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Genetic differentiation and overexploitation history of the critically endangered Lehmann’s Poison Frog: Oophaga lehmanni

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Species conservation with fragmented and endangered populations must be based on a prior and thorough knowledge of the structure and population dynamics. Oophaga lehmanni is a dendrobatid species endemic of Colombia and is restricted to its type locality. This species has a fragmented distribution and is considered as critically endangered mainly due to habitat destruction and overexploitation. Oophaga lehmanni exhibits phenotypic variation in the dorsal color pattern (red and yellow morphs). We reconstructed the overexploitation history that this species has faced in the last 40 years. In addition, we collected genetic and morphological data for the first time in natural populations to describe genetic diversity between and within populations, and to evaluate morphological and genetic differences between red and yellow morphs. Overexploitation data suggest that more than 80.000 (Min = 60.047–Max = 102.236) frogs were extracted from the field in the last four decades, probably generating the local extirpation or population decline from the type locality. Genetic data showed reduced genetic diversity. Observed heterozygosity (mean ± s.d. = 0.599 ± 0.165) is lower than expected (mean ± s.d. = 0.867 ± 0.082). We did not find differences in body size and heterozygosity between the two morphs; however, individuals analyzed were assigned to two genetic clusters, which corresponded to the O. lehmanni-yellow and O. lehmanni-red. In addition, FST (0.209) and Nei genetic distance (0.18) values indicated genetic differentiation between the two morphs; therefore, red and yellow morphs should be treated as independent management units. This information will help to define appropriate and long-term conservation units, as a useful tool to mitigate the extinction risk of this species.
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Conservation Genetics
https://doi.org/10.1007/s10592-020-01262-w
RESEARCH ARTICLE
Genetic dierentiation andoverexploitation history ofthecritically
endangered Lehmann’s Poison Frog: Oophaga lehmanni
MileidyBetancourth‑Cundar1 · PabloPalacios‑Rodríguez1· DanielMejía‑Vargas2· AndreaPaz3,4 ·
AdolfoAmézquita1
Received: 8 February 2019 / Accepted: 22 February 2020
© Springer Nature B.V. 2020
Abstract
Species conservation with fragmented and endangered populations must be based on a prior and thorough knowledge of the
structure and population dynamics. Oophaga lehmanni is a dendrobatid species endemic of Colombia and is restricted to
its type locality. This species has a fragmented distribution and is considered as critically endangered mainly due to habitat
destruction and overexploitation. Oophaga lehmanni exhibits phenotypic variation in the dorsal color pattern (red and yel-
low morphs). We reconstructed the overexploitation history that this species has faced in the last 40years. In addition, we
collected genetic and morphological data for the first time in natural populations to describe genetic diversity between and
within populations, and to evaluate morphological and genetic differences between red and yellow morphs. Overexploita-
tion data suggest that more than 80.000 (Min = 60.047–Max = 102.236) frogs were extracted from the field in the last four
decades, probably generating the local extirpation or population decline from the type locality. Genetic data showed reduced
genetic diversity. Observed heterozygosity (mean ± s.d. = 0.599 ± 0.165) is lower than expected (mean ± s.d. = 0.867 ± 0.082).
We did not find differences in body size and heterozygosity between the two morphs; however, individuals analyzed were
assigned to two genetic clusters, which corresponded to the O. lehmanni-yellow and O. lehmanni-red. In addition, FST (0.209)
and Nei genetic distance (0.18) values indicated genetic differentiation between the two morphs; therefore, red and yellow
morphs should be treated as independent management units. This information will help to define appropriate and long-term
conservation units, as a useful tool to mitigate the extinction risk of this species.
Keywords Conservation genetics· Oophaga lehmanni· Poison frogs· Endangered species· Wildlife trade
Introduction
Amphibians are probably the most threatened group of ver-
tebrates worldwide with about 43% of the species having
declined or come under threat in the past 60years (Houlahan
etal. 2000; Stuart etal. 2004; Shaffer etal. 2015; Powers and
Jetz 2019). Declines may be associated with single factors or
complex synergistic interaction among them (Young etal.
2001; Collins and Storfer 2003). The main threats are habi-
tat loss, fragmentation (Stuart etal. 2004; Cushman 2006;
Becker etal. 2007), climate change (Pounds 2001; Bosch
etal. 2007; Lawler etal. 2009; Warren etal. 2013), emer-
gent diseases (Pounds etal. 2006; Lips etal. 2006; Fisher
etal. 2009; Scheele etal. 2019) and overexploitation (Gor-
zula 1996; Gibbon etal. 2000; Stuart etal. 2004). Many
studies have tried to explain the observed declines at indi-
vidual localities, and, for single or multiple species (Berger
etal. 1998; Lips 1999; Young etal. 2001; Ron etal. 2003;
Microsatellite dataare available from the Dryad Digital
Repository:https ://doi.org/10.5061/dryad .0zpc8 66tx
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1059 2-020-01262 -w) contains
supplementary material, which is available to authorized users.
