Folia Primatologica 2019 Lagothrix Lagotricha Tschudii

Original Research Article
Folia Primatol Received: April 19, 2018

DOI: 10.1159/000497251 Accepted after revision: January 24, 2019
Published online: May 10, 2019
First Molecular Phylogenetic Analysis of the

Lagothrix Taxon Living in Southern Peru
and Northern Bolivia: Lagothrix lagothricha
tschudii (Atelidae, Primates), a New
Subspecies
Manuel Ruiz-García a Aymara Albino a Myreya Pinedo-Castro a
Horacio Zeballos b, c Aurita Bello d Norberto Leguizamon d
Joseph Mark Shostell e
a Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética,
Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana,
Bogotá DC, Colombia; b Museo de Historia Natural de la Universidad Nacional de San
Agustín de Arequipa (MUSA), Arequipa, Peru; c Instituto de Ciencias de la Naturaleza,
Territorio y Energías Renovables, Pontificia Universidad Católica del Perú (PUCP), Lima,
Peru; d Secretaria Distrital del Ambiente (SDA), Bogotá DC, Colombia; e Math, Science and
Technology Department, University of Minnesota Crookston, Crookston, MN, USA
Keywords
Lagothrix lagothricha tschudii · Mitochondrial genes · Phylogenetic analyses ·
Andean mountains · Pleistocene · Peru
Abstract
We sequenced mitochondrial COI and COII genes (1,377 base pairs) of 166 woolly
monkeys (Lagothrix) to determine the phylogenetic relationships of tschudii in reference
to the other taxa within the genus Lagothrix, to provide the first genetic diversity level
estimates for tschudii, and to reconstruct the historical demographic evolution of this
taxon. The sample set included, for the first time, 10 individuals of the elusive tschudii
taxon sensu Groves from southern Peru and northern Bolivia. Our phylogenetic analyses
showed that these 10 exemplars formed a statistically significant and differentiated
(molecularly and morphologically) monophyletic clade relative to other traditional sub-
species of Lagothrix lagothricha. Therefore, tschudii should be recognized as a fifth sub-
species: Lagothrix lagothricha tschudii. The temporal divergence of the ancestors of
tschudii and L. l. cana was estimated to have occurred around 1.8 million years ago
(MYA). Additionally, mitochondrial diversification within tschudii started no later than
0.96 MYA (Bayesian Inference) or 0.88 MYA (Median Joining N etwork), respectively. In
Downloaded by: M. Ruiz-García - 442282
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© 2019 S. Karger AG, Basel Manuel Ruiz-García

Unidad de Genética, Departamento de Biología
E-Mail karger@karger.com Facultad de Ciencias, Pontificia Universidad Javeriana
www.karger.com/fpr Cra 7A, No. 43–82, Bogotá DC (Colombia)
E-Mail mruizgar @ yahoo.es, mruiz @ javeriana.edu.co
contrast to the phylogenetic trees, the FST statistic and the gene flow estimates showed
L. l. lugens to be the least differentiated taxon of L. lagothricha from L. l. tschudii. Based
on genetic distances, L. l. tschudii had the smallest average genetic distance from the
other subspecies of L. lagothricha. It was also the taxon within L. lagothricha that had the
smallest genetic distance from L. flavicauda. It should be related to L. l. tschudii as the
first original taxon in L. lagothricha. Furthermore, the Andean mountains were extreme-
ly important in the original diversification of the Lagothrix genus and in the original di-
versification of L. lagothricha. Although L. l. tschudii has the smallest geographical range
of all the taxa of L. lagothricha, its genetic diversity is even higher than in other taxa with
wider geographical ranges, such as L. l. lagothricha and L. l. cana. L. l. tschudii showed a
very slight demographic increase during the Pleistocene with a decrease of females in
the last 10,000 Y, similar to that found for L. l. lugens in a previous study.
