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Genetic diversity and differentiation of the Western Leopard Toad (Sclerophrys pantherina) based on mitochondrial and microsatellite markers


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Intraspecific genetic diversity provides the basis for evolutionary change and is therefore considered the most fundamental level of biodiversity. Mitochondrial DNA (mtDNA) and microsatellite loci are the markers most typically used in population-level studies; however, their patterns of genetic variation are not always congruent. This can result in different interpretations of the data, which can impact on management decisions, especially for threatened species. Consequently, in this study, we developed and analysed novel microsatellite markers for the Endangered Western Leopard Toad (WLT), Sclerophrys pantherina, and compared the results to previously published mtDNA data to compare the level of genetic diversity between the two molecular markers. The microsatellite evidence showed signs of a past bottleneck, yet relatively high levels of genetic diversity and low genetic differentiation between two sampling sites. In contrast, the mtDNA revealed moderate to low levels of diversity between sampling sites, and strong genetic differentiation. An explanation for the conflicting patterns may be that the current genetic signature, as depicted by the microsatellite data, is not yet reflected in the mitochondrial dataset; and, as such the data are depicting a timeline for genetic variation within the WLT. Both markers revealed important information about the two sampling sites, which can help inform conservation management of the species.
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African Journal of Herpetology
ISSN: 2156-4574 (Print) 2153-3660 (Online) Journal homepage:
Genetic diversity and differentiation of the
Western Leopard Toad (Sclerophrys pantherina)
based on mitochondrial and microsatellite
Jessica M. da Silva , Kevin A. Feldheim, G. John Measey , Stephen Doucette-
Riise, Ryan J. Daniels, Lucas F. Chauke & Krystal A. Tolley
To cite this article: Jessica M. da Silva , Kevin A. Feldheim, G. John Measey , Stephen
Doucette-Riise, Ryan J. Daniels, Lucas F. Chauke & Krystal A. Tolley (2017) Genetic
diversity and differentiation of the Western Leopard Toad (Sclerophrys pantherina) based on
mitochondrial and microsatellite markers, African Journal of Herpetology, 66:1, 25-38, DOI:
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Genetic diversity and differentiation of the Western
Leopard Toad (Sclerophrys pantherina) based on
mitochondrial and microsatellite markers
Kirstenbosch Research Centre, South African National Biodiversity Institute, Private Bag X7, Claremont, Cape
Town, South Africa;
Department of Botany & Zoology, University of Stellenbosch, Private Bag X1, Matieland
7602, Stellenbosch, South Africa;
Pritzker Laboratory for Molecular Systematics and Evolution, The Field
Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA;
Centre for Invasion Biology, Department of
Botany and Zoology, Stellenbosch University, Natural Sciences Building, Matieland, Stellenbosch, South Africa;
Department of Biological Sciences, University of Cape Town, Private Bag X3, Rondebosch 770, Cape Town,
South Africa
Abstract.Intraspecic genetic diversity provides the basis for evolutionary change and is
therefore considered the most fundamental level of biodiversity. Mitochondrial DNA (mtDNA)
and microsatellite loci are the markers most typically used in population-level studies; however,
their patterns of genetic variation are not always congruent. This can result in different
interpretations of the data, which can impact on management decisions, especially for
threatened species. Consequently, in this study, we developed and analysed novel microsatellite
markers for the Endangered Western Leopard Toad (WLT), Sclerophrys pantherina, and
compared the results to previously published mtDNA data to compare the level of genetic
diversity between the two molecular markers. The microsatellite evidence showed signs of a
past bottleneck, yet relatively high levels of genetic diversity and low genetic differentiation
between two sampling sites. In contrast, the mtDNA revealed moderate to low levels of
diversity between sampling sites, and strong genetic differentiation. An explanation for the
conicting patterns may be that the current genetic signature, as depicted by the microsatellite
data, is not yet reected in the mitochondrial dataset; and, as such the data are depicting a
timeline for genetic variation within the WLT. Both markers revealed important information
about the two sampling sites, which can help inform conservation management of the species.