* Mileidy Betancourth-Cundar
cm.betancourth2026@uniandes.edu.co
1 Department ofBiological Sciences, Universidad de Los
Andes, Cra. 1 #18A-12, 111711Bogotá, Colombia
2 Independent Researcher, San Antonio del Tequendama,
Cundinamarca, Colombia
3 PhD Program inBiology, The Graduate Center, City
University ofNew York, NewYork, USA
4 Biology Department, City College ofNew York, NewYork,
USA
Conservation Genetics
1 3
Burrowes etal. 2004; La Marca etal. 2005). Even though
these studies provide evidence about the patterns of declines,
they do not provide a detailed picture of their causes or spe-
cific risk factors such habitat requirements. The dramatic and
fast decline of amphibian populations emphasizes the need
to prioritize field monitoring and natural history studies of
declining species to improve our understanding of their pop-
ulation dynamics and ecology to guide conservation actions
(La Marca etal. 2005; Becker etal. 2010; Zumbado-Ulate
etal. 2010). In this context, research on endemic species that
by definition have restricted geographic ranges and that can
be especially vulnerable to anthropogenic intervention and
habitat destruction should be considered a priority.
Poison frogs of the family Dendrobatidae occur in Cen-
tral and tropical South America (Kahn etal. 2016); with
diversity peaking in the tropical Andes, more specifically in
Colombia where about 43% of these frogs (84 species) are
distributed (Acosta Galvis 2017; Frost 2018). This group of
anurans stands out because of a variety of ecological and
phenotypic characteristics, such as diurnal habits, alkaloid
sequestration, bright coloration (Myers and Daly 1976;
Kahn etal. 2016) and, very elaborate and conspicuous social
behaviors like territoriality (Wells 1977, 2007; Summers and
McKeon 2004; Summers and Tumulty 2014). Similarly to
other amphibians, dendrobatids may undergo a rapid decline
by a synergistic interaction among habitat loss and emer-
gent diseases (Rosser and Mainka 2002; Stuart etal. 2004;
Flechas etal. 2017). In addition, unsustainable capture for
the pet trade is a great threat for these frogs (Rosser and
Mainka 2002), particularly for the Oophaga genus and other
colorful frogs (Gorzula 1996; Schlaepfer etal. 2005; Nij-
man and Shepherd 2010), with about 30–40 species of this
group found in the international wildlife trade (Nijman and
Shepherd 2010).
The demand for these species probably depends on fac-
tors such as endemism, rarity, scarcity in the pet market, cap-
tive breeding difficulty and the presence of attractive or new
colorations. Lehmann’s Poison Frog, Oophaga lehmanni
(Myers and Daly 1976) meets all these characteristics. This
frog is endemic to Colombia, restricted to its type locality
in the south-facing versant of upper Río Anchicayá drain-
age, Department of Valle del Cauca, Colombia (Fig.1a)
and listed as critically endangered (IUCN SSC Amphibian
Specialist Group 2019). Within its small distribution, O.
Fig. 1 Data about the extraction places and estimated number of O.
lehmanni frogs removed from the field. a The map shows the frog
extraction areas (differently colored polygons) of Oophaga lehmanni
frogs during 1977–2009. Red circles indicate current localities of O.
lehmanni, pink circles show localities where the population is decline
and blue circles represent localities where O. lehmanni is not present
anymore. Black circles indicate putative hybrid lineages (pHYB).
Red line show the distribution range reported by International Union
for Conservation of Nature-IUCN (IUCN SSC Amphibian Specialist
Group 2019). b Cumulative frogs extracted from the field at six-time
frames. Data for 2018 and 2019 comes from confiscation events at El
Dorado International Airport in Bogota
Conservation Genetics
1 3
lehmanni presents a variation in the coloration pattern, with
two morphs, red and yellow. Even before its formal descrip-
tion in the 70s, O. lehmanni had been subject to massive
overexploitation for commercial purposes. In addition, con-
stant destruction of its natural habitat mainly from logging
for agricultural and ranching purposes is another factor that
has contributed to its population decline (Myers and Daly
1976; Castro-Herrera and Amézquita 2004).
Overexploitation for commercial purposes has led to the
risk of extinction of different taxa (Fuller 2003; Minteer
etal. 2014). The intensive collection decreases genetic vari-
ability and represents a real threat to the ecological viability
of populations. Reduction in population size can enhance
the probability of extinction (Allee Effect) due to inbreeding
depression, impoverishment of its genetic variation or risk
of stochastic factors (Allee and Bowen 1932; Courchamp
etal. 1999). For instance, bottlenecks reduce genetic diver-
sity dramatically and can happen even from one genera-
tion to another. Furthermore, overexploitation in particular
areas can lead to local extirpation of populations, which can
generate a loss of connectivity between them. Although no
quantifiable information is available, O. lehmanni is declin-
ing. Historical reports (Myers and Daly 1976) contrast with
current data and its abundance in most localities is very
low (Velásquez etal. 2009) and has even been locally extir-
pated in some (Betancourth-Cundar and Palacios-Rodríguez
2018). For these reasons, Lehmann’s Poison Frog is a good
model to evaluate the effect of artificial and natural pressures
in the reduction of genetic diversity.
Despite an intensive search, very few locations and indi-
viduals have been registered (Velásquez etal. 2009; Vargas-
Salinas and Amézquita 2013). Fortunately, some of these
records are located within a protected area, the Farallones de
Cali National Park (FCNP). This represents a valuable find-
ing given the threat status of this poison frog in its natural
environment. However, biological information of this spe-
cies is limited to taxonomic and toxin data of its original
description (Myers and Daly 1976), reports of reproductive
behavior in captivity (Zimmermann and Zimmermann 1986;
Lötters etal. 2007), description of geographic variation of
the advertisement call (Lötters etal. 1999) and assessment
of their conservation status based on a few registered indi-
viduals (Velásquez etal. 2009). A conservation plan tar-
geting this endemic poison frog requires population genetic
information to correctly assess risk and ensure long-term
viability.