© 2019 S. Karger AG, Basel
Introduction
The woolly monkeys are classified as part of the Lagothrix genus [Geoffroyi,
1812] and are 1 of 4 Neotropical primate genera of the Atelidae family. These pri-
mates have thick woolly fur, strong prehensile tails, and occupy a critically important
niche linked to the successful dispersal and recruitment of trees [Levi and Peres,
2013]. Rightly so, conservation biology depends on the accurate identification and
description of the species and infra-specific taxonomic categories within this genus.
Fooden [1963] determined that this genus integrated 2 species. One was Lago-
thrix flavicauda (yellow-tailed woolly monkey), an endemic species in Peru. The sec-
ond was Lagothrix lagothricha (Humboldt’s woolly monkey), distributed in Colom-
bia, Venezuela, Ecuador, Peru, Brazil, and Bolivia [Wallace and Painter, 1999].
Fooden [1963] determined 4 subspecies within L. lagothricha: (1) L. l. lugens distrib-
uted on the Eastern slope of the Eastern Colombian Cordillera northward to the
Guayabero and Apuré rivers near the border between Colombia and Venezuela.
There are also fragmented populations in the upper Magdalena River region and in
the Central Cordillera along the Magdalena River (Bolivar, Córdoba, Antioquia, and
Santander Departments). This subspecies is found 100–3,000 meters above sea level
(masl). (2) L. l. lagothricha is found from the north bank of the Amazon River, includ-
ing the north bank of the Napo and Aguarico rivers in Ecuador and Peru, to the west
side of the Negro River in Brazil. Its distribution also extends to the upper Orinoco
in Venezuela, including the northern Colombian Amazon, up to about 400 masl. (3)
L. l. poeppigii is found on the south side of the Napo and Aguarico rivers across a large
fraction of the Ecuadorian Amazon and of the Loreto and Ucayali Departments in the
Peruvian Amazon to the Juruá River in the Brazilian Amazon, reaching an elevation
range as high as 1,200 m in parts of the Ecuadorian and Peruvian Andes. (4) L. l. cana
is found on the east side of the Juruá River to the west side of the Tapajós River (Bra-
zil) as well as by the Madre de Dios River in Peru and in northern Bolivia and north-
ward along the left bank of the upper Ucayali River to the lower Pachitea River, in-
habiting a wide elevation ranging from sea level to 1,800 masl in the Andes near
Cusco. Within this last subspecies, Fooden [1963] morphologically differentiated the
animals of the Cusco Department (southern Peru) from the animals of the upper and
middle Madeira River, the upper Ucayali River, and the upper Pachitea River. He de-
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2 Folia Primatol Ruiz-García et al.

DOI: 10.1159/000497251
scribed the specimens from the Comberciato River (Cusco) as the darkest pelage se-
ries in L. l. cana. However, he did not taxonomically differentiate this population
from L. l. cana.
Groves [2001] elevated all of these L. lagothricha taxa to the status of different
species. Thus, this author considered the existence of 2 woolly monkey genera, Ore-
onax, with a unique species, O. flavicauda, and Lagothrix with 4 species: L. lugens, L.
lagothricha, L. poeppigii, and L. cana. Groves [2001], in contrast to Fooden [1963],
recognized tschudii as a differentiable taxon but as a subspecies within L. cana. He
described the individuals of tschudii (L. cana tschudii) as much darker than the other
specimens of cana (L. cana cana), with a deep blackish-gray pelage that is tinged with
red. Its limbs, tail, and head are all completely black.
