Mitochondrial DNA Part A

DNA Mapping, Sequencing, and Analysis

ISSN: 2470-1394 (Print) 2470-1408 (Online) Journal homepage: https://www.tandfonline.com/loi/imdn21

Genetics of the Andean bear (Tremarctos ornatus;

Ursidae, Carnivora) in Ecuador: when the Andean
Cordilleras are not an Obstacle

Manuel Ruiz-García, Armando Castellanos, Jessica Yanina Arias-Vásquez &

Joseph Mark Shostell

To cite this article: Manuel Ruiz-García, Armando Castellanos, Jessica Yanina Arias-Vásquez
& Joseph Mark Shostell (2020) Genetics of the Andean bear (Tremarctos�ornatus; Ursidae,
Carnivora) in Ecuador: when the Andean Cordilleras are not an Obstacle, Mitochondrial DNA Part
A, 31:5, 190-208, DOI: 10.1080/24701394.2020.1769088

To link to this article: https://doi.org/10.1080/24701394.2020.1769088

Published online: 29 May 2020.

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MITOCHONDRIAL DNA PART A
2020, VOL. 31, NO. 5, 190–208
https://doi.org/10.1080/24701394.2020.1769088

RESEARCH ARTICLE

Genetics of the Andean bear (Tremarctos ornatus; Ursidae, Carnivora) in Ecuador:

when the Andean Cordilleras are not an Obstacle
Manuel Ruiz-Garc
ıaaa,b, ,Armando Castellanosb,c
ArmandoCastellanos
b,c
, Jessica Yanina
, Jessica Yanina Arias-V
asquezaaand
Arias-Vasquez Joseph Mark Shostell dd
and Joseph
a
Laboratorio de Gen
etica de Poblaciones Molecular-Biolog
ıa Evolutiva, Unidad de Gen
etica, Departamento de Biolog
ıa, Facultad de Ciencias,
Pontificia Universidad Javeriana, Bogot
a, Colombia; bInstituto Nacional de Biodiversidad (INABIO), Quito, Ecuador; cAndean Bear Foundation,
Quito, Ecuador; dMath, Science and Technology Department, University of Minnesota Crookston, Crookston, MN, USA

ABSTRACT ARTICLE HISTORY

One of the top carnivores in the Andean mountains is the Andean bear (Tremarctos ornatus, Ursidae), Received 9 February 2020
the only bear in South America. This is a flagship and key umbrella species in Ecuador because its con- Accepted 10 May 2020
servation has a positive impact on the conservation of many other species in the Andes. But to pre-
KEYWORDS
serve, first one must know the genetic characteristics of a species, among other things. For this, we
Andean bear; Cordilleras;
analyzed six mitochondrial genes and seven nuclear DNA microsatellites of 108 Andean bear specimens Ecuador; DNA
sampled throughout Ecuador. We adopted three strategies for analyzing the data: by Province, by microsatellites; mitochon-
Region (north vs south), and by Cordillera. Four main results were obtained. First, the mitochondrial drial genes; spatial patterns;
genetic diversity levels were elevated, but there were no differences in genetic diversity by Province or Tremarctos ornatus
by Cordillera. By Regions, southern Ecuador had higher genetic diversity levels than to northern
Ecuador. The genetic diversity for the microsatellites was only medium for the Andean bear at this
country. Second, there was clear and significant evidence of female population expansions, for the
overall sample, by Province, Region, and Cordillera. This population expansion was determined to have
occurred in the time interval of 30,000–20,000 years ago (YA), during the last phase of the Pleistocene.
We detected a population decrease to have occurred more recently, within the last 5000 years. It con-
tinued until about 300–200 YA when a population increase was again detected. Third, there were, prac-
tically, no phylogeographic pattern nor genetic differentiation among Andean bear populations in
Ecuador by Province or by Cordillera for either mitochondrial or microsatellite markers. There was a lit-
tle more genetic differentiation between northern and southern areas. Fourth, there was no trace of
significant spatial genetic structure for the Andean bear in Ecuador in agreement with the genetic dif-
ferentiation analyses. This shows that the Andean Cordilleras in this country did not present an obs-
tacle to the dispersion of this species. Therefore, all of the Andean bear specimens in Ecuador should
be treated as a unique Management Unit (MU) for conservation purposes, differently to that deter-
mined for other countries as Colombia.

Introduction origin of this specimen, as coming from the Libertad

The Andean or spectacled bear (Tremarctos ornatus, Ursidae,
Carnivora, Mammalia; other common names, Frontino, Careto
Enjaquimado, Congo, Ucumar
ı, Manaba, Mashiramo, Wii) is
currently the only living bear in South America. From a
molecular and karyotype perspective, Tremarctinae subfamily
is unique and separated from the living bears around 11 mil-
lions of years ago (MYA) (Kumar et al. 2017). The Andean
bear has a chromosome number of 2n ¼ 52, whereas the
Ursus genus has 2n ¼ 74 (Nash and O’Brien 1987; O’Brien
and Knight 1987; Goldman et al. 1989; Waits et al. 1999).
Cuvier (1825) became the first to describe an Andean bear
specimen. He had obtained it from a Chilean port and classi-
fied it as Ursus ornatus. Gervais (1855) described the genus
Tremarctos, where the Andean bear was included. As this
species is not present in Chile, Cabrera (1957) clarified the
Department, near the city of Trujillo, in Peru.
The Andean bear has been classified as Vulnerable
(A3c þ 4c) by the IUCN and in Appendix I (endangered situ-
ation) by CITES. This is a flagship (it is an icon and symbo for
the people living in the Andes because it is very charismatic)
and umbrella (its protection indirectly protects many other
species of its ecological community) species in the Andean
ecosystems in Venezuela, Colombia, Ecuador, Peru, Bolivia,
and possibly a small fraction of northern Argentina (Del
Moral and Bracho 2009; Del Moral and Lameda 2011).
This species lives in a wide variety of Andean ecosystems
(Rodr
ıguez et al. 1986, 2003) within an elevation range
between 200 and 4750 m above sea level (masl) and across
approximately 260,000 km2 (7.71%) of the 3371 million km2
of the Andes cordillera. The great variety of ecosystems
inhabited by this bear species in the Andes includes dry

CONTACT Manuel Ruiz-Garc
ıa mruizgar@yahoo.es, mruiz@javeriana.edu.co Laboratorio de Gen
etica de Poblaciones Molecular-Biolog
ıa Evolutiva, Unidad
de Gen
etica, Departamento de Biolog
ıa, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogot
a, Colombia
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