Key words.Africa; Amietophrynus; Bufonidae; conservation; endangered; genetic
Intraspecic genetic diversity provides the basis for evolutionary change and is therefore
considered the most fundamental level of biodiversity (May 1994). This diversity is gov-
erned by the loss and gain of alleles, which can be the result of mutations, random genetic
drift, natural and sexual selection or migration. In our human-mediated world, ecological
*Corresponding author. Email:
African Journal of Herpetology,
Vol. 66, No. 1, 2017, 2538
ISSN 2156-4574 print/ISSN 2153-3660 online
© 2017 Herpetological Association of Africa
disturbance is proving to be a key driver of changes to the effective population size (N
a species (for an in-depth review, see Banks et al. 2013); and, over time, these changes can
greatly affect a speciesgenetic diversity (Alcala & Vuilleumier 2014; Ellegren & Galtier
2016). It is well established that a reduction in genetic diversity compromises the adaptive
potential of a species and, therefore, decreases its long-term survival (Lacy 1997; Markert
et al. 2010). This is especially true for threatened species, which can sometimes be charac-
terised by low levels of genetic variation, often due to inbreeding, population bottlenecks,
or the disproportionate effects of genetic drift on small populations (e.g. England et al.
2003; Spielman et al. 2004; Allendorf 2005; Willoughby et al. 2015).
Molecular markers, such as mitochondrial DNA (mtDNA) and microsatellite loci,
are powerful tools for estimating the diversity among individuals and populations
(e.g. Avise et al. 1987; Schwartz et al. 2007). However, patterns of genetic variation
are not always congruent between the two. At times, greater population differentiation
has been found using microsatellite markers (e.g. Lu et al. 2001; Johnson et al. 2003;
De Oliveira Francisco et al. 2013; Kolleck et al.2013; da Silva et al. 2016); while the
reverse has also been documented (e.g. Castella et al. 2001; Yang & Kenagy 2009).
Some possible explanations for these discrepancies include: (i) differences in the selec-
tion intensities acting on each marker; (ii) the different mutation rates between markers;
and, (iii) the N
for maternally-inherited markers, such as mtDNA, differ from those of
microsatellites which are biparentally inherited markers (Johnson et al. 2003). Genetic
diversity is also inuenced by patterns of mating, sex-biased dispersal and other demo-
graphic parameters (Chesser & Baker 1996; Johnson et al. 2003; Yang & Kenagy 2009;
De Oliveira Francisco et al. 2013; Kolleck et al. 2013), which can further contribute to
the discrepancies in genetic diversity estimated between mtDNA and microsatellite
markers. In general, mtDNA is most often used to analyse phylogeographic events,
while microsatellites typically provide more ne-scale resolution of more recent demo-
graphic events (e.g. Avise et al. 1987). Consequently, researchers must carefully deter-
mine what questions they want to address, so that the appropriate markers can be
utilised (Hoban et al. 2013). This is especially critical for research on threatened
species that have small and/or declining populations, such as the Western Leopard
Toad (WLT: Sclerophrys pantherina; previously Bufo pantherina and Amietophrynus
The WLT is endemic to the winter rainfall region of the southwestern Cape of South
Africa where it falls into two historically disjunct areas: (1) to the west of False Bay,
including the Cape metropolitan area, and (2) to the east of False Bay (De Villiers
2004; Measey & Tolley 2011;Fig. 1). Its Endangered status is based on its small distri-
bution (area of occupancy [AOO] 440 km
) and the ongoing reduction in quantity and
quality of habitat associated with urbanisation and agricultural expansion throughout its
range (de Villiers 2004; SA-FRoG 2016; Measey 2011). Adults are explosive breeders,
moving to breeding sites during the antipodean winter (late July to early September)
where they lay large clutches of spawn in strings (Cherry 1992b; de Villiers 2004). Tad-
poles take around three months to reach metamorphosis, and males are known to breed
after one year and females after two (Cherry 1992a).
In order to investigate the structure and distribution of S. pantherina across its range, a
recent study utilised the mitochondrial marker, ND2 (Measey & Tolley 2011), as it was
thought to have sufcient population-level variation (Cunningham & Cherry 2004).