Until recently, there were no published works on the molecular systematics of the
woolly monkeys [Botero et al., 2010, 2015; Ruiz-García and Pinedo-Castro, 2010;
Botero and Stevenson, 2014; Ruiz-García et al., 2014; Di Fiore et al., 2015]. The first
work analyzed the mitochondrial (mt) COII gene of 97 Lagothrix lagothricha speci-
mens, belonging to the lugens, lagothricha, poeppigii, and cana taxa [Ruiz-García and
Pinedo-Castro, 2010]. The authors documented exact geographical origins of their
samples, because they came from hunted animals or from those adopted locally as pets
by indigenous communities. This study showed that poeppigii and lugens had the high-
est levels of genetic diversity for the mt gene they sequenced. Based on these data, the
ancestor of poeppigii could have been the first to begin mitochondrial diversification
within L. lagothricha. In the case of lugens, the high levels of genetic diversity were due
to the high degree of fragmentation of the current distribution range of this taxon. In
contrast, lagothricha and cana showed the lowest genetic diversity levels. They also
detected recent hybridization events and historical introgression between some of
these taxa pairs (lagothricha and lugens; lagothricha and poeppigii; lugens and poeppi-
gii). It was the first molecular validation that these taxa are subspecies and not full spe-
cies as claimed by Groves [2001]. Following these authors, the beginning of the mito-
chondrial haplotype diversification in L. lagothricha began around 2.5 million years
ago (MYA), coinciding with the beginning of the Pleistocene.
The second work analyzed 16 samples collected from Colombian zoos [Botero
et al., 2010]. Unfortunately, as often occurs with these kinds of samples, the authors
could not determine the exact geographical origins of their exemplars, and, therefore,
their conclusions were less robust than in the previous work. Additionally, only indi-
viduals belonging to lugens and lagothricha were included in their study. The authors
sequenced 2 mitochondrial genes, analyzed the karyotypes, and provided 3 main con-
clusions. First, they determined that there were no significant differences in the vari-
ation of karyotype between lugens and lagothricha. Second, they obtained a phyloge-
netic tree with lugens and lagothricha haplotypes intermixed. These 2 conclusions
agree quite well with what was first reported by Ruiz-García and Pinedo-Castro
[2010]. Third, these authors detected a very recent split between lugens and lagothri-
cha at the beginning of the Holocene. This last conclusion was probably an artifact of
the zoo specimens in their study, since some of them were likely hybrids originating
while in captivity.
The third work [Ruiz-García et al., 2014] was the first to include mtCOI and COII
genetic data of the very scarce and endemic Peruvian L. flavicauda. These authors
studied 141 Neotropical woolly monkeys (including 8 individuals of L. flavicauda) and
showed that L. flavicauda yielded a genetic diversity of zero. Their findings included
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Lagothrix lagothricha tschudii, First Molecular Folia Primatol 3

Study DOI: 10.1159/000497251
many cases of genetic introgression and recent hybridization suggesting that all wool-
ly monkeys could be included in one unique genus Lagothrix. They divided the genus
into 2 species, L. flavicauda and L. lagothricha (the last one was divided into at least 4
subspecies in agreement with Fooden’s [1963] classification, but not with the classifi-
cation proposed by Groves [2001]). This study also provided strong evidence that
poeppigii and lagothricha were the taxa that showed the highest population expansions
in different Pleistocene periods. In contrast, lugens experienced a population declina-
tion in the last 25,000 Y. The same study also dated the temporal split between the
ancestors of L. flavicauda and L. lagothricha to have occurred around 2.4 MYA.
The next 2 studies [Botero and Stevenson, 2014; Botero et al., 2015] were very
similar. In the former, the authors analyzed 4 populations of L. l. lugens and 2 popu-
lations of L. l. lagothricha, all of them in Colombia. The analysis was at a higher micro-
geographical level than in previous studies. In the latter work, the populations and
samples were the same plus the inclusion of 2 populations of L. l. poeppigii from Ec-
uador. However, the conclusions were basically the same as those reached by Ruiz-
García and Pinedo-Castro [2010] and Ruiz-García et al. [2014]. The phenotypes of
the subspecies were not perfectly delimited. Therefore, incomplete lineage sorting,
historical introgression, and recent hybridization cases were likely underlying causes
of a lack of perfect reciprocal monophyly among these subspecies.
The last work [Di Fiore et al., 2015] used mitogenomics and Bayesian dating
analyses with different calibrations and suggested a temporal divergence of L. flavi-
cauda from other Lagothrix during the Pleistocene, around 2.1 MYA. This estimate
was very similar to that obtained by Ruiz-García et al. [2014] (2.4 MYA) with a dif-
ferent procedure. Also, similarly to Ruiz-García et al. [2014], these authors concluded
that L. flavicauda and other Lagothrix taxa fall well within the range of between-spe-
cies divergences and not as different genera, refuting the point of Groves [2001] that
flavicauda is a species within a different genus than Lagothrix (Oreonax).