forests, moist lowland and montane forests, dry and moist
shrub lands, and high altitude shrub land, grasslands, and
paramos (Garc
ıa-Rangel 2012).
In Ecuador, the Andean bear is threatened by habitat deg-
radation, habitat fragmentation, hunting, conflict with cattle
ranchers, road construction and illegal trade. Destruction and
fragmentation of the habitat are probably the leading threats
to this species. The expansion of the agricultural frontier,
together with inadequate agricultural practices have been
the main drivers of the loss of natural ecosystems (Peyton
et al. 1998; Yerena 1998; Portillo-Quintero et al. 2012). Mining
and oil exploitation are becoming an increasing menace for
Andean ecosystems and their species, with loss of habitat
connectivity (including road building) as well as contamin-
ation of water and soil (Bebbington et al. 2008). Furthermore,
as a consequence of habitat loss, human-Andean bear con-
flicts are likely to increase with dangerous consequences for
this species (S
anchez-Mercado et al. 2008; Garc
ıa-Rangel
2012; Zukowski and Ormsby 2016). Illegal killing is also an
important threat for Andean bears. Many of these bears are
killed for retaliation against crop or livestock depredations as
well as for medical beliefs, and for commercial trade
(Orejuela and Jorgenson 1999; Peyton 1999; Yerena
et al. 2007).
Additionally, projections of effects of climate change show
a general pattern toward upslope displacement of the moun-
tain biome, suggesting that the Tropical Andes is among the
most vulnerable region to climate change (Beaumont et al.
2011; Tovar et al. 2013 ). With an increment of 0.74  C in the
last century, and with a projected increase of around 3.6–5  C
by 2100 (IPCC 2013), extensive changes in Andean habitat
areas are expected. Henceforth, all ecosystems associated
with Andean bears will probably exhibit reductions in the
near future.
To date, there have been ten population genetics studies
of this species (Ruiz-Garc
ıa 2003, 2006, 2007, 2013; Ruiz-
Garc
ıa et al. 2003, 2005, 2020a, 2020b; Viteri and Waits 2009;
Cueva et al. 2018). The major part of these studies used
nuclear microsatellite markers, and mainly focused on the
genetic diversity levels of populations of this species, popula-
tion analysis for possible Hardy-Weinberg equilibrium, geo-
graphical assignment, and the possible historical effective
population sizes for the overall range of the species as well
as for the Andean bear populations in Venezuela, Colombia,
and Ecuador. The work of Viteri and Waits (2009) was based
exclusively on Ecuadorian Andean bears, but basically
repeated and obtained the same results previously obtained
by Ruiz-Garc
ıa (2003) and Ruiz-Garc
ıa et al. (2005) in Ecuador.
Only recently, mitochondrial markers have been used to
determine the phylogeographic patterns of this species.
Cueva et al. (2018) analyzed 38 samples of hairs in a very
restricted area in the Metropolitan District of Quito at the mt
control region. Ruiz-Garc
ıa et al. (2020a) analyzed 302 speci-
mens of Andean bears at three mt genes (ND5, 12 s rRNA,
and COI) including specimens from Venezuela, Colombia,
Ecuador, Peru, and Bolivia and describing geographical pat-
terns at a macro-geographic level. More recently, Ruiz-Garc
ıa
et al. (2020b) carried out a study, using the same microsatel-
lite and mitochondrial genes as here employed, to describe
MITOCHONDRIAL DNA PART A 191
the genetic structure of the Andean bear in Colombia, where
some evidence of significant differences and spatial structure
was found. Thus, the current study is the second one to sim-
ultaneously apply mitochondrial and nuclear markers to
describe the genetic structure and spatial patterns of the
Andean bear in a country, in this case Ecuador.
Mitochondrial DNA sequences (mtDNA) are interesting for
phylogenetic and phylogeographic tasks because they
include a rapid accumulation of mutations, rapid coalescence
time, lack introns, have a high number of copies per cell, a
negligible recombination rate, and haploid inheritance (Avise
et al. 1987). Despite representing a single linked locus, selec-
tion pressures and evolutionary rates are highly heteroge-
neous across the mtDNA (Galtier et al. 2005; Nabholz et al.
2013). For all of these reasons, mtDNA sequences are more
precise in reconstructing the divergence history within spe-
cies than other molecular markers (Moore 1995). We also
analyzed seven nuclear microsatellite DNA markers to deter-
mine possible significant genetic heterogeneity among the
allele frequencies of these microsatellites. Nuclear microsatel-
lites are composed of tandem, repetitive units of 2–6 base
pairs in length (Weber and May 1989). Microsatellites are ran-
domly distributed, highly polymorphic and frequently found
inside eukaryotic nuclear genomes.
Thus, we addressed four objectives: (1) to determine the
mitochondrial genetic diversity levels for the Andean bear in
Ecuador as a whole, and in different areas of this country
(Provinces, Regions, Cordilleras); (2) to estimate the possible
existence of historical demographic changes for this species
in Ecuador as a whole, and in different areas of this country;
(3) to determine possible phylogeographic patterns and tem-
poral splits among different Andean bear populations in
Ecuador as well as to estimate the levels of genetic hetero-
geneity and gene flow among Andean bears among different
areas of Ecuador by means of mt and microsatellite markers;
and (4) to determine the possible spatial autocorrelation gen-
etic structure for both microdatellite and mitochondrial genes
in the Andean bear in Ecuador.
Methods
Study area
In Ecuador, there are three Cordilleras where the Andean
bear lives. The Western Andean Cordillera (WAC) has the
highest peaks with Chimborazo at 6267 m above the sea
level (masl), Illiniza Sur at 5263 masl, Illiniza Norte at 5116
masl, Carihuairazo at 5018 masl, and Cotacachi at 4944 masl.
The highest peaks in the Central Cordillera (Cordillera Real;
CAC) are Cotopaxi (5897 masl, the tallest active volcano on
the world), Cayambe (5790 masl), Antisana (5753 masl), El
Altar (5319 masl), Sangay (5230 masl), Tungurahua (5023
masl), and Sincholagua (4873 masl). The Eastern Andean
Cordillera (EAC) is the smallest Andean Cordillera in Ecuador
and has no outstanding peaks. The cordilleras were formed
earlier in the Cenozoic era, when the Nazca Plate subducted
underneath the South American Plate and raised the moun-
tain range. In southern Ecuador, the cordilleras are not well
separated. These three Cordilleras have connection points
192 M. RUIZ-GARCÍA ET AL.
(named ‘nudos transversales’) that split the inter-Andean
region into different ‘hoyas’. Andean bears were sampled in
these three Cordilleras.
Sampling strategy
A total of 108 Ecuadorian Andean bears were sequenced at
six mt genes (ND5, 16S rRNA, 12S rRNA, COI, COII, and control
region). A fraction of the specimens were captured to carry
out studies of radio-telemetry (field projects of A.
Castellanos). These specimens provided samples of blood
and hairs. Another fraction of the samples were pieces of
skins, teeth and bones collected in Andean communities,
where these specimens were hunted by local people.
From the 108 Ecuadorian Andean bears sequenced by
Province, the numbers were as follows: Imbabura (37 speci-
mens), Pichincha (10), Santo Domingo de Ts
achilas (2), Cotopaxi
(4), Tungurahua (3), Bolivar (1), Can ~ar (1), Sucumbios (2), Napo
(25), Azuay (10), Loja (5), Morona Santiago (3), and Zamora-
Chinchipe (5). By North vs. South, the sample sizes were 83 and
25, respectively. By Cordillera, the sample sizes were 53 (WAC),
52 (CAC), and 3 (EAC), respectively. For the microsatellite ana-
lysis, we analyzed 92 specimens (some of the specimens ana-
lyzed for the mtDNA did not work well for all seven
microsatellites and some for some specimens we spent all the
DNA on microsatellites and it was not possible to study the
mtDNA). The specimens represented the three Cordilleras were
60 from CAC, 28 from WAC, and 4 were from EAC. The follow-
ing seven Provinces were represented: Imbabura, Pichincha,
Can~ar, Napo, Azuay, Loja, and Zamora-Chinchipe. Samples from
each of these 92 specimens were genotyped for seven microsa-
tellites. The localities sampled in Ecuador for the 108 specimens
of Andean bears analyzed at the mt genes can be seen in
Figure 1. As out-group, a sample of Giant Panda (Ailuropoda
melanoleuca) was employed.
Laboratory procedures
The DNA from blood and skin was extracted using the phe-
nol-chloroform procedure (Sambrook et al. 1989). Skin sam-
ples were crushed and macerated with a pestle and mortar
and then added to a 300 ll of lysis buffer. Two microliters of
proteinase-k and 6 ll of DTT were added and the solution
digested at 56  C for two days. A solution of phenol-chloro-
form-isoamyl alcohol (25:24:1) was added to the digested
sample to obtain DNA. To extract DNA from hair we added
200 ll of 10% Chelex resin, 7 ll of DDT, and 4 ll of protein-
ase-k and digested at 56  C overnight (Walsh et al. 1991). To
prep osseous tissues for DNA extraction, bone samples were
ground to a powder. We obtained DNA from osseous tissue
by processing one to five grams of bone powder with the
Blood and Tissue Kit (Qiagen, Inc.) following the protocol pro-
vided by the manufacturer.
The conditions of PCR amplifications (Polymerase Chain
Reaction) and the primers at the six mt genes (ND5, 16S
rRNA, 12S rRNA, COI, COII, and control region) follow those in
Folmer et al. (1994), Zhang and Ryder (1993, 1994), Waits
et al. (1999), and in Yu et al. (2004). PCR reactions were
carried out in a BioRad thermocycler. The six mitochondrial
genes we studied equaled up to 2901 base pairs (bp).
All amplifications, including positive and negative controls,
were checked in 2% agarose gels. The gels were visualized in
a Hoefer UV Transilluminator. Both mtDNA strands were
sequenced directly using BigDye Terminator v3.1 (Applied
Biosystems, Inc.). We used a 377 A (ABI) automated DNA
sequencer and sequenced in both directions to ensure
sequence accuracy.
The final PCR volume reaction for nuclear microsatellites
(DNA obtained by the phenol–chloroform procedure) was 25 ll
with 2.5 ll of MgCl2 3 mM, 2.5 ll of buffer 10x, 2 ll of dNTPs
1 mM, 10 pmol of each primer (forward and reverse), 13 ll of
H2O, 2 ll of DNA and one unit of Taq Polymerase. For the PCR
with template DNA extracted from hairs, using 10% Chelex
resin, the overall volume was 50 ll, with 20 ll of DNA and two-
fold amounts of all other reactants. Seven microsatellites were
used: G1D, G10B, G10C, G10L, G10M, G10P and G10X, originally
developed for the black bear (Ursus americanus) by Paetkau and
Strobeck (1994) and Paetkau et al. (1995). The PCR reactions
were carried out in a BioRad thermocycler. The temperatures
were 95  C for 5 min, 35 cycles of 1 min at 95  C, 1 min at the
most accurate annealing temperature (52, 55 or 60  C depend-
ing on the microsatellite) and 1 min at 72  C. Following the
cycles, the sample was kept 5 min at 72  C. The amplification
products were kept at 4  C until used. The PCR amplification
products were run in denaturant 6% polyacrylamide gels and
visualized with a Hoefer SQ3 sequencer vertical camera. They
were stained with silver nitrate. The PCR reactions were
repeated three times for DNA extracted from hairs, teeth and
bones in order to confirm the genotypes obtained from these
tissues. Therefore, genotyping errors due to allelic dropout
were minimized.
The GenBank accession numbers of the different specimens
of Andean bear studied are from MH163059 to MH163166.
Phylogenetic and population genetic analyses
Mitochondrial DNA
Genetic diversity and historical demographic statistics
The following genetic diversity statistics were used for the
overall Ecuadorian sample, Province, Region (North vs.
South), and Cordillera: haplotype diversity (Hd), nucleotide
diversity (p), and h statistic by sequence. These genetic diver-
sity statistics were calculated with DNAsp v5.1 software
(Librado and Rozas 2009).
We relied on three procedures to determine a possible his-
torical female in the overall Ecuadorian Andean bear sample as
well as for Province (where sample sizes were large enough),
Region (North vs. South), and Cordillera. First, we used the Fu
and Li D and F tests (Fu and Li 1993), the Fu FS statistic (Fu
1997), the Tajima D test (Tajima 1989) and the R2 statistic
(Ramos-Onsins and Rozas 2002). A 95% confidence interval and
probabilities were obtained with 10,000 coalescence permuta-
tions. Second, the mismatch distribution (pairwise sequence dif-
ferences) was obtained following the method of Rogers and
Harpending (1992) and Rogers et al. (1996). We used the
 MITOCHONDRIAL DNA PART A 193