Here, our main aim is to evaluate the genetic and pheno-
typic variation of the surviving populations of O. lehmanni
and to understand if overexploitation was one of the fac-
tors leading to population declines and local extirpation
of this frog. This information will help define appropriate
and long-term conservation units, as a useful and viable
tool to mitigate the extinction risk of this species. To do
it, (1) we reconstructed the overexploitation history that
this species has faced in the last 40years using surveys of
local collectors. For the first time in natural populations
of O. lehmanni (red and yellow morphs), (2) we collected
genetic data to describe genetic diversity between and
within populations, and (3) we collected morphological
data to evaluate if the phenotypic variation (body size and
color pattern) is correlated with the genetic variation (i.e.
if red and yellow morphs can be grouped genetically). In
addition, we used confiscated samples of confiscated indi-
viduals to explore potential source sites for pet trafficking.
Materials andmethods
Study system
Lehmann’s Poison Frog (Oophaga lehmanni) was
described in 1976 from the upper Anchicayá River drain-
age in Colombia between 850 and 1200m (Myers and Daly
1976); however, recent data suggest that these populations
have few individuals or are locally extirpated (Velásquez
etal. 2009; Amézquita 2016; Betancourth-Cundar and
Palacios-Rodríguez 2018) (Fig.1a). This species has also
been reported from Alto del Oso near San José del Pal-
mar, near to Santa Cecilia, Alto del Buey and Carmen
de Atrato (Vereda el 12, Sitio el 12) in the Department
of Chocó; however, genetic analyses and phenotypic data
suggest that these records correspond to a closely related
species, Oophaga histrionica (Amézquita etal. in prep.).
In addition, Garraffo etal. (2001) used specimens from
the Rio Taparó drainage near San Jose del Palmar, Chocó,
Colombia, collected in the early 1990s. These specimens
had the same alkaloids profile as those individuals from
the type locality (Garraffo etal. 2001). This dataset sug-
gests that it is O. lehmanni, but we have not been able to
confirm it. Furthermore, one previous study shows that O.
lehmanni can hybridize with its sister species, O. histri-
onica, in areas near the type locality (Medina etal. 2013).
These populations are found in habitats like their puta-
tive parents and occupy an altitudinal range between 700
and 1000m. Within its small range, O. lehmanni exhib-
its phenotypic variation in the dorsal color pattern with
red and yellow morphs (Myers and Daly 1976; Lötters
1992). The red individuals are found in the FCNP and are
the only protected populations. The yellow morph is not
found within protected areas and is very difficult to find in
the field, probably due to its excessive extraction or geo-
graphical rarity. The observed phenotypic variation leads
us to hypothesize that O. lehmanni may exhibit variation
in other morphological and genetic traits.
Conservation Genetics
1 3
Overexploitation data
To explore the massive extraction process that this frog has
faced in the past 40years, we conducted a survey (TableS1
in Supplementary Material) among local people who have
been collecting poison frogs in the study area since the 70s.
The survey was focused on estimating the number of frogs
collected, extraction locality, year and frequency of extrac-
tion. For this, we used photographs of the two O. lehmanni
morphs, red and yellow, some O. histrionica morphs
reported in the area, and a map of the study area for the
respondent to identify the extracted morphs and localities.
Data about extracted frogs were coded as maximum, mini-
mum and average values for each location identified by the
respondent. To estimate the number of extraction events we
divide the total extraction time (months) by the frequency
of the events. Subsequently, we used this value to estimate
the cumulative maximum, minimum and average values
of frogs extracted for each locality. To estimate the total
value, we summed these values for each time frame. In addi-
tion, we built a map using polygons to represent the areas
where the frogs were collected. For this, we used data from
the respondents, historical reports (Myers and Daly 1976;
Velásquez etal. 2009) and our previous field sampling.
Morphological data
To collect genetic and morphological data, we conducted
five field trips at the FCNP and nearby sites between 2015
and 2017 at different times of the year (March 2015, March
and December 2016, March and May 2017). We conducted
visual searches of amphibians during the day thoroughly
surveying the available microhabitats (Rueda etal. 2006).
For the collected individuals (N = 165), we determined the
sex with the presence or absence of vocal slits and took dor-
sal and ventral photographs with a digital camera (Canon
PowerShot), using a scale of size and color. From the pho-
tographs, we measured body length as snout-vent length
(SVL) with an accuracy of 0.1mm using the ImageJ soft-
ware (Schneider etal. 2012). To evaluate differences in body
size between the two morphs, we did an analysis of variance
(ANOVA) in the R software (R Core Team 2013).
Microsatellite genotyping analysis
We obtained genomic DNA from wild populations of O.
lehmanni-red from the FCNP (N = 54) and of O. lehmanni-
yellow from areas near the type locality (N = 15). We do not
provide precise coordinates given the species’ threat levels
(Fig.1a). Additionally, samples from O. lehmanni with yel-
low and red phenotypes were obtained from specimens kept
in captivity at the Cali Zoo (N = 15). These animals were
confiscated between 2006 and 2009. We collected tissue
samples through a noninvasive technique of mouth swabs
(Goldberg etal. 2003; Pidancier etal. 2003; Beebee 2008)
for all individuals. In the field, samples were stored dry at
room temperature. In the laboratory, these were stored at
4°C until extraction. DNA was extracted and purified using
the DNeasy tissue extraction kit (QIAGEN, Valencia, CA),
following the manufacturer’s protocol.