Nevertheless, neither of the aforementioned molecular genetics and systematics
studies included mitochondrial gene sequences of the tschudii taxon [sensu Groves,
2001]. We present new mitochondrial sequence data from an understudied (almost
unstudied) primate taxon.
Mitochondrial genes are powerful markers for phylogenetic tasks because they
lack introns and include a rapid accumulation of mutations, rapid coalescence time,
no recombination rate, and haploid inheritance [Avise et al., 1987]. Moore [1995]
showed that mitochondrial gene trees are more precise in reconstructing the diver-
gence history among related taxa, such as in this case, than nuclear genes. In agree-
ment with this, Cummings et al. [1995] showed that mitochondrial genomes have
higher information content per base than nuclear DNA. However, some care should
be taken when using mitochondrial genes for resolving taxonomic problems because
gene trees do not necessarily correspond well with species trees [Nascimento et al.,
2015]. Furthermore, mitochondrial data show only the evolution of the female lin-
eages, and this could miss hybridization events between close taxa when males are the
gene flow vectors [“mitochondrial capture”; Burrell et al., 2009].
Taking all of this into account, there are 2 main aims of this study. (1) We plan
to determine the phylogenetic relationships of tschudii in reference to the other taxa
within the genus Lagothrix. This will be accomplished by analyzing mt genes COI
and COII, for the first time, of 10 individuals of tschudii from the Cusco Depart-
ment (southern Peru) and northern Bolivia [a population discovered in 1999 by
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DOI: 10.1159/000497251
Table 1. Geographical origins of the 25 new specimens of Lagothrix sequenced at the mtDNA
genes (COI and COII)
Taxa of Lagothrix Geographic origin n

L. lagothricha tschudii 1 Cusco Department (La Convención, Quincemil, 6
(n = 10) SN Megantoni, PN Otishi; Peru)
2 Madre de Dios Department (PN Manu; Peru) 1
3 La Paz Department (Alto Madidi, Apolobamba; Bolivia) 3
L. lagothricha lugens 1 Cundinamarca Department (Cáqueza; Colombia) 2
(n = 3) 2 Meta Department (Puerto Concordia; Colombia) 1
L. lagothricha poeppigii 1 Orellana Province (San José Payamino; Ecuador) 1
(n = 11) 2 Pastaza Province (Sarayaku; Ecuador) 5
3 Loreto Department (Pantoja, Napo River; Peru) 1
4 Loreto Department (Cerros de Orellana; Peru) 2
5 Ucayali Department (Puerto Putaya; Peru) 2
L. flavicauda (n = 1) Amazonas Department (Santiago River; Peru) 1
n = sample size.
Wallace and Painter, 1999]. (2) We plan to provide the first genetic diversity level
estimates for tschudii as well as reconstruct the historical demographic evolution of
this taxon.
Materials and Methods
We sequenced 2 mt genes from samples representing 166 woolly monkeys. Of these, 141 indi-
viduals were phenotypically, genetically, and geographically described by Ruiz-García et al. [2014].
Herein, we analyze 25 new samples, including the 10 samples of tschudii, 1 sample of L. flavicauda,
3 of L. l. lugens, and 11 of L. l. poeppigii. The origins of these new samples sequenced can be seen in
Table 1. The origins of the other 141 samples can be seen in Table 1 from Ruiz-García et al. [2014].
Broken down by taxon, we sampled 10 specimens of tschudii, 29 specimens of L. l. lugens, 30 speci-
mens of L. l. lagothricha, 58 specimens of L. l. poeppigii, 30 specimens of L. l. cana, and 9 specimens
of L. flavicauda. The outgroups used contained 5 specimens of Alouatta seniculus (1 from Leticia,
Amazon Department, Colombia; 3 from Requena, Tapiche River, Peru; and 1 from the Nanay River,
Peru), 1 specimen of Ateles chamek (Beni Department, Bolivia), 1 specimen of Brachyteles hypoxan-
thus (Brazil), and 1 specimen of Aotus azarae boliviensis (Santa Cruz Department, Bolivia).
