Brazilian Journal of Biology https://doi.org/10.1590/1519-6984.

184842
ISSN 1519-6984 (Print)
Original Article
ISSN 1678-4375 (Online)

Genetic variability of populations of the white-eared opossum,

Didelphis albiventris Lund 1840 (Didelphimorphia; Didelphidae)
in Brazil
D. C. Nascimentoa,b, B. A. T. P. Camposc, E. C. Fragaa,b,c and M. C. Barrosa,b,c*
a
Programa de Mestrado em Ciência Animal – CCMA, Centro de Ciências Agrárias – CCA, Universidade Estadual do
Maranhão – UEMA, Cidade Universitária Paulo VI, CP 9, São Luís, MA, Brasil
Centro de Estudos Superiores de Caxias – CESC, Laboratório de Genética e Biologia Molecular – GENBIMOL,
b

Universidade Estadual do Maranhão – UEMA, Praça Duque de Caxias, s/n, Morro do Alecrim, CEP 65604-380,
Caxias, MA, Brasil
Programa de Pós-graduação em Biodiversidade, Ambiente e Saúde – PPGBAS, Centro de Estudos Superiores de
c

Caxias – CESC, Universidade Estadual do Maranhão – UEMA, Praça Duque de Caxias, s/n, Morro do Alecrim,
CEP 65604-380, Caxias, MA, Brasil
*e-mail: [email protected]

Received: September 01, 2017 – Accepted: February 10, 2018 – Distributed: November 30, 2019
(With 4 figures)

Abstract
Didelphis albiventris are found throughout Northeast and Central Brazil to central-southern Uruguay and it was subject
of few studies in a population level. Given this, the present study investigated the genetic variability of the species
using the mitochondrial molecular marker cytochrome oxidase c subunit I. We analyzed samples from the different
biomes within three Brazilian regions: Northeast (Caatinga, Cerrado, and Atlantic Forest), Southeast (Cerrado, Atlantic
Forest, Cerrado/Atlantic Forest, and Cerrado/Caatinga ecotones) and South (Pampa and Atlantic Forest). Software BAPs
retrieved five distinct demes: dm 1, dm 2, and dm 5 that occurs in South, Northeast and Southeast regions respectively
and the dm 3 and dm 4 are wide distributed in Northeast and Southeast. Population analysis performed with AMOVA,
haplotype network and Mantel test estimated the veracity of the demes. The FST shows structuring for the five demes,
with dm 1 (South region) isolated from the others, however the other analysis showed the Northeast/Southeast demes
(dm 2-5) united, diagnosing gene flow between them, mainly at the transitional zones, in areas as far away as areas with
similar latitude interval (Southeast vs South) that was not detected gene flow. In the haplotype network, the mutational
steps was conclusive in split dm1 from dm 2-5 with 15 mutational steps and the Mantel test was moderated, which
is explained by genetic similarity despite the great geographic distances (Northeast/Southeast). Thus, our analysis
recognized two different lineages (South and Northeast/Southeast) and indicate that the biomes were not decisive in
their isolation. The sharing of demes at the transitional zones and in areas with high latitudinal intervals reflects a recent
ancestral polymorphism for D. albiventris. The plasticity in the occupation of the space by this species contributes in
its wide dispersion capability, that is, geographical distribution. Our results revealed important implications for the
management of D. albiventris in these transitional zones areas where demes were shared.
Keywords: marsupials, COI, population diversity, geographic lineages.