Extracted DNA samples were diluted to 12ng/µl and
used as template in polymerase chain reactions (PCR) for
ten di-, tri-, and tetranucleotide microsatellite loci previously
designed: Dpum 110, Dpum 14, Dpum 63, Dpum 44 (Wang
and Summers 2009) and Oop C11, Oop F1, Oop B9, Oop
G5, Oop H5, Oop E3 (Hauswaldt etal. 2009). Microsatel-
lites are suitable to identify genetic differences between indi-
viduals and populations, and these have been widely used in
conservation genetics (Bruford and Wayne 1993; Jarne and
Lagoda 1996). Forward primers for each PCR amplifica-
tion were labeled with a 5-fluorescent tag (6-FAM, NED,
VIC, or PET) for later visualization. We amplified the loci
individually and confirmed the PCR amplification using
an agarose gel electrophoresis for all samples. The PCR
reactions were performed using this thermal cycling pro-
gram: following an initial denaturation at 95°C for 3min,
35 cycles were run with 95°C for 30s, 54°C for 30s and
72°C for 4s. This was followed by a 6-min extension at
72°C and 25min incubation at 60°C. This amplification
cycling was used for seven markers (Oop B9, Oop G5, Oop
H5, Oop E3, Dpum 14, Dpum 63, and Dpum 44). For Dpum
110 annealing temperatures were at 58°C and Oop C11
and Oop F1 amplified at 64°C. For genotyping, we per-
formed a multiplex of three markers. Fragments were sized
with LIZ-500 size standard and then scored genotypes using
GeneMapper V4.1 (Applied Biosystems). We repeated the
scoring procedure two times on all samples to check that
alleles were assigned appropriately. PCR products were run
on an ABI3500 Genetic Analyzer (Applied Bio-systems) in
the Sequentiation Center at the Universidad de los Andes.
To screen our markers, we computed the number of alleles
per locus and the polymorphic information content (PIC) using
Cervus v.3.0.7 (Kalinowski etal. 2007). We used Genepop
vs. 4.5.1 (Raymond and Rousset 1995) to calculate deviations
from Hardy–Weinberg equilibrium (HWE) through an exact
test for heterozygote deficit at each locus and group (Markov
chain parameters: 10,000 dememorizations, 600 batches, 1000
iterations per batch) and to check for linkage disequilibrium
(LD) between all pairs of loci thru a likelihood-ratio test
(10,000 permutations). Considering that in significantly differ-
entiated populations the null alleles may generate overestima-
tions in FST and genetic distances (Pemberton etal. 1995; Van
Oosterhout etal. 2006; Chapuis and Estoup 2007), we checked
and corrected its presence using FreeNA (Chapuis and Estoup
2007) and MICRO-CHECKER 2.2.3. (Van Oosterhout etal.
2004). These software estimates the frequency of null alleles
Conservation Genetics
1 3
and estimating FST values with corrected genotype frequen-
cies restricted to visible allele sizes. MICRO-CHECKER also
allowed us to evaluate genotyping errors and allelic dropout.
To evaluate genetic diversity, we calculated observed (Ho)
and expected (He) heterozygosity with GENALEX v.6.51b2
(Peakall and Smouse 2012) using all data matrix. Next, we
calculated the same values for each group (yellow and red
morphs) and compared all the values of Ho and He among
both groups using a t test in the R software (R Core Team
2013). To evaluate levels of inbreeding in these groups, we
calculate the inbreeding coefficient (FIS: Weir and Cockerham
1984) used Fstat (Goudet 1995).
To evaluate population structuring, we first analyzed the
FCNP samples, because they were collected at three different
sites. Then, we performed the same analyses for all samples.
We used Structure 2.3.4 (Pritchard etal. 2000); this Bayes-
ian model-based clustering algorithm uses the information on
allele frequencies and assumes Hardy–Weinberg and linkage
equilibrium between loci. In addition, this approach assumes
a model with genetic clusters (K), where K is initially fixed
and subsequently estimated. Individuals are then assigned
probabilistically to one or more populations (Pritchard etal.
2000). In our study, we implemented an admixture model with
correlated allele frequencies between populations. This model
assumes that frequencies in the different groups are likely to
be similar due to migration or individuals may have ancestry
in several clusters (Falush etal. 2003). Structure was run with
50,000 generations of burn-in, a Markov chain of 500,000 gen-
erations, from one to ten genetic clusters (K = 1–10). Analyses
were repeated ten times for each value of K. To define the
number of genetic clusters that best fit the data, we used the
method of the second-order rate of change of the log-likeli-
hood proposed by Evanno etal. (2005) using STRU CTU RE
HARVESTER (Earl and vonHoldt 2012). Structure plots were
made with Structure Plot v2.0 (Ramasamy etal. 2014). Finally,
population differentiation was calculated between sampling
sites based on Wright’s FSTΘ estimator (Weir and Cockerham
1984) and their significance (from 10,000 per-mutations) using
Arlequin 3.11 (Excoffier etal. 2005). We also calculated the
Nei genetic distance (Nei 1972) between each pair of popula-
tions using GENALEX v.6.51b2 (Peakall and Smouse 2012).
To evaluate unique alleles, we checked visually for the number
of private alleles for each locus and morphs, and we included
as private alleles those that were in more than two individu-
als. In addition, we checked these alleles in the confiscated
individuals to assess their origin.
