10:564–567
Excoffier L, Smmosue PE, Quattro JM (1992) Analysis of molecular
variance inferred from metric distances among DNA Haplo-
types: applications to Human Mitochondrial DNA Restriction
Data. Genetics 131:479–491
Frankham R (1998) Inbreeding and extinction: island populations.
Conserv Biol 12:665–675
Frankham R (2009) Effective population size/adult population size
ratios in wildlife: a review. Genet Res 66:95–107
Frankham R, Ballou J, Briscoe D (2005) Introduction to conservation
genetics. Cambridge University Press, Cambridge
Funk WC, Tallmon DA, Allendorf FW (1999) Small effective population
size in the long-toed salamander. Mol Ecol 8:1633–1640
Funk WC, Blouin MS, Corn PS, Maxell BA, Pilliod DS, Amish S,
Allendorf FW (2005) Population structure of Columbia spotted
frogs (Rana luteiventris) is strongly affected by the landscape.
Mol Ecol 14:483–496
Gaggiotti OE, Lange O, Rassmann K, Gliddons C (1999) A compar-
ison of two indirect methods for estimating average levels of gene
for using microsatellite data. Mol Ecol 8:1513–1520
Gamble LR, McGarigal K, Jenkins CL, Timm BC (2006) Limitations
of regulated ‘‘buffer zones’’ for the conservation of Marbled
Salamanders. Wetlands 26:298–306
Gamble LR, McGarigal K, Compton BW (2007) Fidelity and
dispersal in the pond-breeding amphibian. Ambystoma opacum:
implications for spatio-temporal population dynamics and con-
servation. Biol Conserv 139:247–257
Garner A, Rachlow J, Waits L (2005) Genetic diversity and
population divergence in fragmented habitats: conservation of
Idaho ground squirrels. Conserv Genet 6:759–774
Garza JC, Williamson EG (2001) Detection of reduction in population
size using data from microsatellite loci. Mol Ecol 10:305–318
Gibbs JP (1998) Distribution of woodland amphibians along a forest
fragmentation gradient. Landscape Ecol 13:263–268
Giordano AR, Ridenhour BJ, Storfer A (2007) The influence of altitude
and topography on genetic structure in the long-toed salamander
(Ambystoma macrodactulym). Mol Ecol 16:1625–1637
Goprenko D, Williams RN, DeWoody JA (2007) Reproductive and
mating success in the small-mouthed salamander (Ambystoma
texanum) estimated via microsatellite parentage analysis. Evol
Biol 34:130–139
Greenwald KR, Gibbs HL, Waite AT (2009) Efficacy of land-cover
models in predicting isolation of marbled salamander popula-
tions in a fragmented landscape. Conserv Biol 25:1232–1241
57
Guillot G, Mortier F, Estoup A (2005) GENELAND: a computer
package for landscape genetics. Mol Ecol Notes 5(3):712–715
Hedrick P (2005) Genetics of populations. Jones and Bartlett,
Sudbury
Hedrick PW, Parker KM, Lee RN (2001) Using microsatellite and
MHC variation to identify species ESUs and MUs in the
endangered Sonoran topminnow. Mol Ecol 10:1399–1412
Hicks NG, Pearson SM (2003) Salamander diversity and abundance
in forests with alternative land use histories in the Southern Blue
Ridge Mountains. For Ecol Manag 177:117–130
Jehle R, Arntzen JW (2002) Microsatellite markers in amphibian
conservation genetics. Herpetol J 12:1–9
Jehle R, Wilson GA, Arntzen JW, Burke T (2005) Contemporary
gene flow and the spatio-temporal genetic structure of subdi-
vided newt populations (Triturus cristatus, T marmoratusi).
J Evol Biol 18:619–628
Johansson M, Primmer CR, Merila J (2006) History vs current
demography: explaining the genetic population structure of the
common frog (Rana temporaria). Mol Ecol 15:975–983
Johnson JR, Johnson BB, Shaffer B (2010) Genotype and temperature
affect locomotor performance in a tiger salamander hybrid
swarm. Funct Ecol 24:1073–1080
Jost L (2008) GST and its relatives do not measure differentiation. Mol
Ecol 17:4015–4026
Kalinowski S, Wagner AP, Taper ML (2006) ML-RELATE: a
computer program for maximum likelihood estimation of
relatedness and relationship. Mol Ecol Notes 6:576–579
Kilpatrick CW (1981) Genetic structure of insular populations. In:
Smith MH, Joule J (eds) Mammalian population genetics.