Two genetically distinct populations were recognised that corresponded to the distribution
of the toads (refer to Fig. 1): population 1 toads within the Cape Metropolitan area
26 DA SILVA ET AL.Genetic diversity in the Western Leopard Toad
(CMA) and population 2 toads east of False Bay (EFB). Moreover,
haplotype and nucleotide diversity were greater for the CMA population compared to
the EFB population, with no shared haplotypes across the two populations (Measey &
Tolley 2011).
The relatively low genetic diversity found among EFB individuals, coupled with local
extinctions and a small number of remaining breeding sites could be an indication that this
population is more vulnerable than the CMA population (Measey & Tolley 2011). With
ongoing development in the EFB lowlands due to agriculture and tourism, the vulnerability
of this population is likely to worsen. Consequently, it has been recommended that this
population be treated as a separate management unit to safeguard the future of the remain-
ing breeding sites (Measey & Tolley 2011).
Given that this previous study only utilised a single mtDNA marker and the two popu-
lations were estimated to have diverged as recently as 1.2 thousand years ago (Measey &
Tolley 2011), the results may not adequately reect the current genetic diversity within the
species. Consequently, in this study, we developed and analysed novel microsatellite
markers for S. pantherina and compared the results to the previously published ND2
sequence data, specically for the EFB population, to compare the level of genetic diver-
sity between the two molecular markers.
Figure 1. Map depicting the distribution of Sclerophrys pantherina (inset) in the southwestern Cape,
South Africa. Shaded blue areas show the disjunct distribution of the species. Circles denote known
sampling sites: black-lled circles denote the two sampling sites included in this study; while the
white lled circles are sampling sites within the Cape Metropolitan Area (refer to Measey &
Tolley, 2011).
Data Collection
Sampling was conducted during the 2010 breeding season in July and August at night
when the toads are most active. Two sampling sites within the EFB were targeted: Uilenk-
raal (S 34° 346E 19° 2757) and Klein Paradijs (S 34° 3910E 19° 321:Fig. 1)
which are 11.25 km apart in a straight line. Both sites are articial impoundments which
S. pantherina have used for oviposition for many years. A single toe clip (the tip to the
rst articulation on the inside toe on the left foot) was collected for each toad using ster-
ilised surgical scissors. In addition to providing tissue for DNA analysis, the toe clip
was used as an identication of previously captured individuals. Each toe clip was
stored in 99% ethanol and stored at -40 °C until DNA was extracted.
Microsatellite Development and Optimisation
Twelve microsatellite markers were developed for S. pantherina (online supplemental
material, Table S1), using an enrichment protocol (Glenn & Schable 2005). Genomic
DNA (gDNA) from one individual was digested with RsaI and XmnI, and SuperSNX24
linkers were ligated onto the ends of gDNA fragments. Linkers act as priming sites for
polymerase chain reactions (PCR) in subsequent steps. Five tetranucleotide [(AAAT)
, (AAGT)
, (ACAT)
, (AGAT)
] and six trinucleotide [(AAT)
, (ACT)
, (AAG)
, (AAC)
] biotinylated probes were hybridised to gDNA in two
separate reactions. Streptavidin-coated magnetic beads (Dynabeads® M-280 Invitrogen,
Carlsbad, CA) were added to the probe-gDNA complexes, and this mixture was washed
twice with 2× SSC, 0.1% SDS and four times with 2× SSC, 0.1% SDS at 52
Between washes, a magnetic particle collecting unit was used to capture the magnetic
beads which are bound to the biotin-gDNA complex. This allows us to capture gDNA con-
taining repeats while other fragments are washed away. Enriched fragments were removed
from the probes by denaturing at 95
C and precipitated with 3 Msodium acetate and 95%
ethanol. To increase the amount of fragments, a recoveryPCR was performed in a 25 µl
reaction containing 1× PCR buffer (10 mMTris-HCl, 50 mMKCl, pH 8.3), 1.5 mMMgCl
0.16 mMof each dNTP, 0.52 µMof the SuperSNX24 forward primer, 10× BSA, 1U Ta q
DNA polymerase, and approximately 25 ng enriched gDNA fragments. Thermal
cycling, performed in a BIO-RAD DYAD, was as follows: 95 °C for 2 min followed
by 25 cycles of 95 °C for 20 s, 60 °C for 20 s and 72 °C for 90 s, and a nal elongation
step of 72 °C for 30 min. PCR fragments were then cloned using the TOPO-TA Cloning®
kit following the manufacturers protocol (Invitrogen). Bacterial colonies containing a
vector with gDNA (i.e. white colonies) were used as a template for subsequent PCR.