Results
Overexploitation data
We surveyed six individuals who have worked in the frog’s
extraction since the 70s. The respondents identified the
same extraction localities, but different extraction dates.
The number of frogs extracted was between 100 and 300
per event and, was consistent among the respondents. The
time between extractions was 1 to 5months and the extrac-
tion events were 1 to 24 per surveyed individual. These
numbers vary with the extraction dates considered. Our
data suggests that there were four periods of heavy frog
extraction, between 1977 and 1980, 1980 and 1985, 1987
and 1992 and the most recent in 2009, 2018 and 2019 (Fig.
S2). The 1987–1992 period refers to a single respondent,
who did not collect in the field but was responsible for the
storage and distribution of frogs. He reports shipments
of 500–1000 frogs per event. Data from 2018 to 2019
come from confiscated shipments at El Dorado Interna-
tional Airport in Bogota (Fig.1b). Approximately 81,000
frogs were extracted from the field (mean = 81,141; mini-
mum = 60,047; maximum = 102,236) from at least eight
localities (Fig.1b, S2). The map shows a reconstruction
of the areas in which frogs were extracted between 1977
and 2009 (Fig.1a). The largest extraction occurred in
areas near the type locality such as Amapola, Cabeceras
Danubio, Cabeceras Rio Blanco and Cruceta, where O.
lehmanni frogs are not currently recorded or their abun-
dance is very low.
Genetic diversity
The sample size was between 55 and 83 individuals for
each locus. The number of alleles per locus was between
10 and 26, these values are higher than previously reported
(Hauswaldt etal. 2009; Wang and Summers 2009; Medina
etal. 2013). The polymorphic information content (PIC)
showed that all the loci were informative (PIC > 63%),
which is similar to previous studies (Hauswaldt etal.
2009) (TableS3). For O. lehmanni-yellow only five mark-
ers were amplified (Oop B9, Dpum 63, Dpum 14, Oop
F1 and Oop C11). Oop E3 did not amplify, and Oop G5,
Oop H5, Dpum 44 and Dpum 110 amplified with less than
four individuals. The Hardy–Weinberg exact test (HWE)
for heterozygote deficit showed equilibrium deviations in
six loci (Dpum63, Dpum110, Oop_H5, Oop_E3, Oop_F1,
and Oop_C11) (p-value < 0.001, df = 20), just for the O.
lehmanni-red population. However, after correction for
null alleles, only three loci (Dpum110, Oop_H5, and
Oop_F1) showed equilibrium deviations. This correction
Conservation Genetics
1 3
was also taken into account for genetic distance analysis
(FST) (TableS4). The linkage disequilibrium test indi-
cated no significant correlation between each pair of loci
(p-value > 0.001). MICRO-CHECKER underlined that no
evidence of scoring error due to stuttering or of large allele
dropout was found.
Our data showed that intra-population genetic diver-
sity is reduced because observed heterozygosity (Ho:
mean ± s.d. = 0.599 ± 0.165) is lower than expected het-
erozygosity (He: mean ± s.d. = 0.867 ± 0.082) for all loci
(Fig.2a, TableS3). Ho was significantly lower than He
for all the loci and samples (t test: t = − 4.54, df = 42.46,
p-value < 0.001). Similarly, our data suggest there are no
differences in observed heterozygosity between the two
groups (t test: t = − 1.66, df = 5.75, p-value = 0.15). The
expected heterozygosity was significantly greater than
observed for O. lehmanni-red (t test: t = − 3.53, df = 15.52,
p-value < 0.01). However, for O. lehmanni-yellow did not
find significant differences between Ho and He (t test:
t = − 0.16, df = 8.50, p-value = 0.87) (Fig.2b). Confiscated
samples were excluded from the analysis since we do not
know their origin; therefore, it would not be comparable
with the other two groups. The inbreeding coefficients
were positive values indicating inbreeding due to excess
homozygotes, but the FIS was higher for O. lehmanni-red
(FIS: mean ± s.d. = 0.289 ± 0.20) than for O. lehmanni-yellow
(FIS: mean ± s.d. = 0.221 ± 0.55).
Phenotypic andgenetic variation
Our data suggest that there are no significant differ-
ences in body size between red and yellow morphs
(p-value = 0.09, F-statistic = 2.74, df = 163), however, the
body length of the sampled red frogs was slightly larger
(mean ± s.d. = 35.4 ± 2.04mm, N = 151) than that of the yel-
low frogs (mean ± s.d. = 34.46 ± 1.85mm, N = 14) (Fig.3).
Genetic structuring analysis shows that FCNP individuals
exhibit no significant genetic structure and were grouped as
one cluster; therefore, we analyze them as a single popula-
tion. Analysis with all the samples indicated that the individ-
uals were assigned most likely to two genetic groups (red and
yellow morphs, respectively). According to Evannos’ ΔK,
the most likely number of clusters obtained in STRU CTU
RE was K = 2. (LnP(D) = − 3016.9, Delta K peak = 12.04)
(Evanno etal. 2005) (Fig.4a). Color variation is associated
with genetic structuring. The yellow frogs sampled in the
field clearly form a genetic group, which is consistent for
all values of K (1–5) (Fig.4a). Likewise, the red frogs from
FCNP constitute another genetic group. With regard to con-
fiscated frogs, some individuals are assigned to the red group
and others to the yellow group, and not always, the phenotype
registered in the database of the Cali Zoo corresponds to the
allocation group (four individuals). It is probable that these
samples come from more than one locality, and that it is dif-
ferent from those of our study. This observation is evident
Fig. 2 Relationship between observed heterozygosity (Ho) and
expected heterozygosity (He) for ten microsatellite molecular mark-
ers. a Ho minus He relationship for all samples obtained from field
and ten microsatellite markers. Negative values indicate that He
is greater than Ho. b Values of observed and expected heterozygo-
sity for yellow and red morphs of O. lehmanni. Kruskal–Wallis test
(K–W) was done to compare Ho and He intra and interspecific. Red
bean indicates values for O. lehmanni-red and yellow bean for O.
