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Jurnal Kelompok 5
Jurnal Kelompok 5
doi:10.1093/jhered/esz049
Original Article
Advance Access publication August 17, 2019
Original Article
Abstract
In this study, we quantified the 3 pivotal genetic processes (i.e., genetic diversity, spatial genetic
structuring, and migration) necessary for a better biological understanding and management of
the singular “living-fossil” and near-threatened mouse opossum marsupial Dromiciops gliroides,
the “Monito del Monte,” in south-central Chile. We used 11 microsatellite loci to genotype 47
individuals distributed on the mainland and northern Chiloé Island. Allelic richness, observed
and expected heterozygosity, inbreeding coefficient, and levels of genetic differentiation were
estimated. The genetic structure was assessed based on Bayesian clustering methods. In addition,
potential migration scenarios were evaluated based on a coalescent theory framework and Bayesian
approach to parameter estimations. Microsatellites revealed moderate to high levels of genetic
diversity across sampled localities. Moreover, such molecular markers suggested that at least 2
consistent genetic clusters could be identified along the D. gliroides distribution (“Northern” and
“Southern” cluster). However, general levels of genetic differentiation observed among localities
and between the 2 genetic clusters were relatively low. Migration analyses showed that the most
likely routes of migration of D. gliroides occurred 1) from the Southern cluster to the Northern
cluster and 2) from the Mainland to Chiloé Island. Our results could represent critical information
for future conservation programs and for a recent proposal about the taxonomic status of this
unique mouse opossum marsupial.
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652 Journal of Heredity, 2019, Vol. 110, No. 6
The analyses of genetic diversity and population structure along Dromiciops gliroides has a restricted geographical distribution
the geographic distribution of species have received substantial at- between 35 and 43°S in south-central Chile and the adjacent re-
tention in the literature (Almeida et al. 2005; Eckert et al. 2008; gions of Argentina (Martin 2010). Within that range, D. gliroides
Castellanos-Morales 2015). Such interest has increased, especially populations are present both on the mainland and on Chiloé Island
in studies focused on threatened taxa due to the detrimental effects in southern Chile. Former molecular analyses based on mtDNA
of human activities resulting in fragmentation, habitat disturb- markers have suggested that populations of D. gliroides are struc-
ance, and connectivity loss among natural populations (Rodriguez tured into 3 phylogeographic clades (Himes et al. 2008). Two of
et al. 2012; Fietz et al. 2014; Napolitano et al. 2014; Li et al. these clades included specimens from the Chilean and Argentinean
2016; Eldridge et al. 2017; Perl et al. 2018). This is because genetic distributional range: a northern clade or clade A (36–39°S) and a
diversity is crucial for the adaptive potential of natural popula- southern clade or clade C (40–43°S) that also included Chiloé Island.
tions in the face of environmental changes (Frankham et al. 2005), A third clade, “B,” was geographically intermediate between clades
and it could be especially reduced in small populations due to the A and C but restricted to the Valdivian coastal range in Chile. A later
stronger effects of genetic drift and inbreeding (Lacy et al. 1997; study that included cranial morphologic variation of the “Monito
Methods were manipulated and released after handling, following the standard
bioethical and biosafety protocols proposed by the American Society
Study Area and Sample Collection
of Mammalogists (ASM: Sikes and Gannon 2011). In addition, we
Samples were collected in native forest fragments along the distribu- obtained muscle tissue of specimens deposited at the “Colección
tional range of D. gliroides in mainland Chile, including the northeast de Flora y Fauna Prof. Patricio Sánchez-Reyes,” Departamento de
end of Chiloé Island (Figure 1; Table 1). Chiloé Island is located in Ecología, Pontificia Universidad Católica de Chile, Santiago, Chile.
Southern Chile between 41 and 43°S, 73 and 74°30′W, in the Los
Lagos Region, and covers an area of approximately 9000 km2. The
island represents the sole insular territory where contemporary
populations of D. gliroides have been observed (Martin 2010). The
Microsatellite Genotyping
northern area of the island is approximately 2.5 km from the closest DNA was extracted with the phenol-chloroform protocol. The
point to the mainland, and it is separated by the Chacao channel pellet was resuspended in 50 mL of ultrapure water and stored at
(Figure 1). Animal trapping and handling were conducted following −20 °C. Samples were genotyped by 12 microsatellite loci designed
the protocol recommended by Celis-Diez et al. (2012). A 1 mm2 piece by Ecogenics, Zurich-Schlieren (Switzerland) in July 2014 (Table 2).
Figure 1. Map of the study area along Dromiciops gliroides distribution in south-central Chile. PNVPR, Parque Nacional Vicente Pérez Rosales.