PCR products were then cleaned using Shrimp Alkaline Phosphatase and Exonuclease I
according to the manufacturers protocol (Affymetrix, Santa Clara, CA). DNA sequencing
was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Bio-
systems, Foster City, CA). Sequencing reactions were precipitated with 125 mM EDTA
and 100% ethanol and run on an ABI 3730 DNA Analyzer. Primer3 (http://frodo.wi. was used to develop microsatellite PCR
Optimisation of the 12 markers was carried out in a 10 µl reaction volume containing
approximately 550 ng of DNA template, 80 µMof dNTPs, and 0.2 µMof each primer.
28 DA SILVA ET AL.Genetic diversity in the Western Leopard Toad
The PCR buffer concentrations, MgCl
concentrations and annealing temperatures (T
varied with the Taq used and the loci targeted (Table S1). Thermal cycling parameters
were as follows: 95 °C for 4 min, followed by 40 cycles at 95 °C for 30 s, T
for 30 s,
72 °C for 45 s and a nal extension at 72 °C for 510 min. Successful products were
then combined in a poolplex format and proled at the Central Analytical Facility at Stel-
lenbosch University using an ABI 3100xl Prism (Applied Biosystems, Foster City, CA),
ROX 500 or LIZ 500 as the internal size standard, and POP-7 as the polymer, as per man-
ufacturers recommendation. Alleles were scored and binned using the microsatellite
plugin in Geneious version 9.1.5 (Kearse et al. 2012).
Data Analysis
Microsatellite genotypes.Microsatellites were screened for genotyping errors and null
alleles using Micro-Checker version 2.2.3 (Van Oosterhout et al. 2004) as loci possessing
either of these would not typically be considered useful in population-level studies as they
may affect the estimation of population differentiation, for example, by reducing the
genetic diversity within populations (e.g. Paetkau & Strobeck 1995). As such, some
studies have attempted to correct for null alleles in population genetic studies by statisti-
cally adjusting the visible allele and genotype frequencies (e.g. Roques et al. 1999;
Chapuis & Estoup 2007). However, the estimation of null alleles can be biased upwards
in populations that are either inbred or consist of closely related individuals (Chybicki
& Burczyk 2009; Campagne et al. 2012), and, conversely, that estimations of inbreeding
can be biased upwards when null alleles or genotyping errors occur (e.g. Björklund 2005).
Consequently, automatically correcting for null alleles might be unnecessary and inap-
propriate. To better assess the likely presence of null alleles, we conducted a simultaneous
estimation of the inbreeding coefcient (f), null allele frequencies (n), and random geno-
typing failure (b) using the software INEst v2 (Chybicki et al. 2011). This method uses a
Bayesian approach to simultaneously estimate the three parameters, which should provide
a more accurate estimation of each parameter because it allows for the relative contribution
of each through selection of the best tting model (i.e. model with the lowest Deviance
Information Criterion [DIC]).
The Markov Chain Monte Carlo (MCMC) was run with 500 000 cycles and 10% burn-in,
saving parameters every 100 cycles. The DICs from each model were then compared, with
the better (lowest) scoring model considered a better t. Given that the effects of null
alleles are locus-specic (Dakin & Avise 2004), their presence should be reected in the
best tting models of each population, or in this case, sampling site. An examination of
the p[j,k] parameter from the INEst results should provide conrmation of the locus/
loci containing null alleles This parameter examines the frequency of the kth allele at
the jth locus, where p[j, 0] denotes the null allele frequency at the jth locus
(Chybicki et al. 2011), For these loci, we manually adjusted allele frequencies using the
Brookeld 1 algorithm implemented in Micro-Checker. This method discounts non-
amplifying individuals when calculating null allele frequencies, as opposed to the Brook-
eld 2 algorithm, which treats non-amplications as data and regards them as null homo-
zygotes when calculating null allele frequencies (Brookeld 1996). By correcting for null
alleles in this way, we were able to maximise sample size and retain loci that may have
sufcient variability to detect patterns. All subsequent analyses were conducted on this cor-
rected dataset.