Variabilidade genética de populações do gambá de orelha branca,

Didelphis albiventris Lund 1840 (Didelphimorphia; Didelphidae) no Brasil

Resumo
Didelphis albiventris é encontrada em todo o Nordeste e região central do Brasil até o centro-sul do Uruguai e foi
alvo de poucos estudos em nível populacional. Dessa forma, o presente estudo, investiga a variabilidade genética da
espécie usando o marcador molecular citocromo c oxidase subunidade I. Analisou-se amostras de diferentes biomas
de três regiões brasileiras: Nordeste (Caatinga, Cerrado e Floresta Atlântica), Sudeste (Cerrado, Floresta Atlântica,
ecótonos Cerrado/Floresta Atlântica e Cerrado/Caatinga) e Sul (Pampa e Floresta Atlântica). O software BAPs recuperou
cinco demes distintos: dm 1, dm 2 e dm 5, que ocorrem nas regiões Sul, Nordeste e Sudeste, respectivamente, e os
dm 3 e dm 4, que são amplamente distribuído no Nordeste e Sudeste. Análises populacionais realizadas com AMOVA,
rede de haplótipo e teste de Mantel estimaram a veracidade das demes. O FST mostrou estruturação para as cinco demes,
com dm 1 (região Sul) isolada das demais, entretanto as outras análises mostraram as demes Nordeste/Sudeste (dm 2-5)
unidos, diagnosticando fluxo gênico entre elas, principalmente em zonas de transição, em áreas tão distante quanto
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 Genetic variability of population of the Didelphis albiventris

áreas com similar intervalo de latitude (Sudeste e Sul), onde não foram detectado fluxo gênico. Na rede de haplótipo,
os passos mutacionais foram conclusivos em separar dm 1 do dm 2-5 com 15 passos mutacionais, e o teste de Mantel
foi moderado, o que é explicado pela similaridade genética apesar da grande distância geográfica (Nordeste/Sudeste).
Assim, duas linhagens diferentes (Sul e Sudeste/Nordeste) foram encontradas, indicando que os biomas não foram
decisivos em seus isolamentos. Os compartilhamentos das demes, em zonas de transição e em áreas com elevados
intervalos de latitude, refletem um polimorfismo ancestral recente para D. albiventris. A plasticidade na ocupação
do espaço por esta espécie contribui em sua ampla capacidade de dispersão, ou seja, distribuição geográfica. Nossos
resultados revelam importantes implicações para o manejo de D. albiventris nessas áreas de zonas de transição, onde
as demes são compartilhadas.
Palavras-chave: marsupiais, COI, diversidade populacional, linhagens geográficas.

1. Introduction phylogenetic studies of small mammals (Carvalho et al.,

2011; Agrizzi et al., 2012; Sousa et al., 2012; Muller et al.,
The marsupials of the genus Didelphis Linnaeus,
2013; Rocha et al., 2015). In particular, the COI gene has
1758 are a small non-volant mammals that are an
been used as a DNA barcode for the identification of a
essential component of natural communities and good
number of different vertebrate groups (Hebert et al., 2003),
indicators of environmental changes and its degree of
and has contributed important findings on D. albiventris
conservation (Conde y Vera and Rocha, 2006; Castro,
populations (Sousa et al., 2012).
2012; Quintela et al., 2013). In particular, populations of the Ecological studies use population and genetic data in
white-eared opossum (Didelphis albiventris Lund, 1840) attempt to understand the biology of a species. With the
tends to increase in density in response to environmental scarcity of population genetic information throughout its
disturbance (Cáceres et al., 2008), with associated distribution, especially for the inhabitants of Cerrado and
implications for public health given that this species is a Caatinga, the present work investigated how D. albiventris
natural reservoir for a number of human diseases such as populations are distributed in Brazil using the COI as a
Chagas disease and leishmaniasis (Sherlock et al., 1984; molecular marker: 1. The species possesses populations
Pinho et al., 2000; De Luca et al., 2003; Bern et al., 2011; structured in the Northeast/South axis? 2. There is more
Zetun et al., 2014). than one population per Biome? 3. If yes, they are shared
Due to its generalist habit, D. albiventris is highly flexible between Biomes or are exclusive for one Biome? Ecotone
in the use of space, exploiting a variety of vertical strata areas has a different role in the population structure of the
and occupying large home ranges (Almeida et al., 2008; species? 4. Is there natural selection or are the populations
Sanches et al., 2012). The species is widely distributed selectively neutral? 5. In attempt to answer these questions
in deciduous forests, occurring from northeastern and we conducted genetic variability study of populations of
central Brazil to central and southern Uruguay, as well as the white-eared opossum, D. albiventris.
in Paraguay, Argentina and Bolivia (Cerqueira and Tribe,
2008). In Brazil there are records in Caatinga, Cerrado, 2. Material and Methods
Pantanal, Pampas habitats and transitional zones between
these biomes (Cerqueira and Tribe, 2008; Paglia et al., 2012). 2.1. Description of the samples
Despite the genus Didelphis is common and ecologically Samples of D. albiventris were obtained from the
successful (Durant, 2001), little is known about the Northeast, Southeast and South regions of Brazil, between
relationships among of D. albiventris populations, with latitudes 04°05’ S and 30º01’ S (Figure 1). A  total of
only two studies available: Sousa  et  al. (2012) and 52 samples were obtained in the Northeast region
Rocha et al. (2015). Both works revealed some degree of (Paraíba/PB, Sergipe/SE, Pernambuco/PE, Ceará/CE,
genetic structuring, the former studied the mitochondrial Piauí/PI and Maranhão/MA), from the Atlantic Forest,
gene cytochrome c oxidase subunit I (COI) of populations Caatinga and Cerrado biomes. Eighty four samples from
from southern and southeastern Brazil, and the other the Southeast (Minas Gerais/MG) from the Atlantic Forest
mitochondrial gene cytochrome b from few localities in and Cerrado biomes, and the Cerrado/Atlantic Forest
central Brazil. and Cerrado/Caatinga ecotones. Six samples from South
Mitochondrial DNA have revealed degree of population (Rio Grande do Sul/RS) corresponding to the Pampas
structure for many species, being the most used molecular and Atlantic Forest (Figure 1; Supplementary Material).
marker for tracing the geographic distribution of genealogical The samples from the Northeast were obtained in
lineages (Avise et al., 1987). Males and females of the Universidade Estadual do Maranhão, for which the
the D.  albiventris present similar patterns of dispersal collecting data are available in Costa et al. (2012), and
(Sanches et al., 2012), and the sex of the specimen should from collaborations with the scientific collections of other
be irrelevant to the selection of the molecular marker, Brazilian research institutions, such as Universidade
consequently the matrilineal inheritance of the mitochondria Federal da Paraíba (UFPB), Universidade Federal de
should not interfere in the analyses. Notably, mitochondrial Sergipe (UFS), Universidade Federal do Piauí (UFPI),
genes have been used successfully in population-level and and Museu Paraense Emílio Goeldi (MPEG). All samples