The DNA of some tschudii individuals was extracted from hairs obtained from animals
found alive in diverse Indian communities throughout southern Andean Peru and northern An-
dean Bolivia. Another fraction of the DNA was obtained from skins of hunted individuals of
tschudii. We requested permission to collect samples (1–2 cm2) from skins that were already pres-
ent in the Indian communities. Communities were visited only once. All sample donations were
voluntary, and no financial or other incentive was offered for supplying specimens for analysis.
For more information about sample permissions, see the Acknowledgements section.
Molecular Procedures
DNA from skins was extracted using the phenol-chloroform procedure [Sambrook et al.,
1989], whereas DNA in hair was extracted with 10% Chelex resin [Walsh et al., 1991]. Primers
and PCR conditions for the mtCOI and COII genes can be found in Folmer et al. [1994], Collins
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Study DOI: 10.1159/000497251
and Dubach [2000], Ruiz-García and Pinedo-Castro [2010], and Ruiz-García et al. [2014]. The
sequences of both mt genes were concatenated (1,377 bp). These 2 mt genes were selected for
diverse reasons. The mtCOI gene has emerged as the standard barcode region for animals (www.
mammaliabol.org). Hebert et al. [2003, 2004] strongly argued in favor of using a fragment of this
mt gene as a barcoding marker. The mtCOII gene has been studied extensively in primate phylo-
genetics, including Neotropical primates, with a large data bank for this mt gene [Ashley and
Vaughn, 1995; Collins and Dubach, 2000; Plautz et al., 2009; Ruiz-García et al., 2010, 2011, 2014,
2015, 2017]. Andrews and Easteal [2000] and Ascunce et al. [2003] concluded that the mtCOII
gene could be very useful in molecular platyrrhine systematics, both at intra-generic and at intra-
specific levels. It seems that both mtCO genes have an accelerated evolution in primates. This
pattern is consistent with coadaptation between nuclear and mitochondrial genomes to meet the
aerobic demands of elevated brain activity in primate lineages, the “brain-energy” hypothesis
[Grossman et al., 2004; Uddin et al., 2008]. Additionally, both mt genes easily amplify for small
and degraded samples, as were some of the samples that we used.
The double-stranded DNA was directly sequenced in a 377A (ABI) automated DNA se-
quencer. The samples were sequenced in both directions using the BigDyeTM kit, and all the sam-
ples were repeated to ensure sequence accuracy. We deposited the sequences in GenBank. It is
possible that some of the sequences represent numts (mitochondrial DNA fragments inserted
into the nuclear genome) rather than true mtDNA [Chung and Steiper, 2008]. We note that all
amino acid translations of the obtained sequences showed initial start and terminal stop codons
and the absence of premature stop codons. Additionally, protein translation was checked to eval-
uate the possible presence of numts. All the mutations we observed were synonymous changes.
This agrees quite well with the fact that there were no numts in our sequence data.
The GenBank accession numbers of the different Lagothrix taxa and specimens analyzed
are: from BankIt2143794-1 to BankIt2143794-8 (L. l. tschudii), from BankIt2143525-1 to
BankIt2143525-11 (L. l. lugens), from BankIt2143784-1 to BankIt2143784-14 (L. l. cana),
from BankIt2143787-1 to BankIt2143787-16 (L. l. lagothricha), from BankIt2143791-1 to
BankIt2143791-44 (L. l. poeppigii), and from BankIt2143781-1 (L. flavicauda).
Data Analysis
Phylogenetics Procedures
The sequence alignments were carried out manually as well as with the DNA Alignment
program (Fluxus Technology Ltd.). The MrModeltest v2.3 software [Nylander, 2004] and the
MEGA v6.05 software [Tamura et al., 2013] were applied to determine the best evolutionary mu-
tation model. The Akaike information criteria [Akaike, 1974] were used to determine the best
evolutionary nucleotide model in the Lagothrix sequence set analyzed.