Genepop on the Web (Raymond & Rousset 1995; Rousset 2008) was used to test for
linkage disequilibrium (LD) between loci using the log-likelihood ratio test (Cockerham &
Weir 1977). Analyses for LD were run with a MCMC dememorisation of 10 000 for 100
batch runs of 1 000 iterations. For each locus, the number of alleles (allelic richness: A
allelic size range, observed (H
) and expected (H
) heterozygosities (Nei 1987), and devi-
ations from HardyWeinberg equilibrium (HWE) were also estimated for the two sampling
sites using ARLEQUIN version (Excofer & Lischer 2010). Deviations from HWE
were tested using 1.0 ×10
Markov chains and 100 000 dememorisation steps. To minimise
the possibility of Type I errors, tests for linkage and HardyWeinberg disequilibria were
corrected for multiple comparisons by applying Holms sequential Bonferroni correction
(Holm 1979; Rice 1989).
ARLEQUIN was used to test for signatures of genetic bottlenecks in the two sampling
sites using the GarzaWilliamson M-ratio (G-W: Garza & Williamson 2001) and to deter-
mine the level of gene ow between sites using R
(Slatkin 1995). The M-ratio is esti-
mated according to the equation M= k/ R+1, where k represents the number of alleles at
a locus and R is the associated allelic range (Garza & Williamson 2001; Excofer &
Lischer 2010). Populations that have experienced a reduction in their effective population
size exhibit a larger reduction in allele numbers than range (Excofer & Lischer 2010).
Accordingly, an M-ratio less than 0.68 (value derived from stable wild populations)
would indicate that the sampling site has been through a bottleneck at the locus under
examination, whereas a value closer to one is indicative of stationary/stable populations
(Garza & Williamson 2001; Peery et al. 2012). Gene ow was measured using R
instead of F
because it relies on a stepwise mutation model which is better suited for
the high mutation rates and memory dependent allele mutations found within microsatellite
loci (Di Rienzo et al. 1994; Slatkin 1995). In contrast, F
relies upon the innite allele
model, which assumes low mutation rates and a mutation process independent of the
prior allelic state (Weber & Wong 1993; Slatkin 1995). The number of migrants (N
between populations was then calculated from the equation, N
= 0.25*(1- F
)/ F
(Slatkin & Barton 1989), using R
in place of F
. Genetic differentiation can be cate-
gorised as great if R
> 0.15, moderate if R
= 0.050.15, and little if R
< 0.05
(Wright 1978). Similarly, the level of gene ow between sampling sites is categorised as
high if N
>1, intermediate if N
= 0.25 to 0.99, and low if N
< 0.25 (Wright 1978).
mtDNA sequences.The ND2 sequence data for Uilenkraal and Klein Paradijs data from
Measey & Tolley (2011) was used to estimate genetic diversity within S. pantherina.
Specically, the number of haplotypes (h), haplotypic diversity (hd), nucleotide diversity
(π), the number of polymorphic sites (S), and the total number of mutations (Eta) were cal-
culated using DnaSP version 5.10.1 (Librado & Rozas, 2009). Haplotype diversity (or gene
diversity) is dened as the probability that two randomly sampled alleles are different; and,
nucleotide diversity is dened as the average number of nucleotide differences per site in
pairwise comparisons among DNA sequences (Nei 1987). DnaSP was also used to deter-
mine the level of genetic differentiation and gene ow between sampling sites; and, Φ
which is analogous to F
, was calculated in ARLEQUIN. F
is an allelic calculation that
assumes all alleles are equidistant from each other; whereas Φ
(the nucleotide diversity
calculation) allows for different distances between different alleles. Lastly, a median-
joining network was created using NETWORK, version 5.0 (Bandelt et al. 1999) to visu-
alise the relationships among the mtDNA haplotypes between the two sampling sites.