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 Nascimento, D.C. et al.

Figure 1. Localities from which the samples were obtained for the present study. The demes were defined according to the
BAPs software and are represented by geometric figures with numbers (designated in Supplementary Material); Biomes are
indicated in gray scale.

from Southeast and South were obtained at GenBank from at 72ºC for 3 min. We used the BigDye Terminator v3.1
Sousa et al. (2012). Cycle Sequencing Kit (Applied Biosystems) for sequencing
reactions. The sequences were obtained using an ABI 3500
2.2. DNA extraction, amplification and sequencing
automatic DNA sequencer with associated Sequencing
DNA was extracted from muscle, liver, and ear tissue Analysis software (Applied Biosystems).
using the Phenol-Chloroform protocol of Sambrook and
Russel (2001). We amplified and sequenced a fragment 2.3. Data analysis
of the mtDNA COI gene using the primers LCO1490 The sequences were aligned using Clustal W
and HCO2198 (Folmer et al., 1994). The COI gene was (Thompson et al., 1994) in BioEdit v7.0.5.2 (Hall, 1999).
amplified in 25 μl reactions of PCR, consisting of 4.0 μl of The analysis of the variable sites was performed in Mega
dNTP (1.25 nM), 2.5 μl of buffer (10X), 0.5 μl of MgCl2 v6.06 (Tamura et al., 2013). Haplotype and nucleotide
(25 nM), 0.25 μl of each primer (200 ng/μl), 0.2 μl of Taq diversity were estimated in DnaSP v4.9 (Rozas et al., 2003),
DNA Polymerase 10 x 250 μl (GE Healthcare), and 1 μl and the groups used for the population analyses were defined
of template DNA (100 ng/μl), with the final volume being using the BAPs software (Corander et al., 2008). In this
completed by purified water. The samples were amplified method, the number of populations is treated as unknown
in a Veriti 96 well thermal cycler (Applied Biosystems) parameter and is directly inferred from the data set without
with an initial denaturation (94ºC for 3 min), 40 cycles of defining a prior estimate, a dendogram were generated
denaturation (94ºC for 45 s), annealing (48ºC for 45 s), and using Unweighted Pair Group Method using Arithmetic
extension (72ºC for 1 min and 30 s), and a final extension averages - UPGMA. The populations identified by this