lehmanni-yellow. Black points represent raw data, vertical bar shows
the mean, the bean is a smoothed density curve showing the full data
distribution, and the rectangle represents the uncertainty around the
mean using a 95% Bayesian highest density interval. *** indicates
relationship is statistically significant and NS not significant
Conservation Genetics
1 3
when we use K = 3, since the confiscated frogs form an inde-
pendent cluster, and only three individuals remain consist-
ently assigned to the red cluster (Fig.4a). From K = 4 almost
all individuals, except O. lehmanni-yellow, were admixed
indicating no true genetic structure.
Our genetic distance data suggest that there is a moder-
ate to great genetic differentiation between the O. lehmanni
lineages. We include the individuals confiscated in this
analysis as a group, given that although we do not know
their origin, they represent O. lehmanni samples that may
exist in the field. FST values between O. lehmanni-red and
confiscated individuals (FST = 0.064, p-value < 0.005) and O.
lehmanni-yellow and confiscated individuals (FST = 0.130,
p-value < 0.005) suggest a moderate differentiation genetic
(Hartl and Clark 1997). In addition, high genetic differentia-
tion is founded between the field individuals of O. lehmanni
red and yellow (FST = 0.209, p-value < 0.005) (Har tl and
Clark 1997). Even, this genetic differentiation is higher com-
pared to other lineages closely related to O. lehmanni like
O. histrionica and a hybrid lineage (pHYB) (Fig.4b). These
same relationships remain after correcting for null alleles
(TableS4). FST values are consistent with the Nei genetic
distance; thus, populations with greater distance and genetic
structure are O. lehmanni-red and O. lehmanni-yellow
Fig. 3 Body length of the red and yellow O. lehmanni morphs meas-
ured from field collected individuals. Red bean indicates values for
O. lehmanni-red and yellow bean for O. lehmanni-yellow. Black
points represent raw data, vertical bar shows the mean, the bean is
a smoothed density curve showing the full data distribution, and the
rectangle represents the uncertainty around the mean using a 95%
Bayesian highest density interval
Fig. 4 Results of structure analysis and genetic distances. O. leh-yel-
low is the samples of the yellow frogs sampled from the field. Con-
fisc. represents the confiscated individuals of the Cali Zoo. FCNP
are the samples of O. lehmanni-red of the Farallones Park, and
pHYB is a hybrid lineage previously published (Medina etal. 2013).
a Bar plots of assignment probability to each genetic cluster for all
O. lehmanni samples using different K values (2–5). b Black num-
bers show the Wright’s FST estimator values and their significance
(***), and blue numbers indicate Nei genetic distance values between
populations. Values closer to one indicate less genetic distance. For
populations of O. histrionica and pHYB, FST values were taken from
Medina et al. (2013) and Nei genetic distance data ware calculate
with their data
Conservation Genetics
1 3
(Fig.4b). Finally, we found 28 private alleles. Of these, O.
lehmanni-yellow had four of three loci (Oop B9, Opum F1,
and Oop C11) and O. lehmanni-red had 24 private alleles of
five loci (Oop B9, Dpum 63, Dpum 14, Oop F1, Oop C11)
(TableS5). Confiscated individuals only had five private
alleles of one loci from O. lehmanni-red (162, 143, 184, 147
and 160 of Oop C11 locus); for this reason, it is unlikely that
they come from the FCNP.
Discussion
The overexploitation of Lehmann’s poison frog during the
evaluated time could have caused the observed population
declines for two reasons. First, due to their territorial behav-
ior and acoustic conspicuity, males are easier to encounter
and probably collected in higher numbers than females.
Males are not only important for fertilization, but also for
offspring development (Tumulty etal. 2013). Therefore, dif-
ferential extraction would likely affect reproduction rates.
In this study, the number of registered males (117) was 3.4
times greater than that of females (34). Poison frogs have
very elaborate reproductive behaviors (Weygoldt 1980,
1987); for example, tadpoles are deposited in bromeliads
within the male territory and they depend on the unfertilized
eggs deposited by their mothers (Brust 1993; Crump 1996;
Roland and O’Connell 2015). This behavior is described
as maternal care (Summers and Tumulty 2014). However,
for O. lehmanni, we have observed that the male performs
advertisement calls while the female feeds the larvae and
that females live within the male territory (for at least two
months) (Obs. Pers. M. Betancourth-Cundar). These behav-
iors have been described as biparental care in Ranitomeya
imitator (Brown etal. 2010) and R. vanzolinii (Caldwell
1997; Caldwell and de Oliveira 1999). In addition, in cou-
ples where the males are removed, there is lower tadpole
growth and lower survival for widowed females compared
with control couples (Tumulty etal. 2013). Second, data
about the reproductive potential in Oophaga pumilio esti-
mate that this species takes a minimum of 30days to get one
tadpole forward from eight eggs (Pröhl and Hödl 1999). If
we extrapolate this data at O. lehmanni using the informa-
tion of individuals in captivity (Com. Pers. B. Santamaria),
we found that a mean of 24 eggs per month (SD ± 18.37)
are produced, which would allow three tadpoles to be fed
simultaneously. Now, considering the calculation of 182
individuals collected in a single site (Myers and Daly 1976)
and assuming an operational sex ratio 1:1 would produce
approximately 1092 tadpoles per year. If we extrapolate this
to six years, there would be approximately 6552 tadpoles.