654 Journal of Heredity, 2019, Vol. 110, No. 6
Table 1. Details of geographic area and sample size (n) of localities analyzed in this study
0.8 R primer, 0.8 µL M13 primer, 0.1 µL HotStarTaq (Taq Platinium), and Rousset 1995; Rousset 2008). Genetic diversity was assessed by
and 2.0 µL genomic DNA at 60 ng/µL (total PCR volume = 10 µL). calculating rarefied average allelic richness (Ar) to control for un-
The thermocycling protocol (suggested by Ecogenics) consisted of equal population sample size with the program HP-Rare v. June-6-
a denaturation phase of 95 °C for 15 min; 30 cycles of 95 °C for 2006 (Kalinowski 2005). In addition, observed heterozygosity (HO),
30 s, annealing (56 °C) for 45 s, and extension of 72 °C for 45 s; expected heterozygosity (HE), and inbreeding coefficient (FIS) were
then, a final amplification round of 8 cycles of 95 °C for 30 s, an- estimated with the program Arlequin v3.5 (Excoffier et al. 2005).
nealing temperature (56 °C) for 45 s, and 72 °C for 45 s, with a final The significance of FIS was calculated by 100 000 rounds of permu-
extension of 72 °C for 30 min. Microsatellite genotyping was per- tation tests in the same program.
formed at “Servicio de Secuenciación y Genotipado,” Departamento
de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad Population Structure
Católica de Chile, Santiago, Chile. Allele scores were obtained with We evaluated the degree of genetic differentiation of the populations
the program PEAK-SCANNER v1.0 (Applied Biosystems 2006). by calculating pairwise FST indices in GenoDive v2.0b23 (Meirmans
and Van Tienderen 2004). A permutation test with 1000 iterations
was performed to evaluate the significance of FST scores. Population
Microsatellite Data Analyses
genetic structure across the D. gliroides distribution was assessed
Genetic Diversity by applying the Bayesian clustering approach implemented in the
We assessed evidence of genotyping errors, large allele dropouts or program STRUCTURE v.2.3.4 (Pritchard et al. 2000). STRUCTURE
null alleles in our dataset using the program MICRO-CHECKER was carried out with an admixture model and correlated allele fre-
v2.2.3 (Van Oosterhout et al. 2004). Deviations from Hardy– quencies, a length of the burn-in period of 1 000 000 steps and
Weinberg equilibrium (HWE) and evidence for disequilibrium 10 000 000 Markov Chain Monte Carlo (MCMC) repetitions after
linkage across the loci were tested in GENEPOP v.4.2 (Raymond burn-in (Napolitano et al. 2014; Li et al. 2016). STRUCTURE was
Journal of Heredity, 2019, Vol. 110, No. 6 655
run setting the probable number of genetic clusters or “K” present in heating scheme at 4 temperatures (1.0, 1.5, 3.0, 1 000 000) for a
our data from 1 to 6, with 10 iterations of each K. Later, following better search in the genealogy parameter space. The marginal likeli-
Eldridge et al. (2014), we inferred the best value of K applying on hoods of each model were estimated by mean thermodynamic inte-
the STRUCTURE output the maximum posterior probability or gration, which was used for the model comparison through the use
L[K] (Pritchard et al. 2000) and maximum delta log likelihood or of the Bayes Factor (Kass and Raftery 1995).
DK (Evanno et al. 2005) methods. Both of these algorithms were
integrated into the program STRUCTURE HARVESTER v0.6.94
(Earl and vonHoldt 2012). In addition to STRUCTURE analyses, we Results
also applied the Bayesian algorithms implemented in GENELAND Genetic Diversity
v3.1.5 (Guillot et al. 2005) to assess the number of genetic popula- MICROCHECKER did not detect the presence of genotyping errors,
tions present in our dataset and to delineate its spatial boundaries. allele dropouts, or null alleles in our data, except in the population
The latter analysis was performed based on the null allele and uncor- of Caramávida, where one null allele was detected (Drogli_04369).
related frequency model, with 5 000 000 MC iterations, 100 thin- Significant deviation from the HWE was only observed in the
Ar, allelic richness; HO, observed heterozygosity; HE, expected heterozygosity; FIS, inbreeding coefficient, P, P values for significance of FIS after a 1000 permu-
tation test. Localities with low sample sizes are not shown (Vilches Alto, Panguipulli, PNVPR, and Llanquihue).
656 Journal of Heredity, 2019, Vol. 110, No. 6
Figure 2. Genetic structure analysis of Dromiciops gliroides in STRUCTURE. The STRUCTURE output is shown for K= 2. Each vertical bar represents a different
individual. The depth of color is proportional to the probability of belonging to the “Northern cluster” (dark gray) or to the “Southern cluster” (light gray).
PNVPR,Parque Nacional Vicente Pérez Rosales. The arrow indicates the limit between the mainland and Chiloé Island.
the northernmost sampled distribution of the “Monito del Monte” ranging from 0.016 to 0.074 (FST level interpretation followed that
on the mainland. This “Northern cluster” included individuals from of Freeland et al. 2011).