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 Genetic variability of population of the Didelphis albiventris

software were used to structure the subsequent population 31 were variable, and 21 were informative for parsimony
analyses. The haplotype networks were produced in NetWork analyses, corresponding to 19 haplotypes.
v4.6.1.2 (Bandelt et al., 1999). An Analysis of Molecular The sequences plotted in the BAPs software (Figure 2)
Variance (AMOVA) was performed in Arlequin v3.5.2.1 indicated the existence of five Brazilian demes (dm), as
(Excoffier and Lischer, 2010) to obtain the index of allelic can be seen in the haplotype network and in the map
(Figure 1 e 3). The dm 1 occurs in the Pampas and Atlantic
fixation (FST). The neutrality tests D (Tajima, 1989) and
Forest biomes (South region; H1 and H2), dm 2 in the
Fu’s FS (Fu, 1997) were run in Arlequin v3.5.2.1 (Excoffier
Caatinga and Atlantic Forest biomes (Northeast region;
and Lischer, 2010). The Mantel test, which relates genetic H3-H8) and dm 5 Cerrado, Caatinga, Atlantic Forest
distance to geographic distance, was run in the Alleles in and ecotones (Southeast regions; H16-H19). The dm 3
Space software with 10,000 replications (Miller, 2005). (H 9-H13) and dm 4 (H14 and H15) are more widely
distributed, being found simultaneously in the Cerrado
3. Results and Caatinga (Northeast region) and Cerrado, Caatinga,
Atlantic Forest (Southeast region). We can observe that some
3.1. Molecular characterization and neutrality tests
localities present multiple demes: Xacriabá Amerindian
The study included a total of 142 sequences for the Reservations/MG that contains dm 3, dm 4 and dm 5;
mitochondrial COI gene of D. albiventris, made up of a Caxias/MA dm 3 and dm 4, Belo Horizonte/MG and Nova
fragment of 655 bps, of which 624 bps were conserved, Lima/MG dm 4 and dm 5 (Table 1).

Figure 2. Didelphis albiventris demes defined through the BAPs software for the COI gene. (a) UPGMA tree using the Nei
model. (b) Mixture among the demes obtained, the size of the color bars is proportional to the number of specimens in the
populations.

Figure 3. Haplotype network for the COI gene in the D. albiventris demes. The numbers represents the mutational sites between
demes, while the size of the circles is proportional to the frequency of occurrence of the haplotype. Biomes: PA - Pampa;
AF - Atlantic Forest; CA - Caatinga; CE - Cerrado; CE/CA - Ecotone Cerrado/Caatinga; CE/AF - Ecotone Cerrado/Atlantic
Forest. The polygons delimit the haplotypes that comprises each deme.

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 Nascimento, D.C. et al.

The total haplotypic diversity (h) was h = 0.846, and and contribute to the establishment of management and
varied from h = 0.387 for dm 5 to h = 0.614 for dm 3, conservation measures along its geographical distribution.
while total nucleotide diversity (π) was π = 0.00615, and Were analyzed the largest sample for this specie to date,
varied from π = 0.00078 for dm 4 to π = 0.00152 for dm and high levels of haplotype diversity (h) and nucleotide
3 (Table 1). No significant p values were found in any diversity (π) were found. Similar results were retrieved
deme for the D test (Tajima, 1989), and a significant in Sousa et al. (2012); h = 0.7235; π = 0.0065) for COI
value for Fu’s FS (Fu, 1997) was only recorded for dm 2, and Rocha  et  al. (2015) for Cytochrome b (h = 0.876;
indicating that only this deme is influenced by demographic π = 0.0062), reflecting the high genetic diversity of this
parameters (Table 1). species. According to Grant and Bower (1998) high values
3.2. Genetic differentiation among demes of h and π combined (above 0.5 and 0.005 respectively)
AMOVA analyses based on the five demes retrieved
by the BAPs analysis (Table 1) indicated that 87.47% of
the variation was due to differences among the demes,
and 12.53% within the demes, with a highly significant
(p = 0.00000) FST value of 0.87 (Table 2). The pairwise
FST values were higher, ranging from 0.7, for dm 2 vs. dm
3, to 0.97 for dm 4 vs. dm 1 (Table 3).
The results of the Mantel test for the five demes
indicated a moderate degree of correlation between genetic
and geographic distances (r = 0.54; p = 0.00001; Figure 4).
When dm 1 (r = -0.31; p = 0.19), dm 2 (r= -0.13; p = 0.79),
and dm 5 (r= -0.04; p = 0.61) were analyzed separately the
correlation were low, although dm 3 (r = 0.90; p = 0.001)
and dm 4 (r = 0.89; p = 0.001) returned high correlations.