Andean Bears sampled

by Cordilleras

Imbabura (n=37)

Pichincha (n=10)
Sucumbíos (n=1)
Santo Domingo
(n=2)
Napo (n=25)
Cotopaxi (n=4)

Tungurahua
(n=3)

Bolívar (n=1)
Morona SanƟago
(n=3)
Cañar n=1)
Azuay (n=10)

Loja (n=5)

Western Andean Cordillera (n=53) (WAC)

Zamora Chinchipe Central Andean Cordillera (n=52) (CAC)

(n=5) Eastern Andean Cordillera (n=3) (EAC)

Figure 1. Map of Ecuador showing the geographical points (by Provinces, by North vs. South, and by Cordilleras) where 108 Andean bears (Tremarctos ornatus)
were sampled to sequence six mitochondrial genes.

raggedness rg statistic (Harpending et al. 1993; Harpending
1994) to determine the similarity between the observed and
the theoretical curves generated in this procedure. Stirling
(1993) determined that the female sexual maturity ranged from
four to seven years. Based on this, to determine the time when
possible population expansions began, we used six years for a
generation in this species. Third, a Bayesian Skyline Plot (BSP)
was obtained for the concatenated six mt genes by means of
the BEAST v1.8.1 and Tracer v1.6 software programs. The
Coalescent-Bayesian skyline option in the tree priors was
selected with four steps and a piecewise-constant skyline model
with 30,000,000 generations (the first 3,000,000 discarded as
burn-in), kappa with log Normal [1, 1.25] and Skyline population
size with uniform [0, infinite; initial value 80]. In the Tracer v1.6,
the marginal densities of temporal splits were analyzed and the
Bayesian Skyline reconstruction option was selected for the
trees log file. A stepwise (constant) Bayesian skyline variant was
selected with the maximum time as the upper 95% high poster-
ior density (HPD) and the trace of the root height as the tree
Model root Height.
Phylogenetic analyses
jModeltest v2.0 (Darriba et al. 2012) and Mega v6.05 software
(Tamura et al. 2013) were applied to determine the best
evolutionary mutation model for the sequences analyzed of
the six mt genes studied in the Andean bears. The Akaike
information criterion (AIC; Akaike 1974; Posada and Buckley
2004) and the Bayesian information criterion (BIC; Schwarz
1978) were used to determine the best evolutionary nucleo-
tide model.
A Maximum Likelihood (ML) tree was constructed by using
RAxML v7.2.6 software (Stamatakis 2006). We used the parti-
tioning scheme and best-fit models chosen by the
PartitionFinder software (Lanfear et al. 2012). This program
was used to objectively determine the optimal model of evo-
lution and partitioning scheme simultaneously (for the parti-
tions, codons 1 þ 2 combined, and codon 3, for each gene,
and including the control region). Best-fit models were
selected using Bayesian information criteria under a ‘greedy’
search scheme using a subset of models specific to RAxML.
We estimated support for nodes using the rapid-bootstrap-
ping algorithm ( f a -x option) for 1000 non-parametric
bootstrap replicates (Stamatakis et al. 2008).
To estimate possible divergence times among the haplo-
types, we constructed a Median Joining Network (MJN)
(Bandelt et al. 1999) using Network v4.6.0.1 software (Fluxus
Technology Ltd). Additionally, the q statistic (Morral et al.
1994) and its standard deviation (Saillard et al. 2000) were
estimated and transformed into years. The q statistic is
unbiased and highly independent of past demographic
194 M. RUIZ-GARCÍA ET AL.
events. This approach is named ‘borrowed molecular clocks’
and uses direct nucleotide substitution rates inferred from
other taxa (Pennington and Dick 2010). We used an evolu-
tionary rate of 1.22% per one MY, which represented one
mutation each 28,257 years for the 2,901 bp analyzed. This
evolutionary rate is that estimated by Culver et al. (2000) in
some species of carnivores for the genes herein employed.
One advantage of the MJN procedures compared to trad-
itional trees is that they explicitly allow for the co-existence
of ancestral and descendant haplotypes, whereas traditional
trees treat all sequences as terminal taxa (Posada and
Crandall 2001). Use of the MJN procedures allows us to
observe which current haplotypes began to evolve first and
to identify the more recently derived haplotypes.
Genetic heterogeneity
Mitochondrial analyses
Some statistical heterogeneity procedures (table of contin-
gency, HST, KST, KST, cST, NST and FST, Hudson et al. 1992)
were applied to the overall sample, by Province, Region
(North vs. South), and Cordillera. Some indirect gene flow
estimates were obtained assuming an infinite island model in
both cases (Wright 1965). Significance was estimated with
permutation tests using 10,000 replicates. We also estimated
the genetic heterogeneity by Province pairs, North vs. South,
and by Cordillera pairs. For this task, we used the FST statistic
with permutation tests using 10,000 replicates. All of these
heterogeneity statistics were calculated with the DNAsp v5.1
(Librado and Rozas 2009) and Arlequin v3.5.1.2 programs
(Excoffier and Lischer 2010).
Nuclear microsatellite DNA analyses
We performed genic differentiation analyses to determine if
the Ecuadorian Andean bear specimens by Provinces, North
vs. South, and by Cordilleras had significantly different signa-
tures of population differentiation using nuclear markers. This
was done for each microsatellite individually as well as all
together. Exact probability tests were applied. For this, we
relied on the Markov chain method (with Genepop v4.2.1
software), with a 10,000 dememorization number, 50 batches
and 5000 iterations per batch (Raymond and Rousset 1995).
The probability value for all the loci taken together was
obtained with Fisher’s method. Also, we analyzed genetic
heterogeneity among Province pairs and among Ecuadorian
Andean Cordillera pairs for genic differentiation with exact
probability tests with the same parameters as aforemen-
tioned. With this same software, we used the private allele
model to measure possible theoretical gene flow estimates
among the different Ecuadorian Provinces studied as well as
between North vs. South, and among the different
Ecuadorian Andean Cordilleras (Slatkin 1985; Barton and
Slatkin 1986).
Spatial Genetic statistics applied to mitochondrial gene
sequences and microsatellites
A Mantel’s test (Mantel 1967) was used to detect possible
overall relationships between a genetic matrix among the
individuals of Andean bears sampled in Ecuador (Kimura 2 P
genetic distance) and the geographic distance matrix among
the individuals analyzed for both mitochondrial and microsat-
ellite data. In this study, Mantel’s statistic was normalized
according to Smouse et al. (1986). This procedure transforms
the statistic into a correlation coefficient. Both genetic and
geographic distances were logarithmized. The probability was
estimated by means of 10,000 replicates.
A spatial autocorrelation analysis was done employing the
Ay statistic (Miller 2005) with 10 distance class (DC) for both
mitochondrial and microsatellite data. Ay can be interpreted
as the average genetic distance between pairs of individuals
that fall within a specified DC. Ay takes on a value of 0 when
all individuals within a DC are genetically identical and takes
on a value of 1 when all individuals within a DC are com-
pletely dissimilar. The probability for each DC are obtained
using 10,000 randomizations. The 10 DCs were as follows for
mitochondrial genes: 0–16 km; 16–29 km; 29–46 km;
46–54 km; 54–68 km; 68–82 km; 82–95 km; 95–109 km;
109–132 km; 132–250 km, each DC of equal number of speci-
men comparisons. The 10 DCs were as follows for the micro-
satellite data: 0–22 km; 22–43 km; 43–65 km; 65–87 km;
87–109 km; 109–130 km; 130–152 km; 152–174 km;
174–196 km; 196–218 km, each DC of equal size. The real dis-
tances among the specimens studied are higher than that
used in this analysis because we used geographic coordi-
nates that do not take into the geomorphology of the area
studied. This analysis was carried out with AIS software
(Miller 2005).
A last spatial analysis was carried out with the
Monmonier’s Algorithm Analysis (Monmonier 2010; MAA)
with the AIS software (Miller 2005). This geographical region-
alization procedure is used to detect the locations of putative
barriers to gene flow by iteratively identifying sets of con-
tiguous, large genetic distances along connectivity networks
(Dupanloup et al. 2002; Manel et al. 2003; Manni et al. 2004).
A Delaunay triangulation (Watson 1992; Brouns et al. 2003)
was used to generate the connectivity network among sam-
pling points. A graphical representation of putative ‘‘barriers’’
inferred by the algorithm is superimposed over the connect-
ivity network to detect rapid identification of important geo-
graphical features reflected by the genetic dataset. In this
case, we used this procedure to detect the five most import-
ant geographical barriers to the Andean bears in Ecuador at
the mitochondrial genes sequenced.
Results
Mitochondrial genetic diversity and demographic
trajectories for the Andean bears at Ecuador
The overall mitochondrial genetic diversity for the Andean
bear in Ecuador was high (Table 1). All the Provinces showed
high mitochondrial genetic diversity. Of those Provinces with
 MITOCHONDRIAL DNA PART A 195