University of Georgia Press, Athens, pp 28–59
Kim I, Phillips J, Monjeau J, Birney E, Noack K, Pumo D, Sikes R,
Dole J (1998) Habitat islands genetic diversity and gene flow in a
Patagonian rodent. Mol Ecol 7:667–678
Kinkead KE, Abbott AG, Otis DL (2006) Genetic variation among
Ambystoma breeding populations on the Savannah River Site.
Conserv Genet 8:281–292
Kohn MH, York EC, Kanradt DA, Haught G, Sauvajot RM, Wayne
RK (1999) Estimating population size by genotyping feces. Proc
R Soc Lond B Biol Sci 266:657–663
Lande R (1988) Demographic models of the Northern Spotted Owl
(Strix occidentalis cuurina). Oecologia 75:601–607
Langella O (2002) Populations 1230 Copyright (C) 1999 Olivier
Langella CNRS-UPR9034. Available at http://bioinformaticsorg/
;tryphon/populations/
Lemos-Espinal J, Smith GR, Ballinger RE, Ramı́rez-Bautista A
(1999) Status of protected endemic salamanders (Ambystoma:
Ambystomatidae: Caudata) in the transvolcanic belt of México.
British J Herpetol 68:1–4
Mantel N (1967) The detection of disease clustering and a generalized
regression approach. Cancer Res 27:209–220
Marsh DM, Page RB, Hanlon TJ, Bareke H, Corritone R, Jetter N,
Beckman NG, Gardner K, Selfert DE, Cabe PR (2007)
Ecological and genetic evidence that low order stream inhibit
dispersal by red-backed salamanders (Plethodon cinereus). Can J
Zool 85:319–327
Masters BS, Forester DC (1995) Kin recognition in a brooding
salamander. Proc R Soc Lond B Biol Sci 261:43–48
Michalakis Y, Excoffier L (1996) A generic estimation of population
subdivision using distances between alleles with special refer-
ence to microsatellite loci. Genetics 142:1061–1064
Monroy Vilchis O, Zarco-González M, Domı́nguez-Vega H (2012)
Los verdaderos habitantes del monte Tláloc: diversidad e
importancia de la fauna. Dirección de difusión y promoción de
la investigación y los estudios avanzados. Monte Tláloc II. La
casa del dios del agua. Universidad Autónoma del Estado de
México, Gobierno del Estado de México, In, pp 111–135
123
58
Moritz C (1994) Defining ‘‘Evolutionary significant units’’ for
conservation. Trends Ecol Evol 9:373–375
Moritz C (1995) Uses of molecular phylogenies for conservation.
Philos Trans R Soc Lond 349:113–118
Myers EM, Zamudio KR (2004) Multiple paternity in an aggregate
breeding amphibian: the effect of reproductive skew on
estimates of male reproductive success. Mol Ecol 13:1951–1963
Naughton GP, Henderson CB, Foresman KR, McGraw RL II (2000)
Long-toed salamanders in harvested and intact Douglas-fir
forests of western Montana. Ecol Appl 10:1681–1689
Nei M (1972) Genetic distance between populations. Am Nat
106:283–292
Nei M (1973) Analysis of gene diversity in subdivided populations.
Proc Natl Acad Sci USA 70:3321–3323
Newman RA, Squire T (2001) Microsatellite variation and fine-scale
population structure in the wood frog (Rana sylvatica). Mol Ecol
10:1087–1100
Newman D, Tallmon DA (2001) Experimental evidence for beneficial
fitness effects of gene flow in recently isolated populations.