Vilches Alto, Caramávida, and Panguipulli. The second cluster, or
“Southern cluster,” grouped all individuals from the southernmost Migration Scenarios
distribution of D. gliroides, including mainland and insular local- The analysis with Migrate-n suggested that there would be asym-
ities: 1) on the mainland: Parque Nacional Vicente Pérez Rosales or metric gene flow between the Northern-Southern cluster and the
PNVPR and Parque Oncol (Valdivian coastal range); and 2) Chiloé Mainland-Chiloé Island cluster. In fact, the most likely migration
Island: Senda Darwin, Caulín, Quilar, and Llanquihue. scenario for the set of models that evaluated migration between
The GENELAND analysis recovered 4 genetic clusters (Figure 3). the Northern and Southern clusters was the “Southern-Northern”
Cluster 1 corresponded to individuals from the Vilches Alto locality, model. On the other hand, the most likely migration scenario for the
with a posterior probability of 0.8; Cluster 2 grouped together set of models that evaluated migration between the Mainland and
individuals from Chiloé Island, Parque Nacional Vicente Pérez Chiloé Island was the “Mainland-Chiloé Island” model (Table 4).
Rosales and Parque Oncol (Valdivia), with a posterior probability
of 0.9; Cluster 3 corresponded to individuals from Caramávida
(Nahuelbuta mountains), with a posterior probability of 0.9; Cluster
4 corresponded to individuals from Panguipulli, with a posterior Discussion
probability of 0.7 (Figure 3). To our knowledge, this is the first study that uses microsatellite
The FST value indicated little genetic differentiation between markers to assess geographic genetic variation (i.e., genetic diversity,
the “Northern” and “Southern” clusters (FST = 0.034, P ≤ 0.001). spatial genetic structuring and migration) in D. gliroides. Former
Moreover, when evaluating FST values between pairwise localities, the mtDNA studies assessed the secular processes of historical biogeog-
latter score showed low to moderate levels of genetic differentiation raphy and the postglacial recolonization dynamics of this species
Journal of Heredity, 2019, Vol. 110, No. 6 657
Table 4. Evaluation of the best migration models (values in bold) ac- consistent in detecting a major “Southern cluster” (39°42′–41°55′S
cording to the log-probability of data indicating“marginal likelihood” approximately). This cluster suggested a genetic connection between
(log ML), Bayes factor (BF), and model probability (MP) individuals from Chiloé Island and from the southernmost mainland
Migration models Log ML BF MP distribution of D. gliroides, including the Valdivian coastal range.
Nevertheless, with respect to the “Northern cluster” detected by
a) Between the Northern and Southern cluster STRUCTURE, the GENELAND analysis showed a more complex
Panmictic −1356.91 −222.26 5.5E-49 pattern of structuring in the northernmost distribution of the
Southern-Northern −1245.78 0.00 1 “Monito del Monte.” For example, individuals from Vilches Alto,
Northern-Southern −1270.65 −49.74 1.6E-11
Caramávida and Panguipulli (the “Northern cluster” according to
Full −3336.64 −4181.72 0
the STRUCTURE analysis) were assigned to 3 different clusters in
b) Between the Chiloé Island and Mainland
Panmictic −1408.02 −122.56 2.4E-27
GENELAND, with each individual assigned to its own cluster. A pos-
Chiloé Island-Mainland −1384.23 −74.98 5.2E-17 sible explanation for this would be that the low prior genetic infor-
Mainland-Island −1346.74 0 1 mation for the Bayesian approach of two Cluster-Localities such as
genetic groups south of 39°S that were obtained with mtDNA not show congruent patterns. Our data suggested that the current
(clades B and C sensu Himes et al. 2008). Rather, our analyses genetic structure of this species could be partially shaped by histor-
were consistent in showing that clade B (Valdivian coastal range) ical processes, such as Pleistocene migrations, as well as an isolation
and clade C (Chiloé Island plus mainland territory south of 40°S) by distance scenario. On the other hand, morphological variation
were grouped in a unique “Southern” genetic cluster by microsatel- could be determined mainly by variation in local environmental
lites. Clade B could correspond to a coastal refuge that might allow conditions (Maldonado et al. 2001, 2004). Additionally, molecular/
populations to genetically intermingle with nearby populations morphological incongruence could result, for example, from similar
after glacial retreat, thus homogenizing any differentiation due to selection pressures at different places leading to similar forms with
isolation. Thus, this is the type of genetic signal of a fast-evolving different genetic backgrounds (Pissard et al. 2008).
genetic marker that could now be detected through microsatellites.
In line with Himes’ et al. (2008) conclusions, genetic similarity be-
tween insular and mainland individuals of D. gliroides detected by Implications of Our Data for the Conservation of the
microsatellite screening could be a remnant of the historical gen- “Monito del Monte” and Recent Discussion About
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