Table 1. Genetic diversity and historical demographic change statistics in the total sample, in two geographical regions (northern and southern Ecuador), in 3
Ecuadorian Andean Cordilleras, and in 12 Ecuadorian Provinces for the Andean bears, Tremarctos ornatus, sampled in Ecuador at 6 mitochondrial genes.
Populations Ramon-
of Andean h Statistic by Onsins and
bear studied Haplotypic Nucleotide sequence Tajima D Fu and Li Fu and Li Fu’s FS Raggedness Rozas
in Ecuador diversity (Hd) diversity (p) (¼ Nel) (1989) D (1993) F (1993) (1997) rg (1993) R2 (2002)
Total 0.845 ± 0.033 0.0104 ± 0.0013 9.706 ± 2.648 2.37þ 4.74þ 4.51þ 44.8þ 0.0123þ 0.032þ
sample
(n ¼ 108)
North 0.818 ± 0.041 0.0082 ± 0.0021 7.014 ± 2.085 2.18þ 4.29þ 4.16þ 30.8þ 0.014þ 0.031þ
(n ¼ 83)
South 0.910 ± 0.045 0.0154 ± 0.0041 7.945 ± 2.876 1.88 2.35 2.59 6.98þ 0.023 0.091
(n ¼ 25)
Western 0.824 ± 0.050 0.0081 ± 0.0012 6.198 ± 2.025 2.14þ 3.76þ 3.79þ 16.2þ 0.014 0.041þ
Andean
Cordillera
(n ¼ 53)
Central 0.863 ± 0.045 0.0104 ± 0.0016 6.860 ± 2.208 1.93þ 2.64 2.84 18.4þ 0.012þ 0.046
Andean
Cordillera
(n ¼ 52)
Eastern 1.000 ± 0.272 0.0049 ± 0.0016 1.333 ± 1.980 – – – – – –
Andean
Cordillera
(n ¼ 3)
Azuay 0.911 ± 0.077 0.0092 ± 0.0036 3.181 ± 1.604 0.86 1.00 1.09 2.51 0.058 0.153
(n ¼ 10)
Ca~nar- 1.000 ± 0.500 0.042 ± 0.026 12.000 ± 9.491 – – – – – –
Bolivar
(n ¼ 2)
Cotopaxi 0.500 ± 0.265 0.0073 ± 0.0039 2.182 ± 1.489 – – – – – –
(n ¼ 4)
Imbabura 0.755 ± 0.072 0.0064 ± 0.0012 3.833 ± 1.444 1.76 2.43 2.61 8.26þ 0.033 0.057
(n ¼ 37)
Loja (n ¼ 5) 1.000 ± 0.126 0.0204 ± 0.0048 6.240 ± 3.446 – – – – – –
Morona- 1.000 ± 0.275 0.0073 ± 0.0026 2.000 ± 1.512 – – – – – –
Santiago
(n ¼ 5)
Napo (n ¼ 25) 0.807 ± 0.078 0.0087 ± 0.0022 4.502 ± 1.767 1.65 2.44 2.57 5.23þ 0.017þ 0.084
Pichincha 0.978 ± 0.054 0.0103 ± 0.0015 3.888 ± 1.895 1.20 1.25 1.40 5.76þ 0.165 0.088þ
(n ¼ 10)
Santo 1.000 ± 0.500 0.0112 ± 0.0054 3.000 ± 2.449 – – – – – –
Domingo
de
Ts
achilas
(n ¼ 2)
Sucumbios 1.000 ± 0.500 0.0180 ± 0.0091 6.000 ± 4.582 – – – – – –
(n ¼ 2)
Tungurahua 1.000 ± 0.272 0.0097 ± 0.0036 2.667 ± 1.919 – – – – – –
(n ¼ 3)
Zamora- 0.700 ± 0.218 0.0029 ± 0.0011 0.960 ± 0.758 – – – – – –
Chinchipe
(n ¼ 5)
Ne : effective female population size; l : mutation rate per generation. Values ± standard deviation; n: sample size; p < 0.05; þp < 0.01.

sufficiently large sample sizes to determine precise genetic
diversity estimates, Pichincha and Azuay showed the highest
levels of genetic diversity. In contrast, the Imbabura Province
had a large sample size and the lowest genetic diversity.
Nevertheless, if we take into account the magnitude of the
different sample sizes, the levels of genetic diversity were
similar for all the Provinces.
The comparison between North vs. South showed a sig-
nificantly higher mitochondrial genetic diversity level for the
southern area of Ecuador.
For the Cordilleras, there was similar genetic diversity lev-
els for the WEC and CEC (Table 1). The genetic diversity esti-
mate for EEC was less precise because of its small sample
size and therefore it is not comparable with the two other
Cordilleras. At least, WEC and CEC should be correlated with
similar evolutionary trajectories in each Cordillera due to their
similar genetic diversity levels.
The overall Ecuadorian sample showed that all the six
tests we used, including the mismatch distribution, agreed
quite well with a significant female population expansion
(Table 1 and Figure 2). For Provinces, many cases of signifi-
cant female populations were also detected. Imbabura
yielded six significant tests, including the tch distribution.
Napo showed five significant tests, including the mismatch
distribution. Azuay showed two significant tests, including
the mismatch distribution, and Pichincha also yielded two
significant tests, but not the mismatch distribution.
The six tests, including the mismatch distribution, were
significant for the North and South. This was also true for the
WEC and CEC—including the mismatch distribution for
196 M. RUIZ-GARCÍA ET AL.

Figure 2. Analysis of significant mismatch distributions (pairwise sequence differences) at six concatenated mitochondrial genes for the Andean bear (Tremarctos
ornatus) in Ecuador. (A) Overall sample; (B) Imbabura Province (northern Ecuador); (C) Napo Province (northern Central Andean Cordillera); (D) Azuay (southern
Central Andean Cordillera); (E) Northern Ecuador; (F) Southern Ecuador; (G) Ecuadorian Western Andean Cordillera; (H) Ecuadorian Central Andean Cordillera.

female population expansions. Thus, there was evidence of We estimated the time when the female population
significant traces of population expansions for the Andean expansions began for those cases where the mismatch distri-
bear across the entire territory of Ecuador. butions were significant. The beginning of the population