Phylogenetic trees were constructed by using 2 procedures: maximum likelihood (MLT)
and Bayesian analysis (Bayesian Inference [BI]). We constructed the MLT with RAxML v.7.2.6
software [Stamatakis, 2006]. To select the best fitting model, 50 independent iterations were run
using 3 data partitions (codon 1, codon 2, and codon 3). Finally, we used the GTR + G model
(general time reversible + gamma distributed rate variation among sites [Tavaré, 1986]) to search
for the MLT. Topologic support was estimated with 500 bootstrap replicates using GTR + G.
The BI tree was completed with the BEAST v1.8.1 program [Drummond et al., 2012]. Four
independent iterations were run using 3 data partitions (codon 1, codon 2, and codon 3) with 6
MCMC chains sampled every 10,000 generations for 30 million generations after a burn-in pe-
riod of 3 million generations. We checked for convergence using Tracer v1.6 [Rambaut et al.,
2013]. We plotted the likelihood versus generation and estimated the effective sample size (>200)
of all parameters across the 4 independent analyses to determine convergence and optimal re-
sults. The results from different runs were combined using LogCombiner v1.8.0 and TreeAnno-
tator v1.8.0 software [Rambaut and Drummond, 2013]. A Yule speciation model and a relaxed
molecular clock with an uncorrelated log-normal rate of distribution [Drummond et al., 2006]
were used. Posterior probability values provide an assessment of the degree of support of each
node on the tree. The tree was visualized in FigTree v1.4 software [Rambaut, 2012]. This BI tree
was used to estimate the time to most recent common ancestor (TMRCA) for the different nodes.
We used some priors of 16.0 ± 1 MYA (95% confidence interval: 17.64–14.36 MYA) for the split
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DOI: 10.1159/000497251
between the ancestors of Alouatta and the other Atelidae genera. We used 11 ± 1 MYA (12.82–
9.88 MYA) for the split between the ancestors of Ateles and Brachyteles + Lagothrix, and of 9.5 ±
0.5 MYA (10.56–8.77 MYA) for the divergence between the ancestors of Brachyteles and Lago-
thrix. These priors followed the results of different paleontological and genetics studies [MacFad-
den, 1990; Meireles et al., 1999; Rosenberger, 2002; Opazo et al., 2006; Wildman et al., 2009;
Perelman et al., 2011; Springer et al., 2012; Jameson Kiesling et al., 2015; Kay, 2015]. We used a
normal parametric distribution.
Following Pennington and Dick [2010], the previous BI temporal estimates belong to 1 of
2 different approaches for inferring divergence times. The first approach is based on fossil-cali-
brated DNA phylogenies. The second approach is named “borrowed molecular clocks” and uses
direct nucleotide substitution rates inferred from other taxa. For this second approach, we used
a Median Joining Network (MJN) with the help of Network v4.6.10 software from Fluxus Tech-
nology Ltd. [Bandelt et al., 1999]. The ρ statistic [Morral et al., 1994] was estimated and trans-
formed into years of divergence between the haplotypes. To determine the temporal splits, it is
necessary to estimate the mutation rate at these mtCOI and COII genes. Ruvolo et al. [1991] de-
termined a mutation rate of 0.85% per million years per lineage for Hominoidea at mtCOII. This
represents an average of 1 mutation every 199,402 years. This mutation rate was practically iden-
tical to that determined by Ruiz-García and Pinedo-Castro [2010] for Lagothrix (an average of 1
mutation every 191,000 years). Similarly, for Aotus, Ashley and Vaughn [1995] and Ruiz-García
et al. [2011] calculated an average of 1 mutation per 199,000 years at this same mitochondrial
gene. For the current work, we estimated a range from 1.034 to 0.678% per million years for the
Platyrrhini at the mtCOI. We have used an average of 0.856% per million years, which represents
an average of 1 mutation every 189,600 years. Thus, we have used an average of 1 mutation each
190,300 years for both mtCOI and COII genes. The networks are more appropriate for intraspe-
cific phylogenies (which is the current situation) than tree algorithms because they explicitly al-
low for the coexistence of ancestral and descendant haplotypes, whereas trees treat all sequences
as terminal taxa [Posada and Crandall, 2001].