4. Discussion
Due to the ecological importance of D. albiventris, the Figure 4. Results of the Mantel test for the correlation
investigation of the dynamics of dispersion of its populations between genetic (y) and geographic (x) distances in the COI
through a genetic approach will improve the knowledge gene among the D. albiventris demes analyzed.

Table 1. Genetic diversity and the results of the neutrality tests for the COI gene in the D. albiventris demes analyzed.
Molecular diversity Neutrality tests
Demes Localities N NH
h π Test D Test Fs
Dm1 27 - 30 6 2 0.533 0.00087 0.85057 0.62543
Dm2 1 - 13, 16 e 17 40 6 0.529 0.00099 -1.14587 -2.65922*
Dm3 14, 15, 18, 19 e 25 18 5 0.614 0.00152 -1.13016 -1.43002
Dm4 19, 20, 23 e 25 20 2 0.479 0.00078 1.26176 1.31130
Dm5 20-26 58 4 0.387 0.00094 -0.72085 -0.38003
Pooled demes 142 19 0.846 0.00615 -0.17691 -0.50651
N: Sample size; NH: Number of haplotypes; h: Haplotype diversity; π: Nucleotide diversity; (*) = p < 0.05.

Table 2. Results of the AMOVA for the COI gene for the five Didelphis albiventris demes retrieved from BAPs.
Source of variation Variance component % Variation FST p*
Among demes 2.18911 87.47 0.87465 0.0000
Within demes 0.31372 12.53
*Values of P, calculated randomly through 1023 permutations.

Table 3. Pairwise FST values for the COI gene for five demes of D. albiventris.
Deme Dm 1 Dm 2 Dm 3 Dm 4 Dm 5
Dm 1 0.00000
Dm 2 0.96492 0.00000
Dm 3 0.94811 0.71389 0.00000
Dm 4 0.97321 0.87873 0.85555 0.00000
Dm 5 0.96605 0.87873 0.81081 0.72012 0.00000
P = 0.0000.

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 Genetic variability of population of the Didelphis albiventris