Figure 3. Bayesian skyline plot analysis (BSP) to determine possible demographic changes across the natural history of the Andean bear (Tremarctos ornatus) in
Ecuador for six mitochondrial genes.
expansion for the overall sample (representing all the current
Ecuadorian territory) was dated to 23,537 YA. For Provinces,
Imbabura showed a female population expansion dated
around 21,634 YA, Napo around 16,567 YA, and Azuay
around 34,799 YA. On the other hand, the Andean bear pop-
ulations from North and South also showed significant
female population expansions around 25,037 YA and 12,096
YA, respectively. Also, WEC and CEC showed significant mis-
match distributions, with population expansions around
29,434 YA and 29,011 YA, respectively. Therefore, the popula-
tion expansions in both Cordilleras occurred at practically the
same time.
The BSP analysis for the overall Ecuadorian sample (Figure
3) showed a continuous female population declination in the
last 4500 Y, during the Holocene, but especially in the last
2500 Y with a considerable population increase in the last
200–300 Y.
In whatever case, all of these temporal estimates were in
the last phase of the Pleistocene and in the Holocene, which
shows that the Andean bear colonization of the current
Ecuadorian lands, was a relatively recent process.
Phylogeographic analyses
The best nucleotide substitution model found for the overall
Ecuadorian Andean bear data analyzed was T92 þ G
(14,192.670) for BIC and TN93 þ G (12,306.944) for AIC.
The ML tree (Figure 4) showed that the most differenti-
ated specimen was one from Can ~ ar. In the remaining tree,
only five small significant clades (bootstrap percentages
higher than 50%) were found.
The first small cluster was formed by two specimens from
Gualaquiza (Morona-Santiago; CEC) (52% bootstrap). The
second small cluster was integrated by two specimens from
Tandapi (Pichincha; WEC) (51%), and the third had two speci-
mens, one from Santo Domingo (WEC) and another from
MITOCHONDRIAL DNA PART A 197
Imbabura (WEC) (51%). The fourth cluster contained one spe-
cimen from Oyacachi (Cayambe-Coca National Park; Napo;
CEC) and another near Antisana (Pichincha; CEC) (57%). In
addition, the fifth contained two specimens from Cotacachi
(Imbabura; WEC) (83%).
Therefore, the mitochondrial genes of the Ecuadorian
Andean bear have no obvious genetic structure.
The MJN procedure (Figure 5) showed that the two more
frequent haplotypes (H1 and H2) were expanded, one (H1),
for the three Ecuadorian Cordilleras, and the other (H2) for
two Cordilleras (WEC and CEC). The haplotypes 14 and 19
were also recorded in the three Ecuadorian Cordilleras. There
were many other less frequent haplotypes detected in WEC
or in CEC, but intermixed across the MJN. Henceforth, such
as it was found with the ML tree, the haplotypes detected
were intermixed independently if they were classified by
Province, North vs. South, or by Cordillera. This agrees quite
well with the absence of genetic structure of the Andean
bear in Ecuador.
This last procedure estimated the temporal split between
the two more frequent haplotypes (H1 and H2) to have
occurred around 53,651 ± 23,344 Y. Other temporal splits esti-
mated were those between H1 and the five haplotypes dir-
ectly derived from it (104,085 ± 56,774 YA), and similarly
between H2 and its six derived haplotypes (227,930 ± 65,603
YA). Additionally the star-like form of the MJN was indicative
of a population expansion for the overall sample of the
Ecuadorian Andean bears. This agrees quite well with the
data obtained with the six procedures to detect possible
demographic changes.
Genetic heterogeneity among Ecuadorian Andean bear
populations with mitochondrial sequences
The overall genetic heterogeneity for all of the Provinces
taken together for the Ecuadorian Andean bears did have
some significant statistics (Table 2). Nonetheless, some
198 M. RUIZ-GARCÍA ET AL.

Figure 4. Maximum likelihood tree (ML) with 108 Andean bears (Tremarctos ornatus) sequenced at six mitochondrial genes sampled in Ecuador. Numbers in the
nodes are bootstrap percentages. A sequence of Ailuropoda melanoleuca (Giant Panda) was used as the outgroup. Only the clusters with bootstraps higher than
50% are shown.

statistics (GST, cST, NST, and FST) offered insignificant differen-
ces. The gene flow estimates were substantially higher than 1
(Nm-GST ¼ 15.74; Nm-cST ¼ 2.15; Nm-FST and NST ¼ infinite),
which is correlated with a high gene flow among the
Andean bear populations from different Provinces across
Ecuador. Thus, there was certain overall significant genetic
heterogeneity among Ecuadorian Andean bear populations,
but they were highly connected from a genetic and repro-
ductive point of view by considerable gene flow.
We used the FST statistic to determine possible genetic dif-
ferences by Province pairs (Table 3). Thirteen out of 66 com-
parison pairs (20.63%) showed significant differences.
 MITOCHONDRIAL DNA PART A 199

Figure 5. Median Joining Network (MJN) with haplotypes found at six concatenated mitochondrial genes for the Andean bear (Tremarctos ornatus) sampled in
Ecuador. Yellow ¼ Western Andean Cordillera; blue ¼ Central Andean Cordillera; green ¼ Eastern Andean Cordillera. Small red circles indicate missing intermedi-
ate haplotypes.

Table 2. Genetic heterogeneity and gene flow statistics across 12 Provinces Azuay seems to be the more differentiated population. Thus,
from Ecuador where specimens of Andean bear, Tremarctos ornatus, were mitochondrial genetic heterogeneity was scarce among the
sampled and analyzed at six mitochondrial genes.
Ecuadorian Provinces.
Genetic heterogeneity
and gene flow statistics Values Probabilities The gene flow comparison pairs (Table 3) showed 5 out of
v2 612.39 df ¼ 394 0.0001 66 comparison pairs (7.58%) with gene flow values lower
HST 0.0393 0.0176 than 1. In contrast, we estimated 19 out of 66 comparison
KST 0.0229 0.0394
KST 0.1029 0.0005
pairs (28.79%) with infinite values of gene flow. This agrees
ZS 2625.537 0.0007 with the fact that the Ecuadorian Andean bear population is
ZS 7.6055 0.0017 basically a genetic unity.
Snn 0.2654 0.0009
cST 0.1886 0.123 The North vs. South mitochondrial genetic differentiation
NST 0.0000 0.147 was significant with the FST statistic (¼ 0.059; p ¼ 0.000001),
FST and GST 0.0000 and 0.0308 0.151 and 0.347 although it was a relatively low value. Related with this, the
Nm-cST 2.15
Nm-NST Infinite gene flow estimate was considerably high (Nm ¼ 7.94). With
Nm-FST and Nm-GST Infinite and 15.74 other statistics, such as GST (¼ 0.011) and cST (¼ 0.031), the
p < 0.05;  p < 0.01, df: degree of freedom; Nm: gene flow statistics. genetic differentiation was not significant, with elevated esti-
mates of gene flow (Nm ¼ 46.37 and 15.50, respectively).
The overall genetic heterogeneity for the three Ecuadorian
However, when the Benjamini-Hochberg’s correction was Andean Cordilleras did not show any case of significant pair
applied, only 6.1% of the Province pairs were significant (4/ differentiation. Due to this very low genetic differentiation, all
66). They were the cases of Imbabura vs. Azuay (p ¼ 0.00001), of the gene flow estimates were extremely high (Nm-GST ¼
Imbabura vs. Morona Santiago (p ¼ 0.00001), Napo vs. Azuay 19.78; Nm-cST ¼ 36.68; Nm-FST ¼ infinite). All of the Cordillera
(p ¼ 0.000001), and Pichincha vs. Azuay (p ¼ 0.000001). Thus, pair comparisons with the FST statistic were null. This puts
 200 M. RUIZ-GARCÍA ET AL.

forward that the Andean Cordilleras in Ecuador exerted no

Table 3. FST test pairs (below main diagonal) and gene flow estimates (Nm) (above main diagonal) among 12 Ecuadorian Provinces where the Andean bear, Tremarctos ornatus, were sampled and analyzed at six mito-

Cotopaxi

Infinite
Infinite

Infinite

Infinite
143.64
16.77

19.17
influence on the ability to generate genetic heterogeneity in

4.00

5.32
1.27
2.00
the Ecuadorian Andean bear populations.
Of the three sampling schemes (Provinces, North vs.
Zamora-Chinchipe

South, and Cordilleras), the North vs. South yielded slightly

more genetic heterogeneity. Therefore, for the mitochondrial
markers, the Andean bears constituted one unique popula-
Infinite
Infinite
40.00

60.00

21.13

0.025
tion in Ecuador.
1.25

5.90
1.57

0.67
1.23
Morona-Santiago

0.291
0.200
1.32
1.93
4.85
2.14
2.62
2.51
2.63
67.50
2.40

Genetic heterogeneity among Ecuadorian Andean bear

populations with nuclear microsatellites
Similar to the mtDNA, analysis of the nuclear DNA microsatel-
lites, indicated a low degree of significant genetic heterogen-
nar-Bolivar

eity. By Province, for the exact probability test with genic

differentiation (Table 4), three out of the seven microsatellites
Infinite

Infinite

0.172
0.427
0.283

(G10M: p ¼ 0.000412; G10P: p ¼ 0.0464; G10X: p ¼ 0.000566) as

0.26
0.40
3.05

3.35
0.70

0.57
Ca~

well as the overall seven loci (v2 ¼ infinite, 14 df,

p ¼ 0.000002) showed significant heterogeneity. With the
0.467
Azuay

0.007
0.023
0.086

Benjamini-Hochberg’s correction, only two out of the seven

2.66
3.13
3.75
2.13
7.59
3.38
5.91

microsatellites (G10M and G10X) and the set of seven micro-

satellites were significant. The level of gene flow estimated
Santo Domingo

using microsatellites for all the Ecuadorian Provinces studied

was high (Nm ¼ 4.16), which agrees quite well with that
Infinite
Infinite
Infinite
infinite
20.74

0.078
0.000
0.160
0.242
0.000

observed for the mt markers.