30 DA SILVA ET AL.Genetic diversity in the Western Leopard Toad
Microsatellite Variation
All 12 loci were polymorphic with the number of alleles ranging from 3 to18 (Table 1). There
was no evidence of large allele dropout or scoring errors due to stutter peaks; however, the
likely presence of null alleles due to homozygote excess was detected in both Klein Paradijs
and Uilenkraal sampling sites (Tables 1 and 2). Because null alleles are locus-specic,
WLT16 and WLT39 likely represent truenull alleles. Although the best-tting INEst
models also indicated the inuence of inbreeding, with average F
= 0.0247 for Uilenkraal
and 0.0588 for Klein Paradijs sampling sites, the effects are minimal. An examination of the
95% posterior probability intervals for F
included zero for Uilenkraal, indicating that there
is no signicant inbreeding in this population (Table 2).
No signicant linkage disequilibrium was detected for any of the loci; however, two
loci were found to deviate from HWE (WLT39 & WLT42: Table 1). Because locus
WLT39 was identied as possibly having null alleles across populations, this is the
most probable explanation for its deviation from HWE. Locus WLT42, on the other
hand, only showed deviations from HWE for Klein Paradijs, indicating that these devi-
ations are most likely attributed to some biological factor such as the Wahlund effect or
inbreeding (Chakraborty et al. 1992). Given the highly fragmented nature of the WLT
habitat, it is possible that small populations have become xed for diverse alleles.
Evidence of a genetic bottleneck was found across all loci for both sampling sites (M-ratio:
Table 1), which also show fairly similar levels of microsatellite genetic diversity for all indices
examined (Table 3) and exhibit little differentiation (R
: 0.082, P< 0.001; N
mtDNA Variation
Unlike the microsatellite results, analysis of the mtDNA data revealed differing levels of
diversity between Uilenkraal and Klein Paradijs sampling sites (Table 3). In particular,
Uilenkraal was found to have moderate haplotype diversity (hd = 0.567 ± 0.051), yet
low nucleotide diversity (π= 0.00095 ± 0.00022); while Klein Paradijs has low haplotype
diversity (hd = 0.154 ± 0.126) and very low nucleotide diversity (π= 0.00019 ± 0.0016).
The two populations share one haplotype, possessed by the majority of individuals
sampled (Fig. 2), indicating a previously continuous population or past gene ow
between populations; however, the two populations were found to be strongly differen-
tiated (Φ
= 0.41991, P=0.002).
Although the mtDNA and microsatellite evidence presented here reveal different patterns
with respect to the genetic diversity within the two sampling sites of S. pantherina, simi-
larities were also present with both markers showing signs of population bottlenecks and
the presence of gene ow between Uilenkraal and Klein Paradijs. A possible explanation
for the conicting patterns of genetic diversity may be that the current genetic signature (as
depicted by the microsatellite data) is not yet reected in the mitochondrial dataset due to
the slower evolution of the mtDNA markers. The data may, therefore, be depicting a time-
line for genetic variation within the WLT, with the mtDNA data revealing the more
Table 1. Descriptive statistics of genetic variability for 12 Sclerophrys pantherina microsatellite loci across two sampling sites in the southwestern Cape, South
Uilenkraal (n=39) Klein Paradijs (n=41)
HWE Null M
WLT1 13 134194 0.78 0.89 0.0937 Maybe 0.230 11 138182 0.70 0.76 0.2801 No 0.244
WLT2 13 119179 0.81 0.88 0.2772 No 0.230 10 123167 0.80 0.80 0.5150 No 0.222
WLT16 14 142358 0.62 0.92 0.0006 Maybe 0.065 15 142358 0.74 0.91 0.0411 Maybe 0.069
WLT25 11 167219 0.92 0.90 0.8701 No 0.226 9 159207 0.69 0.81 0.0443 No 0.204
WLT36 14 216276 0.90 0.89 0.6666 No 0.230 9 216280 0.75 0.82 0.1783 No 0.138
WLT38 16 275387 0.89 0.92 0.1888 No 0.150 18 266387 0.91 0.93 0.4804 No 0.148
WLT39 5 157181 0.42 0.71 0.0031 Maybe 0.200 3 165181 0.32 0.56 0.0087 Maybe 0.176
WLT40 10 152200 0.61 0.85 0.0054 Maybe 0.204 9 148200 0.71 0.85 0.0643 No 0.170
WLT42 6 179211 0.39 0.43 0.1636 No 0.182 5 187211 0.27 0.60 <0.001* Maybe 0.240
WLT44 15 195347 0.74 0.87 0.0260 Maybe 0.118 8 195343 0.75 0.75 0.4391 No 0.054
WLT76 4 174183 0.57 0.54 0.8948 No 0.400 4 174183 0.73 0.67 0.5540 No 0.400
WLT88 7 197245 0.67 0.74 0.7256 No 0.163 6 197245 0.63 0.78 0.0237 Maybe 0.142
, Number of observed alleles; S, allele size range; H
,observed and H
,expected heterozygosity; HWE, HardyWeinberg equilibrium Pvalue; Null, presence of null alleles; M,
GarzaWilliamson index. Signicant deviations from HWE are denoted by *, based on a Bonferroni signicance value of 0.0042 (P< 0.05/12).