reflects long evolutionary history. In the present study our Studies of didelphids have shown population
results shows the haplotypes H11, H14 and H16 widely structuring among different Brazilian biomes, for example
geographic distributed, suggesting long dispersion time Carvalho et al. (2011) and Caramaschi et al. (2011), found
and consequently long evolutionary history, corroborating population structuring of Monodelphis domestica between
Grant and Bower (1998). the Cerrado and Caatinga. In the present study this
By randomly treating allelic frequencies of genetic relationship was not verified, as the demes are not restricted
marker and genetic divergence between groups, the BAPS to one specific biome and are dispersed among a number of
software retrieved five demes, which are divided between different biomes. This fact may be associated in differences
the three regions analyzed, with overlap for demes 3 and 4 in home ranges and in ecological characteristics between
in the Northeast and Southeast Brazil. The BAPS was not M. domestica and D. albiventris that could be possible
unanimous in considering the geographic proximity or the directly related with body size. Didelphis albiventris
biomes to delimit the demes, consequently other population is the largest Didelphidae weighting 500-2,750g while
analyzes were carried out to verify if they correspond to M. domestica weight 80-120g only. This size difference
different lineages. reflects in greater dispersion capacity (Reis et al., 2006;
In order to identify if there is selective neutrality, tests Wang et al., 2009) of D. albiventris that increases the gene
were performed for the five demes. The results of Tajima’s flow, making population structuring more difficult. In this
D were not significant and Fu’s Fs was only significant study, only dm 1 was isolated from the others, but this fact
for dm 2, which contains haplotypes from the Northeast. is not associated with biomes, since that deme occurs in
Significant results for these tests indicate the presence of Atlantic Forest and Pampa’s biomes.
natural selection or demographic processes among the When comparing the mutational sites among the
variants of a nucleotide sequence in a population. Dm 2 is demes, the haplotype network also shows two lineages
distributed over a wide area, with different kind of biomes with different geographic distributions for D. albiventris
which could be interfering in the selective neutrality in this study: one Southern lineages (dm 1) and the other
(Fu and Li, 1993; Nielsen 2001). No significant results comprising Northeast/Southeast (demes 2-5) showed shared
were obtained for these same tests by Sousa et al. (2012) haplotypes. These two principal lineages are separated by
and Rocha et al. (2015), which indicates that this species 15 mutational steps. Northeast and Southeast dm’s are
is relatively constant. It is important to mention that these separated by few mutational steps (maximum of five, see
authors did not study northeastern populations. Figure 3), have distribution overlapping between biomes
The results of the AMOVA for the five demes indicate and therefore should be considered as a single lineage.
structuring (FST = 0.87). This structuring is evident in the The gene flow shown in the haplotype network represents
case of dm 1, which has pairwise FST values of 0.95 to 0.97, an ancestral polymorphism, since the shared haplotypes
indicating possible processes of allele fixation and genetic are distributed over long geographic distances, as can be
isolation. Sousa  et  al. (2012) also found evidence of seen in dm 3 (H11) that occurs in Rio Grande do Piauí/PI
population structuring (FST> 91%) in the Southeast and (Cerrado) and Xacriabá Amerindian reservations/MG
South populations. (Cerrado/Caatinga ecotone) (latitudes 07º 45’ S - 14° 53’ S).
The remaining demes analyzed in the AMOVA But the results of the haplotype network did not reveal
(dms 2, 3, 4 and 5) had also high pairwise FST values connection between dm 5 (H16 and18) in Piracema/MG
(0.71 to 0.88). Despite the high FST, the same demes are (Atlantic Forest) and dm 1 (H1) Machadinho/RS (Atlantic
distributed in different localities, for example: dm 3 are in Forest) which has similar latitude interval (latitudes 20º 30’ S
Caxias/MA and Xacriabá Amerindian Reservations/MG; dm - 27º 34’ S) (Figure 1 and 3; Table S1), another indication
4 in Caxias/MA, Xacriabá Amerindian Reservations/MG, of the isolation of these two strains.
Belo Horizonte/MG and Nova Lima/MG; dm 5 in Xacriabá The Mantel test indicated a moderate degree of
Amerindian Reservations/MG, Belo Horizonte/MG correlation between genetic and geographic distances in the
and Nova Lima/MG. We can also notice that different five demes. In this study, with the addition of D. albiventris
demes are in sympatry, for example: dm 3 and dm 4 in samples from the Northeast, we obtained a reduction in the
Caxias/MA; dm 3, dm 4 and dm 5 in Xacriabá Amerindian correlation obtained by Sousa et al. (2012). This reduction
Reservations/MG; dm 4 and dm 5 in Belo Horizonte/MG in the correlation reinforces the similarity found between
and Nova Lima/MG. demes 2-5, not diagnosed by these authors, and indicates
These sympatric demes show that FST are considering that the Northeast/Southeast lineage is genetically similar
different structured population in the same localities. although geographically distant.
This condition could be explained by the wide distribution With the addition of northeastern D. albiventris
of D. albiventris (Figure 1) and by the highly adaptable samples, were possible to conduct broader analyses
and generalist habit of the specie, since it is known that encompassing a more complete distribution range of the
altitude and environment do not interferes in the specie specie. Sousa  et  al.  (2012), found patterns of isolation
distribution (Aragona and Marinho-Filho, 2009). Thus, between Southeast and South regions as well as ours
although FST shows structuring, the connection between analyses, but it represents just a small fraction of the
demes allows that Northeastern and Southeastern regions distribution. The addition of Northeast does not interfere
belong to the same widespread lineages. in the isolation of the South region from the others, but the

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 Nascimento, D.C. et al.

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Supplementary Material
Supplementary material accompanies this paper.
Table S1. List of the D. albiventris specimens included in this study, their field numbers (BC, PR, FNI, ALN,
TNM, T, PNSC, URC, LLLS, and DCN), GenBank accession number (JN), demes, haplotype number (H), locality,
geographical coordinates and origin of the samples.
This material is available as part of the online article from https://drive.google.com/open?id=17CmDgYMAmH
LH0VTD_0c2AM476u2fJRrx

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