3.86

The exact probability tests for genic differentiation with

the Andean bears by Province pairs can be seen in Table 5.
Pichincha

0.120

There was eight out of 21 comparisons (38.09%) as significant

Infinite

0.166
11.11

0.000

0.416

0.078
0.004
4.50
8.10

9.19

(although with the Benjamini-Hochberg’s correction, this per-

centage was notably reduced; 2/21 ¼ 9.52%). The most out-
standing Province comparison difference pairs were Loja vs.
Tungurahua

Azuay (p ¼ 0.0012), and Loja vs. Napo (p ¼ 0.00019). Such as

Infinite
Infinite
Infinite
Infinite

p < 0.00011 (Benjamini-Hochberg’s correction).

0.000
0.000
0.062
0.129
0.160
0.008
0.000

it was also found for mtDNA, there was scarce genetic differ-
entiation among populations of Andean bear throughout the
Ecuadorian Provinces where this species lives.
Sucumbios

We did not detect evident genetic differentiation by

Infinite

0.000
0.052
0.000
0.189
0.000
0.189
0.286
0.111
1.31
3.06

Cordillera with the exact probability test with genic differenti-

ation (Table 6). Two out of the seven microsatellites (G10M:
p ¼ 0.0084; G10P: p ¼ 0.0261) as well as the overall seven loci
0.118
Loja

0.000
0.000
0.043
0.000

0.141
0.093
0.012
0.000
2.95
7.26

(v2 ¼ 29.771, 14 df, p ¼ 0.0082) showed significant hetero-

geneity. With the Benjamini-Hochberg’s correction, however,
neither microsatellite nor the set of the seven microsatellites
0.138
Infinite

0.058

0.555
0.206
Napo

0.064
0.140
0.000

0.024

0.000
0.000

showed significant genetic heterogeneity.

With microsatellites, we estimated an elevated level of
p < 0.05 (Benjamini-Hochberg’s correction), 

gene flow for all three Ecuadorian Andean Cordilleras

Imbabura

0.158

0.274
0.145

0.099

0.657

(Nm ¼ 4.09). This was lower than what was observed for the
0.000

0.277
0.000

0.115

0.000
0.029

mtDNA. However, it is a high gene flow for conserving gen-

etic connectivity among these Andean bear populations.
Ecuadorian Provinces analyzed

The exact probability tests for genic differentiation by cor-

dillera pairs showed one case of significant heterogeneity
(WAC vs. CAC, p ¼ 0.0177). Nevertheless, if we applied the
for the Andean bear

Benjamini-Hochberg’s correction, no cases of genetic differen-

Zamora-Chinchipe
Morona-Santiago
chondrial genes.

Santo Domingo

tiation were detected. Henceforth, the Andean Cordilleras did

nar-Bolivar
Tungurahua
Sucumbios

not introduce any significant genetic heterogeneity for mt

Imbabura

Pichincha

Cotopaxi

nor nuclear genes in the Andean bears that inhabit

Azuay
Napo
Loja

Ca~

that country.
 MITOCHONDRIAL DNA PART A 201

Spatial structure of the Andean bear in Ecuador
The Mantel test showed a very low non-significant correlation
between the geographic and genetic distances for this spe-
cies for both mitochondrial and microsatellite loci (r ¼ 0.044,
p ¼ 0.146, and r ¼ 0.029, p ¼ 0.242, respectively). Thus, there
was no global spatial structure for the Andean bears in
Ecuador for both mitochondrial and microsatellite data.
The spatial autocorrelation analysis with 10 DCs showed a
global non-significant correlogram for the mitochondrial
sequences (p ¼ 0.256) (Figure 6), which ratified the inexis-
tence of spatial structure of this species in Ecuador. The cor-
relogram showed a ‘crazy-quilt’ pattern without any spatial
significance (Sokal and Oden 1978). Only the 5 DC (54–68 km;
p ¼ 0.0246) and the 7 DC (82–95 km; p ¼ 0.029) showed sig-
nificant negative autocorrelation. The overall spatial autocor-
relation analysis with microsatellites was also non-significant
(p ¼ 0.752; Figure 7). Therefore, both mitochondrial and
nuclear loci showed the non existence of spatial structure for
the Andean bear in Ecuador.
Although the spatial structure was minimal, we applied a
MAA for detecting which areas presented some minimal spa-
tial differences for the mitochondrial dataset (Figure 8). The
first geographical point with some minimal gene flow restric-
tion was the area of Cosanga, Napo (blue barrier; CAC). The
second point was the area of Oyacachi in the Cayambe-Coca
National Park in Napo (green point; CAC). The third point cor-
responded to Azuay (bluish green barrier; CAC), and the
fourth gene flow restriction point (lilac barrier) was the area
of Bolivar-Can~ar (WAC).
In general, the main message of these last results is the
absence of spatial genetic structure for the Andean bear in
Ecuador, at least, in mitochondrial genes.
Discussion
Genetic diversity
Mitochondrial genetic diversity was elevated for the
Ecuadorian Andean bear population. This level was similar in
those areas where the sample sizes were sufficiently large.
Also, this genetic diversity was similar in the three
Ecuadorian Andean Cordilleras analyzed. The values obtained
for the overall sample in Ecuador of 108 Andean bears (43
haplotypes, Hd ¼ 0.845 ± 0.033, and p ¼ 0.0104 ± 0.0013) were
very similar to that detected for a sample of 115 Andean
bears in Colombia (41 haplotypes, Hd ¼ 0.895 ± 0.018;
p ¼ 0.0129 ± 0.0015; Ruiz-Garc
ıa et al. 2020b), although the
extension of the Colombian territory studied is considerably
higher than the Ecuadorian one.
Cueva et al. (2018) claimed to have found low levels of
genetic diversity, based on haplotype (H ¼ 0.705 ± 0.037) and
nucleotide (p ¼ 0.00197 ± 0.00156) diversity statistics, and low
pairwise genetic distances between haplotypes for the 38
specimens of Andean bear studied near to Quito for the D-
loop region. They concluded the need to establish or revise
the current conservation strategies in the region because of
their results. Nonetheless, with our current results, we believe
that these results were misinterpreted by Cueva et al. (2018).
The question is that they analyzed a very local population in
a very restricted area of Ecuador and they had not a more
global view of the population genetic characteristics of the

Table 4. Genic differentiation among the Andean bears from seven Ecuadorian Table 6. Genic differentiation among the Andean bears from three Ecuadorian
Provinces using seven nuclear DNA microsatellites (GID, G10B, G10C, G10L, Cordilleras using seven nuclear DNA microsatellites (GID, G10B, G10C, G10L,
G10M, G10P, and G10X) with exact probability tests. G10M, G10P, and G10X) with exact probability tests.
Locus p-Value SE Locus p-Value SE
GID 0.05442 NS 0.00164 GID 0.75293 NS 0.00177
G10B 0.15676 NS 0.00495 G10B 0.06206 NS 0.00222
G10C 0.18049 NS 0.00206 G10C 0.30625 NS 0.00179
G10L 0.25504 NS 0.00800 G10L 0.95545 NS 0.00153
G10M 0.000412 0.00031 G10M 0.008402 0.00064
G10P 0.046434 0.00472 G10P 0.026098 0.00167
G10X 0.000566  0.00016 G10X 0.11441 NS 0.00291
All loci taken together 0.000002 – All loci taken together 0.00820 –
(v2 ¼ 52.778; 14 degree of freedom) (v2 ¼ 29.771; 14 degree of freedom)
p < 0.05; p < 0.0001, NS: No significant; p-value: Probability value; SE: p < 0.05; p < 0.01, NS: No significant; p-value: Probability value; SE:
Standard Error. Standard Error.

Table 5. Exact probability tests among seven Ecuadorian Province pairs using seven nuclear DNA microsatellites (GID, G10B, G10C, G10L, G10M, G10P, and G10X)
for the Andean bear, Tremarctos ornatus.
Microsatellite comparisons
among Andean bears in
diverse Ecuadorian Provinces Azuay Pichincha Imbabura Loja Zamora-Chinchipe Napo ~ar
Can
Azuay
Pichincha 0.45285
Imbabura 0.39211 0.39987
Loja 0.00125 0.03417 0.01281
Zamora-Chinchipe 0.04929 0.16918 0.08165 0.02897
Napo 0.00892 0.00756 0.12434 0.00019 0.19839
Ca~nar 0.83817 0.78556 0.88315 0.69605 0.48449 0.34083
p < 0.05, p < 0.0024 (Benjamini-Hochberg’s correction).
202 M. RUIZ-GARCÍA ET AL.