32 DA SILVA ET AL.Genetic diversity in the Western Leopard Toad
Table 2. Results of Bayesian individual inbreeding model for Sclerophrys pantherina. Models were run using all 12 microsatellite loci. Model parameters include
inbreeding (f), null alleles (n) and random genotyping errors (b).
Uilenkraal (n=39)
Klein Paradijs (n=41)
) 95% HPD DIC ΔDIC Avg (F
) 95% HPD
nfb 3294.720 1.274 0.0247 0.00000.0619 nfb 3084.212 18.488 0.0588 0.0156-0.1069
nb 3295.994 19.807 NA NA nb 3102.700 3.919 NA NA
fb 3315.801 19.398 0.1053 0.05960.1504 fb 3106.619 14.333 0.1254 0.07850.1674
b 3335.199 50.288 NA NA nf 3120.952 22.009 0.0805 0.03250.1261
nf 3385.487 1.369 0.0372 0.00000.0819 n 3142.961 14.834 NA NA
n 3386.856 NA NA b 3157.795 NA NA
DIC, Deviance Information Criterion; ΔDIC, difference in DIC from the best model; Avg (F
), average inbreeding coefcient for the population; HPD, highest probability density.
ancestral relationships and the microsatellite data the most recent demographic structuring.
Both markers revealed important information about the two sampling sites, which can help
inform conservation management of the species.
The mtDNA data uncovered different levels of diversity between Uilenkraal and Klein
Paradijs, with the former showing moderate haplotype diversity and low nucleotide diversity,
and the latter showing low levels of both diversity measures. The low nucleotide diversity esti-
mates suggest that both populations underwent a bottleneck in the recent past. There may have
been a single WLT population in the EFB region, or possibly even across the entire range of
S. pantherina, during the last glacial maxima when areas of the continental shelf were exposed
by lowered sea levels (Schreiner et al.2013; Mokhatla et al. 2015). Then, during the Holo-
cene, the rising sea levels and a distinct drying period may have caused a signicant retraction
in the distribution of the species, creating a population bottleneck (Measey & Tolley, 2011),
and through time differentiation between the two sampling sites.
Evidence of a bottleneck is also reected in the microsatellite results; however, the data
also indicate strong gene ow between sites and moderate to high levels of overall genetic
variation as indicated by the allelic richness (the most robust indicator for monitoring
Table 3. Mean microsatellite and mitochondrial DNA genetic diversity indices for Sclerophrys
pantherina from two sampling sites.
Genetic diversity Uilenkraal Klein Paradijs
Sample size (n)3941
Allelic richness (A
) 10.667 8.911
Observed heterozygosity (H
) 0.693 0.667
Expected heterozygosity (H
) 0.795 0.770
Sample size (n)2213
Length (bp) 741 741
No. haplotypes (h)32
Haplotypic diversity (hd) 0.567 ± 0.051 0.154 ± 0.126
Nucleotide diversity (π) 0.00095 ± 0.00022 0.00019 ± 0.00016
No. polymorphic sites (S)3 1
Total no. mutations (Eta)3 1
Figure 2. A median-joining haplotype network based on the ND2 mitochondrial gene for Scler-
ophrys pantherina. Haplotypes are represented by circles and show the proportion of toads from
Uilenkraal (white; n=22) and Klein Paradijs (black; n=13). The area of the circle is proportional
to the number of individuals with that haplotype, and the length of the connecting lines is pro-
portional to the number of base changes between haplotypes (refer to Table 3).