Conserv Biol 15:1054–1063
Noël S, Lapointe FJ (2010) Urban conservation genetics: study of a
terrestrial salamander in the city. Biol Conserv 143:2823–2831
Noël S, Ouellet M, Galois P, Lapointe FJ (2007) Impact of urban
fragmentation on the genetic structure of the eastern red-backed
salamander. Conserv Genet 8:599–606
Paetkau D, Calvert W, Stirling I, Strobeck C (1995) Microsatellite
analysis of population structure in Canadian polar bears. Mol
Ecol 4:347–354
Palo JU, O’Hara RB, Laugen AR (2003) Latitudinal divergence on
common frog (Rana temporaria) life history traits by natural
selection evidence from a comparison of molecular and quan-
titative genetic data. Mol Ecol 12:1963–1978
Palsbøll P, Berube M, Allendorf F (2007) Identification of manage-
ment units using population genetic data. Trends Ecol Evol
22:11–16
Parra-Olea G, Recuero E, Zamudio KR (2007) Primer note:
polymorphic microsatellite markers for Mexican salamanders
of the genus Ambystoma. Mol Ecol Notes 7:818–820
Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel
Population genetic software for teaching and research. Mol Ecol
Notes 6:288–295
Pfennig DW, Sherman PW, Collins JP (1994) Kin recognition and
cannibalism in polyphenic salamanders. Behav Ecol 5:225–232
Piry S, Luikart G, Cornuet JM (1999) BOTTLENECK: a computer
program for detecting recent reductions in the effective popu-
lation size using allele frequency data. J Hered 90:502–503
Piry S, Alapetite A, Cornuet JM, Paetkau D, Baudouin L, Estoup A
(2004) GeneClass 2: a software for genetic assignment and first-
generation migrant detection. J Hered 95:536–539
Purrenhage JL, Niewiarowski PH, Moore FBG (2009) Population
structure of spotted salamanders (Ambystoma maculatum) in a
fragmented landscape. Mol Ecol 18:235–247
Queller DC, Goodnigh KF (1989) Estimating relatedness using
genetic markers. Evolution 43:258–275
Raymond M, Rousset F (1995) GENEPOP v 12: population genetics
software for exact test and ecumenicism. J Hered 86:248–249
Rhoads EA (2011) Landscape genetics of the small-mouthed
salamander (Ambystoma texanum) in a Fragmented Habitat:
impacts of landscape change on breeding populations in Hardin
county Ohio forests. PhD Thesis. University of Dayton
Rowe G, Beebee TJC (2004) Reconciling genetic and demographic
estimators of effective population size in the anuran amphibian
Bufo calamita. Conserv Genet 5:287–298
Rowe G, Beebee TJC, Burke T (2000) A microsatellite analysis of
natterjack toad Bufo calamita metapopulations. Oikos 88:641–651
123
Conserv Genet (2014) 15:49–59
Savage WK, Zamudio KR (2005) Species account: Ambystoma
maculatum. In: Lannoo MJ (ed) Amphibian declines: the
conservation status of United States species. University of
California Press, Berkeley, California, pp 621–627
Savage WK, Fremier AK, Shaffer HB (2010) Landscape genetics of
alpine Sierra Nevada salamanders reveals extreme population
subdivision in space and time. Mol Ecol 19:3301–3314
Searcy CA, Shaffer HB (2008) Calculating biologically accurate
mitigation credits: insights from the California tiger salamander.