The estimated minimum number of frogs removed in that
same period is more than 15,000 frogs, which more than
doubled the tadpoles produced. These estimations could
indicate that the frog’s massive extraction caused impacts
in the local populations that led to almost complete extirpa-
tion of the type locality and nearby places.
Our data show a reduction of genetic diversity for all sam-
ples and markers evaluated. The expected heterozygosity is
greater than that observed, and three of the markers show a
heterozygous deficit for O. lehmanni-red, even after correct-
ing for null alleles. In general, a deficiency in heterozygotes
is an indication of inbreeding and our data suggests that O.
lehmanni is in this situation. Furthermore, O. lehmanni-red
set out significant differences between Ho and He and has
a positive FIS value, which indicates inbreeding depression.
However, other processes like the Wahlund effect (Wahl-
und 1927) can be operating. This effect is defined as the
excess of homozygotes or the deficit in heterozygotes caused
by subpopulation structure, even when the populations are
randomly mating or are in Hardy–Weinberg equilibrium
(Wahlund 1927). The mass extraction of frogs along with
high deforestation may have isolated the populations of the
FCNP, leading to subpopulation structure, inbreeding, and
heterozygous deficiency. Indeed, our data shows high genetic
structure between the red and yellow morphs. Moreover, to
evaluate the hypothesis of fine-scale genetic structuring, it is
necessary to include more samples from each of the locali-
ties within the FCNP, unfortunately, in this study; we only
have three individuals for one of the localities.
Overall, the yellow morph has been rare in the field and
a little more sought-after than the red in the pet market with
populations locally extirpated. The O. lehmanni-yellow
locality that we sampled is a population remnant where we
only registered 15 individuals in two days of exhaustive
search. In addition, this population is little known to local
collectors, and although it has had extraction events, these
are recent, probably among one or two generations. Our data
do not show significant differences between Ho and He for
this morph. This high heterozygosity may be associated with
this population still conserving alleles of the ancestral popu-
lation that may have had a larger effective population size
or high genetic flow with other populations of O. lehmanni
or even O. histrionica in the area. However, our data do not
allow discrimination between these hypotheses. Finally, it
is important to highlight that for these individuals we could
only amplify five markers. It is likely that these markers do
not reflect what is happening throughout the genome; there-
fore, it is necessary to use other molecular techniques. For
example, transcriptome-based exon capture or sequencing
the entire genome to get a better understanding of the history
and current status of this population.
We found no differences in heterozygosity (Ho) between
the two morphs, but high structuring and genetic distance.
This differentiation is also supported by the presence of pri-
vate alleles for each morph (4 for O. lehmanni-yellow and
24 for O. lehmanni-red). The FST is high because there are
Conservation Genetics
1 3
many differences between populations and little variation
within them. Genetic differentiation may be associated with
two factors. First, a loss of structural connectivity between
the two populations generated by continuous and acceler-
ated deforestation processes (Myers and Daly 1976). Sec-
ond, it is likely that the massive extraction of frogs at several
sites along the species distribution has fractured the genetic
flow between the two morphs, reducing its population sizes
and increasing inbreeding depression through Allee effects
(Allee and Bowen 1932; Courchamp etal. 1999). In addi-
tion, some studies suggest that amphibians often exhibit
strong site fidelity (Berven and Grudzien 1990; Reading
etal. 1991; Gascon etal. 1998; Shaffer etal. 2000; New-
man and Squire 2001) and this behavior may limit exchange
among populations, which can generate significant differen-
tiation even at a fine scale (< 1–2km) (Shaffer etal. 2000).
In dendrobatids, due to their territorial behavior, this fidelity
can be greater since they choose their territory for multiple
reproductive events (Pröhl 2005; Brown etal. 2009). We
observed this territorial behavior in O. lehmanni, where
some males have maintained their territory for at least four
years (Betancourth-Cundar etal. In prep.).
Our body size data suggests there are no statistically sig-
nificant differences between the two morphs, but high struc-
turing and genetic distance. In some lineages of the Oophaga
genus, similarly contrasting evidence has been found. For
example, Roland etal. (2017) demonstrated a high level of
genetic polymorphism and phenotypic variation in popula-
tions of northern Ecuador in Oophaga sylvatica. However,
southern populations show big phenotypic but little genetic
differentiation (Roland etal. 2017). In view of genetic differ-
ences, it is necessary to evaluate other ecological, behavioral
and phenotypic traits that may be indicating some differ-
entiation beyond the genetic criterion (Vargas-Salinas and
Amézquita 2013). For example, traits associated with sexual
selection that generate some type of associative mating, as
suggested by mate-choice experiments between O. histri-
onica, O. lehmanni-yellow and pHYB (Medina etal. 2013).
In the same way, discrete coloration patterns are different
and are associated with genetic structuring, but confiscated
individuals move a little away from this hypothesis. These
individuals probably come from one or more different loca-
tions than the sampled FCNP and O. lehmanni-yellow local-
ity and therefore some cannot be assigned to these groups.