34 DA SILVA ET AL.Genetic diversity in the Western Leopard Toad
genetic diversity and decline: Hoban et al. 2014)ofeachsamplingsite(A
10.7 and 8.9 for
Uilenkraal and Klein Paradijs, respectively; Tab le 3), compared to other toad species (e.g. Wu
&Hu2010; Roth & Jehle 2016;daSilvaet al. 2016). These microsatellite data tend to depict
the more recent genetic diversity for the species; and, on the surface, it could be interpreted that
the two sampling sites are now healthy and thriving after the bottleneck. However, high levels
of genetic diversity do not always equate to viable populations (Habel & Schmitt 2012).
Levels of genetic diversity differ greatly among species and especially between eco-
logical generalists and specialists. Generalist species tend to have high genetic diversity
within their populations and low genetic differentiation among them due to the absence
of bottlenecks, strong gene ow, and little effects of genetic drift. Specialist species tend
towards low genetic diversity and strong genetic differentiation due to small or uctuating
populations that experience bottlenecks (e.g. Hughes et al. 1999; Schmitt et al. 2005;
Habel & Schmitt 2009,2012). Based on these distinctions, S. pantherina is probably
not a true habitat specialist nor generalist, but might be an ecologically intermediate
species. Such species must maintain high levels of diversity within their populations,
which is facilitated by gene ow, thereby forcing them to exist in metapopulations
(Habel & Schmitt 2012). Without a network of populations, the species might suffer
greatly from the consequences of population bottlenecks, which result in shifts in allele fre-
quencies, often resulting in the loss of genetic adaptations (Luikart et al. 1998).
Unlike many of the Cape lowland endemics (e.g. Xenopus gilli,Microbatrachella
capensis), the WLT appears to have a high tolerance for disturbance with most, if not
all, of its populations currently breeding in anthropogenically altered habitats (Mokhatla
et al. 2015). This adaptability will be essential to the long-term survival of this species,
which will need to counter predicted fragmentation due to newly created impoundments
in both urban and rural areas (Mokhatla et al. 2015). In addition, this species faces the
threat of an invasive congener currently spreading in the CMA range (Measey et al.
2017). However, there might be a threshold of disturbance beyond which populations
are not able to recover. This might very well explain the loss of other sampling sites in
the EFB region, which were known prior to the 1990s (i.e. Pringle Bay, Bettys Bay and
Kleinmond: Measey & Tolley 2011;Fig. 1).
Given that the EFB is subject to ongoing development, the two sampling sites exam-
ined here, especially Klein Paradijs, may also be at risk. It is, therefore, imperative that the
current genetic diversity within these two sampling sites be maintained over time, with
conservation efforts focused on preserving connectivity between sites to ensure adequate
gene ow between sampling sites. Future studies using these novel microsatellites could
then be used to monitor the frequency of dispersal between sampling sites and, ultimately,
the extent of genetic erosion/preservation within the EFB.
We would like to thank the South African National Biodiversity Institute and the Field
Museums Pritzker Laboratory for Molecular Systematics and Evolution for providing
logistical support and funding for this study. Additional funding was provided by the
SANBI-NORAD Threatened Species Programme. Many thanks also to the landowners,
who gave permission to access their property, Susanna Fuchs and Aletta Groenwald.
Sampling was conducted under permit from the Western Cape provincial conservation
authority, CapeNature (#AAA004-00090-0035) and an ethical clearance certicate
(# 0001/08 from SANBI Ethics Committee).
Supplemental data for this article can be accessed at http://dx.doi.org10.1080/21564574.
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Received: 29 September 2016; Final acceptance: 2 February 2017
38 DA SILVA ET AL.Genetic diversity in the Western Leopard Toad
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