Spatial Autocorrelation Analysis Results

0.011
0.011
0.01
0.01
0.009
0.009
0.008
0.008
0.007
0.007
0.006
0.006
Ay

0.005
0.005
0.004
0.004
0.003
0.003
0.002
0.002
0.001
0.001
0.000
0
0 20 40 60 80 100 120 140 160 180 200 220 240
Geographical Distance
Figure 6. Spatial autocorrelation analysis with ten distance classes (DC) for 108 Andean bears (Tremarctos ornatus) sampled in Ecuador and sequenced at six mito-
chondrial genes.

Spatial Autocorrelation Analysis Results

0.75

0.7

0.65

0.6

0.55

0.5

0.45

0.4
Ay

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
0 20 40 60 80 100 120 140 160 180 200 220
Geographical Distance
Figure 7. Spatial autocorrelation analysis with ten distance classes (DC) for 92 Andean bears (Tremarctos ornatus) sampled in Ecuador and analyzed at seven nuclear
microsatellites.
 MITOCHONDRIAL DNA PART A 203

Figure 8. Monmonier’s algorithm analysis (MAA) to detect the fourth most important geographical barriers for 108 Andean bears (Tremarctos ornatus) sampled in
Ecuador and sequenced at six mitochondrial genes. First barrier (blue) ¼ Cosanga, Napo Province (Central Andean Cordillera); second barrier (green point) ¼
Oyacachi, Cayambe-Coca National Park, Napo Province (Central Andean Cordillera); third barrier (green-bluish) ¼ Azuay Province (Central Andean Cordillera); fourth
~ar Provinces (Western Andean Cordillera).
barrier (lilac) ¼ Bolivar-Can

Andean bear across all Ecuador. This species has not low
mitochondrial genetic diversity levels at this country as they
claimed but, effectively, the degree of differentiation among
the haplotypes found is very low (in this result agrees quite
well both works) because all the Andean bear sampled in
Ecuador came from an unique genetic source and the colon-
ization process of this species in the current Ecuador was
temporally very recent as we detected in the historical demo-
graphic and in the temporal split analyses. Therefore, the
scarce nucleotide differentiation among the haplotypes found
in Ecuador was not caused by recent genetic drift originated
by human habitat fragmentation or other negative human
influences, but it was caused by how and when the Andean
bear lineages colonized that small area of South America.
Thus, the mitochondrial genetic diversity is relatively high for
both populations in Ecuador and Colombia. Still, in the pre-
sent instant, habitat reduction has no effect on the genetic
diversity of this species. This is good news from a conserva-
tion point of view.
Another interesting important point is how microsatellite
diversity within the Andean bear is moderate compared to
mitochondrial genetic diversity. Here, we did not show the
levels of microsatellite genetic diversity in the Results
because this was a topic extensively commented on in Ruiz-
Garc
ıa (2013), and Ruiz-Garc
ıa et al. (2005, 2020a). For
Ecuador, the average overall microsatellite genetic diversity
was H ¼ 0.584 ± 0.188. Indeed, this value was lower that the
value obtained for the Andean bear in Colombia
204 M. RUIZ-GARCÍA ET AL.
(H ¼ 0.645 ± 0.211). The Ecuadorian value is below to those
values found in other bear species. For example, Waits et al.
(2000) showed that the genetic diversity values for several
brown bear studies across North America and Scandinavia
with the same microsatellites employed had an average of
H ¼ 0.74. Paetkau and Strobeck (1994) determined the micro-
satellite genetic diversity for two continental Canadian popu-
lations of the American black bears, to be around 0.8.
However, the genetic diversity level for microsatellites of the
Andean bear in Ecuador was considerably higher than that
estimated for the population of brown bear with the lowest
genetic diversity reported in the Kodiak Island (H ¼ 0.265)
(Paetkau et al. 1998).
A possible explanation to this paradox (high mitochondrial
genetic diversity, low or moderate microsatellite genetic
diversity) is the effect of ‘ascertainment bias’ (Ellegren et al.
1995, 1997; Amos et al. 1996), in which the species to whose
microsatellites were applied, usually shows a lower genetic
diversity than the target species. This effect cannot be dis-
carded because the microsatellites used in this study for the
Andean bear were designed for the American black bear and
the split for the common ancestor of the two species has
been estimated to have occurred around 11 MYA (Kumar
et al. 2017). In order to resolve this question, it is necessary
to design specific microsatellites for the Andean bear.
Genetic differentiation and spatial structure for the
Andean bear in Ecuador
As we have shown, genetic differentiation within the Andean
bear in Ecuador by Province and by Cordillera was practically
nonexistent, showing that the Ecuadorian Andean bear speci-
mens basically conformed one genetic homogeneous popula-
tion. The phylogeographic analyses correlated well with the
genetic differentiation analyses for both mitochondrial and
nuclear genes showing weak genetic structure in the Andean
bear in Ecuador. The spatial genetic analysis confirmed the
existence of a unique population of Andean bear in Ecuador.
Therefore, the entire Ecuadorian Andean bear population
should be treated as a Management Unit (MU; Avise 2000),
meanwhile in Colombia, at least, three MUs should be
defined (one in the Antioquia Department, one in the Norte
de Santander Department, and another for the rest of
Colombia). Thus, the conservation policies of the Andean
bear in Ecuador should be less complex than in Colombia.
The low genetic differentiation degree and the lack of sig-
nificant spatial structure for the Andean bear in Ecuador can
be explained by two non-mutually exclusive events. First,
gene flow estimates were elevated for both mtDNA and
microsatellites. Following Wright (1943), a gene flow estimate
of one is enough to assure reproductive and genetic con-
nectivity among populations. Among Provinces, mt genes
revealed values of gene flow ranging from 2.15 to infinity
and microsatellites estimated a value of 4.16. Among the
Cordilleras, the values were also considerably high with 20 to
infinity for mtDNA and around 4.09 for microsatellites. For
North-South, this sampling scheme offered gene flow esti-
mates ranging from 8 to 46 (due to small sample size, no
microsatellite estimate was completed). These estimates are
even higher than that detected in Colombia (Ruiz-Garc
ıa
et al. 2020b). By Department in Colombia, the values ranged
from 2.56–6.50 for mtDNA and 0.97 for microsatellites, whilst
by Cordillera the estimate was 5.34–21.53 for the mtDNA and
2.01 for microsatellites. The lower gene flow estimates in
Colombia relative to Ecuador can be related with the fact
that the distance and complexity of the Colombian Andean
Cordilleras is considerably higher than in Ecuador.
Furthermore, some Colombian Andean bear populations are
in a peripatric geographical distribution and were isolated
during certain time with some Pleistocene climatic changes.
In contrast, this did not occur in the Ecuadorian territory.
Second, the high gene flow estimates in Ecuador should
agree quite well with an explosive and very recent rapid col-
onization in the territory of this country with no time for dif-
ferentiation, whereas in Colombia this colonization was not
as rapid and/or as recent. The differential action of these two
events in Ecuador and in Colombia is a logical explanation as
to why no spatial structure was found in Ecuador, whilst a
considerable significant spatial structure existed in Colombia
for this species.
Cueva et al. (2018), according to the network they
obtained, detected two haplotypes at greater frequencies
south of the Guayllabamba River, and other two haplotypes,
which share a common with the other two haplotypes, were
more frequent north of the river, with one of them being
found exclusively in the northern zone. They concluded that
this river should be a barrier for the dispersion of the
Andean bear near to Quito. We believe that this result was
not representative of the real dispersion capacity of the
Andean bear and it is a consequence of the small sample
size and the very limited area they analyzed.
Where and when the Andean bear originated
One question of noteworthy importance is where and when
the Andean bear originated. The traditional view has been a
follows. After the Great American Biotic Interchange (GABI),
during the last Pliocene, or the beginning of the Pleistocene
(3–1.8 MYA), one migration of North American bears colon-
ized South America giving rise to Arctotherium (South
American short-faced bears). This branch probably lasted
until the Holocene. A second immigration event should have
given rise to Tremarctos in South America. The most related
fossil, to the current Andean bear, is T. floridianus. It inhab-
ited North America from the late Pliocene (2 MYA) until
about 11,000 years ago. About 14,000 YA, this original
Tremarctos in North America is believed to migrate into
South America (Yerena 1987). The absence of Andean bear
fossils from the Pleistocene has been traditionally explained
by the recent arrival of this species to South America around
the end of the Pleistocene (Soibelzon 2004; Soibelzon and
Prevosti 2013).
Nevertheless, some studies have put in doubt this hypoth-
esis in the last years. Mitchell et al. (2016), from mtDNA
extracted from a fossil of Arctotherium, showed that this fossil
was more closely related to the extant Andean bear than to