Conserv Biol 22:997–1005
SEMARNAT (2010) Norma Oficial Mexicana NOM-059- SEMAR-
NAT-2010, Protección ambiental-Especies nativas de México de
flora y fauna silvestres-Categorı́as de riesgo y especificaciones
para su inclusión, exclusión o cambio-Lista de especies en
riesgo. Diario Oficial de la Federación, 10 diciembre 2010,
México
Semlitsch RD (2008) Differentiating migration and dispersal pro-
cesses for pond-breeding amphibians. J Wildl Manag 72:
260–267
Shaffer B, Parra-Olea G, Wake D (2004) Ambystoma leorae. In:
IUCN 2012. IUCN Red List of Threatened Species. Version
2012.2. www.iucnredlist.org. Accessed on 25 Apr 2013
Slatkin M (1995) A measure of population subdivision based on
microsatellite allele frequencies. Genetics 139:457–462
Spear S, Storfer A (2010) Anthropogenic and natural disturbance lead
to differing patterns of gene flow in the Rocky Mountain tailed
frog, Ascaphus montanus. Biol Conserv 143:778–786
Spear S, Peterson FCR, Matocq MD, Storfer A (2005) Landscape
genetics of the blotched tiger salamander (Ambystoma tigrinum
melanostictum). Mol Ecol 14:2553–2564
Steinfartz S, Stemshorn K, Kuesters D, Tautz D (2006) Patterns of
multiple paternity within and between annual reproduction
cycles of the fire salamander (Salamandra salamandra) under
natural conditions. J Zool 268:1–8
Summitt SD (2009) Determination of dispersal patterns of the small-
mouthed salamander (Ambystoma texanum) in Eagle Creek Park
(Indianapolis, IN). BSc Thesis. Butler University
Taylor EH (1943) Herpetological novelties from Mexico. Univ Kanas
Sci Bull 29:343–361
Templeton A, Read B (1994) Inbreeding: One word several meanings
much confusion. In: Loeschcke, Tomiuk VJ, Jain SK (eds)
Conservation Genetics. Birkhäuser, Basal, pp 91–105
Tennessen JA, Zamudio KR (2003) Early-male reproductive advan-
tage multiple paternity and sperm storage in an amphibian
aggregate breeder. Mol Ecol 12:1567–1576
Trenham PC, Shaffer HB (2005) Amphibian upland habitat use and
its consequences for population viability. Ecol Appl 15:
1158–1168
Valière N (2002) GIMLET: a computer program for analyzing
genetic individual identification data. Mol Ecol Notes 10:
1046–1048
Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004)
MICRO-CHECKER: software for identifying and correcting
genotyping errors in microsatellite data. Mol Ecol Notes
4:535–538
Van Treuren R, Bijlsma R, Van Delden W, Ouborg N (1991) The
significance of genetic erosion in the process of extinction.
I. Genetic differentiation in Salvia pratensis and Scabiosa
columbaria in relation to population size. Heredity 66:181–189
Vázquez-Domı́nguez E, Surárez-Atilano M, Booth W, González-Baca
C, Cuarón AD (2012) Genetic evidence of a recent successful
colonization of introduced species on islands: Boa constrictor
imperator on Cozumel Island. Biol Invasions 14:2101–2116
Vences M, Wake DB (2007) Speciation species boundaries and
phylogeography of amphibians. In: Heatwole H, Tyler M (eds)
Conserv Genet (2014) 15:49–59
Amphibian biology. Surrey Beatty and Sons, Chipping Norton,
pp 2613–2669
Waldman B (1988) The ecology of s kin recognition. Annu Rev Ecol
Syst 19:543–571
Walls SC, Roudebush RE (1991) Reduced aggression toward siblings
as evidence of kin recognition in cannibalistic salamanders. Am
Nat 138:1027–1038
Wang IJ (2009) Fine-scale population structure in a desert amphibian:
landscape genetics of the black toad (Bufo exsul). Mol Ecol
18:3847–3856
Wang IJ, Summers K (2010) Genetic structure is correlated with
phenotypic divergence rather than geographic isolation in the
highly polymorphic strawberry poison-dart frog. Mol Ecol
19:447–458
Wang IJ, Savage WK, Shaffer HB (2009) Landscape genetics and
least-cost path analysis reveal unexpected dispersal routes in the
59
California tiger salamander (Ambystoma californiense). Mol
Ecol 18:1365–1374
Waples RS (2006) A bias correction for estimates of effective
population size based on linkage disequilibrium at unlinked gene
loci. Conserv Genet 7:167–184
Waples RS, Do C (2008) LDNE: a program for estimating effective
population size from data on linkage disequilibrium. Mol Ecol
Resour 8:753–756
Weir BS, Cockerham CC (1984) Estimating F-statistics for the
analysis of population structure. Evolution 38:1358–1370
Yeh FC, Yang RC, Boyle T, Ye ZH, Mao J (1997) POPGENE, the user-
friendly shareware for population genetic analysis. Molecular
Biology and Biotechnology Center, University of Alberta, Edmonton
Zamudio KR, Wieczorek AM (2007) Fine-scale spatial genetic
structure and dispersal among spotted salamander (Ambystoma
maculatum) breeding populations. Mol Ecol 16:257–274
123