This allows us to infer, that an exhaustive search of more
populations of O. lehmanni in both, FCNP and areas near the
type locality is still needed, and, that in spite of the efforts of
researchers and environmental authorities, the illegal extrac-
tion of frogs continues from unknown places.
Genetic results and phenotypic variation lead us to inter-
pret that red and yellow morphs should be considered as
separate management units. The red morph is fortunately
found within a protected area (FCNP), which hopefully
guarantees that at least their populations will not be affected
by habitat fragmentation or deforestation, but other threats,
like emerging diseases, Bd (Batrachochytrium dendroba-
tidis) (Flechas etal. 2012, 2017) and inbreeding depression
cannot be discarded. Therefore, it is necessary to establish a
program of ecological and genetic monitoring and increase
the search for new locations inside and outside the FCNP.
Unfortunately, the yellow morph, or at least the only known
locality, is in a high degree of threat due to fragmentation,
habitat deforestation, agrochemicals, and overexploitation.
This place is located within an area of coca crops (Erythrox-
ylum coca) and thus creating a conservation program in the
area is not a straightforward task. Considering the genetic
data, this population has an exclusive genetic configuration,
which represents a unique biological entity, both, phenotypic
and genotypic. It is very important to establish urgent con-
servation measures on this locality, but it is also important to
evaluate options of captive breeding and reintroduction pro-
grams. Although researchers and environmental authorities
monitor the populations, fighting against illegal trafficking is
very difficult. In November 2018, more than 200 frogs of the
Oophaga genus were confiscated at the El Dorado Interna-
tional Airport, of which 55 frogs were of our focus species,
O. lehmanni, and April 2019 another 415 frogs were seized,
of this near 250 frogs were of O. lehmanni. These frogs were
mostly red (186), but there were also some yellow individu-
als yellow and individuals with different patterns of color-
ing bands or dots (164). It is likely that many of these frogs
come from the locality of putative hybrids, but given the
confiscate amount, they may come from more than one site.
Finally, we must recognize that the scope of micro-
satellite markers to infer the demographic history of
populations is limited, especially in amphibians, because
among terrestrial vertebrates, amphibians show the largest
genome sizes (Portik etal. 2016). It would be interesting
to use new generation sequencing tools that allow us to
solve deeper phylogenetic and population genetics ques-
tions including more localities of O. lehmanni and samples
of intermediate phenotypes between O. lehmanni-red, O.
lehmanni-yellow, and O. histrionica. For an effective man-
agement program, it is important to establish the genetic
limits of the management units at the intraspecific level;
this ensures that we can protect the genetic integrity of
each unit (Shaffer etal. 2000). It is also necessary to incor-
porate captive breeding programs in the long-term man-
agement plans aiming to reintroduce animals in genetic
and phenotypically similar places. Well-designed and
monitored programs can be of great help to many threat-
ened amphibian species (Stockwell etal. 2008; Griffiths
and Pavajeau 2008; Harding etal. 2016). This may be a
viable option to mitigate the extinction risk and maintain
healthy populations of the Lehmann’s poison frog.
Conservation Genetics
1 3
Acknowledgements This work was supported by Asociación Colombi-
ana de Herpetología-ACH—Botas al Campo (Grant 01-2014 to MBC),
Iniciativa de Especies Amenazadas Jorge Ignacio Hernández-Camacho
and Fundación Omacha (Grant 04-2015 to MBC), Consejo Profesional
de Biología—CPBiol (Grant 07-2016 to MBC), Facultad de Ciencias,
Universidad de los Andes—Colombia (Seed Grant 2014-1 to MBC),
Departamento Administrativo de Ciencia, Tecnología e Innovación—
COLCIENCIAS and Empresa de Energía del Pacifico—EPSA (734-
2015). The funders had no role in study design, data collection, and
analysis, decision to publish, or preparation of the manuscript. We are
highly thankful to RF Molina, LA Barragan, A Zarling, M Guayara,
C Amorocho, L Tabares and JD Rueda for their help in the samples
collection and lab work. To V Santamaria and Tesoros de Colombia
for providing us data about poison frogs reproduction in captivity. To
HN Vargas for its friendly management in achieving financing. To R
Marquez for helpful comments and suggestions that greatly improved
this manuscript. To Instituto de Protección y Bienestar Animal de la
Alcaldía de Bogotá for providing us with data on frog confiscations.
Compliance with ethical standards
Conflict of interest The authors have declared that no competing in-
terests exist.
Ethical approval All applicable international, national, and/or insti-
tutional guidelines for the care and use of animals were followed. All
procedures performed in studies involving animals were in accordance
with the ethical standards of Comité Institucional para el Cuidado y
Uso de Animales de Laboratorio (CICUAL) at Universidad de Los
Andes. Procedures for capture, handling and samples collections of live
animals in the field were approved by Parques Nacionales Naturales de
Colombia under research permits 017-2016 and 024-2017 granted to
MBC and Autoridad Nacional de Licencias Ambientales-ANLA (Per-
miso Marco: Resolution 1177 de 2014 to Universidad de los Andes).
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... Several studies have well documented their effect on biodiversity loss [1]. Moreover, also international wildlife trade contributes to the depletion of natural resources; hence, the over-exploitation of natural populations, such as the withdrawal of individuals from the wild, is considered one of the leading causes driving the species into extinction [2][3][4]. It is known that seafood, furniture, and fashion are the main categories requested from the international trade; in addition to this, the commerce of pets affects many individuals. ...
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