the North American short-faced bears. . This study revealed a
major relationship of the Andean bear with the South
American short-faced bears. This implies that the Andean
bear originated ‘in situ’ in South America from a common
ancestor with Arctotherium. The findings of Ruiz-Garc
ıa et al.
(2020a) that the southern Peruvian and Bolivian Andean bear
population could be older than the northern Andean bear
population as well as the southern Andean bear population
with a genetic diversity level higher than in the northern
Andean population. This agrees quite well with Mitchell et al.
(2016) because it means that the current Andean bear spe-
cies may have appeared in an area of the current middle-
southern Peru or northern Bolivia and therefore the species
was originated ‘in situ’ in South America. In relationship with
this, the split between southern and northern Andean bear
populations was estimated to have occurred 500,000–600,000
YA, which supports that the Andean bear did not recently
arrive to South America only 14,000 YA as the traditional
hypothesis mentioned.
The current Ecuadorian data did not also completely
resolve the question. The fact that the southern Ecuadorian
Andean bear populations yielded higher genetic diversity lev-
els than the northern Ecuadorian Andean bear populations is
in agreement with the new hypothesis that the Andean bear
originated ‘in situ’ in South America and migrated from south
to north. There is also the fact of two main haplotypes split-
ting 200,000–100,000 YA with reference to the haplotypes
that they generated in the current Ecuador. This supports an
older presence of the Andean bear in South America, much
earlier than that claimed by the traditional hypothesis.
However, two other points should favor the traditional
hypothesis. On one hand, the times of population expansions
were very similar in Ecuador and in Colombia. In fact, some
Colombian estimates are slightly older than those in Ecuador.
On the other hand, if the southern Andean bear area is older,
then the population expansions should be older in these
populations than in the Andean bear populations of the
northern Andean area. However, this was not the case in
Ecuador. The northern Ecuadorian Andean bear population
showed a population expansion estimate dated to 25,000 YA,
whereas the southern population expansion was dated to
12,000 YA (although the most southern Province where the
analysis was done, Azuay, showed the oldest population
expansion, 35,000 YA). Thus, the current data do not permit
a clear discrimination between the hypotheses. However, a
study similar to the current one in Ecuador and Colombia,
but in Peru and Bolivia, could favor one of these
two hypotheses.
Temporal haplotype diversification and
population expansion
We detected a strong female population expansion for the
Andean bear in Ecuador. This did not agree with that
reported by Cueva et al. (2018). These authors showed the
results of the Tajima’s D (p ¼ 0.87) and the Fu’s Fs (p ¼ 0.59)
tests, which did not detect any significant population change
in the Andean bear they studied. Undoubtedly, these results
MITOCHONDRIAL DNA PART A 205
were the product of a small sample size in a very restricted
geographical point and they were not representative for the
overall area of Ecuador.
The estimates of the temporal splits between the two
more important mt haplotypes found in Ecuador ranged
from 228,000 and 104,000 YA. This period coincided with the
last phase of the Bonaerense (Ameghino 1889). This was a
period between 0.5 and 0.13 MYA. It was characterized by a
great increase of mammals of Holartic origin in South
America (deer and Muridae rodents) and soils supportive of
varied flora. Therefore, this was a period of a certain climatic
stability with a warm and humid environment. The last large
Pleistocene glacial period began at the end of this period,
which could be related to the haplotype diversification
detected in the Andean bear in Ecuador. The last cold period
began approximately 116,000 YA and, around 70,000 YA,
large glaciers (Nevados) were present in the northern Andes,
coinciding with a cold peak during this glacial period
(Wijmstra and Van Der Hammen 1974). Therefore, the begin-
ning of the haplotype fragmentation in the Andean bear in
Ecuador (or proceeding from other areas) occurred before
the beginning of the last large glacial period. Taking into
consideration all of the Ecuadorian territory, the process of
female population expansion with mtDNA was dated to have
occurred around 24,000 YA, exactly the same estimate for
Colombia obtained by Ruiz-Garc
ıa et al. (2020b). These esti-
mates agree quite well with that previously obtained with
microsatellites. Ruiz-Garc
ıa (2003) determined a temporal split
among the Andean bear populations of Venezuela, Colombia
and Ecuador to have occurred around 15,000–24,900 YA. He
also determined a temporal split of these populations to
have occurred around 24,200 YA with another procedure.
Van Der Hammen (1985) determined a retreat of glaciers in
Colombia beginning an estimated 35,000 YA and ending
25,000 YA. The overall Ecuadorian Andean bear population
expansion should be related with that glacial retreat period.
In fact, the population expansions detected in Azuay (35,000
YA) and in the two main Ecuadorian Andean Cordilleras
(29,000 YA) are inside this period of glacial retreat in the
Andes. Nevertheless, the population expansions detected in
Imbabura (22,000 YA) or in Napo (17,000 YA) coincided with
the strongest cold peak of the last Pleistocene glaciation
(25,000–14,000 YA). The average temperature in the northern
Andes dropped 5–8  C relative to current temperatures.
Precipitation levels were 30% lower than today and it turned
to snow at 4000 masl. In the current Savannah of Bogot
a
(2600 masl), there were glaciers and the climatic conditions
were analogous to the current climate at 4200 masl (Dollfus
1999). This was a period of massive mammal extinctions in
North America and Eurasia (Lorenzen et al. 2011). However,
this seems not to have affected the Andean bear in certain
areas of Ecuador and in Colombia (Ruiz-Garc
ıa et al. 2020b).
The BSP detected a continuous Andean bear population
decrease in the last 4500 Y and a sudden population expan-
sion in the last 200–300 Y in Ecuador and also in Colombia
(Ruiz-Garc
ıa et al. 2020b). The Holocene, or the Platense
period (since 10,000 YA until the XVI century), was character-
ized by the temperatures, which increased with a maximum
at 6000 YA. In the first part of the Holocene (9500–6000 YA),
206 M. RUIZ-GARCÍA ET AL.
there was an increase in temperatures and precipitation
(rain), with an optimum climaticum ranging from 7000 to
3000 YA (Kuhry 1988). Around 5000–4500 YA, the level of
Titicaca Lake increased and around the X century AC, the
temperature increased (Dollfus 1999). Paradoxically, although
the climate was more hospitable with a major expansion of
forests, the Andean bear population declined. Maybe this
population reduction is related to human hunting and not
with climatic changes.
The Little Glacial Age (1600–1850 AC) is the most recent
glacier advance. Coinciding with this cooling of the environ-
ment, European settlers arrived to the Andes. With the arrival
of the Spaniards to the Andes, there was a massive introduc-
tion of cattle (bovine, ovine, equine, mule, and swine) as well
as a considerable extension of maize and fruit crops in many
Andean regions (Rostworowski 1981). However, in the last
200–300 Y, the Andean bear population increased in
Ecuador. Again paradoxically, with a colder climate and with
the arrival of the Spaniards and their exploitation of the
environment, the Ecuadorian Andean bear population
increased. There are at least two possible complementary
explanations to this apparent paradox. One, the advances of
glaciers and the concomitant retreat of Andean forests forced
the bears to expand into new areas. Second, the introduction
of cattle and increase of crops by the Spaniards augmented
the quantity of available food and possibly led to an increase
in size of the Andean bear population in Ecuador over the
last centuries. The clear reduction of habitat surface or the
population size by fragmentation or hunting should not still
affect the mitochondrial genome of this species. However,
nuclear markers (as microsatellites) should be more sensitive
to current processes (mitochondrial DNA reconstructed better
the oldest colonization process, meanwhile nuclear DNA
should reconstruct better more recent processes affecting the
genome of the Andean bears).
Looking to the future, it would be valuable to consider
micro-geographic analyses with microsatellites, or other
nuclear markers, of Andean bears in different geographical
regions within Ecuador to estimate current gene flow of
nearby populations.
Acknowledgments
The authors wish to express their gratitude to the Academic Vicerectory
of the Pontificia Universidad Javeriana at Bogot
a (Colombia) and to
Corponarin~o (Aida Delgado, Diana Ocan ~a Saltikova) at Pasto (Narin
~o,
Colombia) for providing monetary resources to carry out this research.
Thanks go also to Termas Papallacta (Ecuador) and to Zoo Conservation
Outreach Group (Kansas City Zoo) as well as to Melchor Ascanta, David
Jackson, and Rodrigo Ascanta for their help to obtain Andean bear sam-
ples across Ecuador.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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