Genetic Diversity and Heterogeneity Analyses

Haplotypic diversity (Hd), nucleotide diversity (π), and the θ statistic by sequence statistics
were used to determine the genetic diversity within the different woolly monkey taxa, with a spe-
cial emphasis on the taxon tschudii.
To determine the overall genetic heterogeneity within Lagothrix lagothricha, we used GST,
γST, NST, and FST statistics [Nei, 1973; Hudson et al., 1992]. Additionally, we relied on the HST,
KST, KST*, Z, Z*, and Snn tests [Hudson, 2000], and the χ2 test on the haplotypic frequencies with
permutation tests using 10,000 replicates. We also estimated the genetic heterogeneity by subspe-
cies pairs within L. lagothricha. For this task, we used 4 procedures: (1) FST and NST statistics; (2)
exact tests with Markov chains, 10,000 dememorizations parameters, 20 batches, and 5,000 itera-
tions per batch; (3) indirect gene flow estimates (Nm) from the FST statistic with a n-dimension-
al island model [Slatkin, 1985; Ruiz-García, 1993, 1994, 1997, 1999; Ruiz-García and Álvarez,
2000]; and (4) Kimura 2P genetic distances [Kimura, 1980]. These genetic diversity and genetic
heterogeneity statistics were calculated with the programs DNAsp v5.1 [Librado and Rozas, 2009]
and Arlequin v3.5.1.2 [Excoffier and Lischer, 2010].
Demographic Changes
We relied on 3 procedures to detect possible historical population changes in the tschudii
taxon: (1) We used Strobeck’s S statistic [Strobeck, 1987], 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 permutations. (2) The mismatch distribution (pairwise sequence differences) was
obtained following the method of Rogers and Harpending [1992] and Rogers et al. [1996]. We
used the raggedness rg statistic to determine the similarity between the observed and the theo-
retical curves. (3) A Bayesian skyline plot (BSP) was obtained by means of the BEAST v1.8.1 and
Tracer v1.6 software. The Coalescent-Bayesian skyline option in the tree priors was selected with
4 steps and a piecewise-constant skyline model with 30,000,000 generations (the first 3 million
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Study DOI: 10.1159/000497251
discarded as burn-in), κ with log-normal (as on the previous page) (1, 1.25) and skyline popula-
tion size with uniform distribution (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 posterior density and the trace of the root height as the
treeModel.rootHeight. To determine the time range for possible demographic changes for
tschudii, we considered that the evolution of this taxon occurred during the last 1 MY.
Results
Phylogenetic Inferences
The Akaike information criteria showed that the best nucleotide substitution
model was GTR + G (15,251.74).
Both phylogenetic trees (Fig. 1, 2) clearly showed that the 10 individuals classi-
fied as tschudii formed a monophyletic clade. Indeed, the bootstrap (MLT) for the
tschudii clade was the highest (66%) compared to other traditionally accepted L.
lagothricha subspecies. For the other subspecies, the values were 52% (cana), 48%
(lugens), 12% (lagothricha), and 6% (poeppigii), respectively. With BI, the posterior
probability was 1 for the clade of tschudii as well as for the 4 traditional subspecies of
L. lagothricha.
The MJN (Fig. 3) also showed the differentiation of tschudii and the 4 tradition-
al subspecies (with cases of introgression and recent hybridization seen in L. l. lagoth-
richa, L. l. lugens, and L. l. poeppigii). Therefore, both phylogenetic trees and the MJN
procedure showed that the tschudii taxon should be elevated to the subspecies level
within L. lagothricha: L. l. tschudii.
It is interesting to note that after the split of the ancestor of L. flavicauda, at least
for the MLT and MJN procedures, the next ancestor to diverge was the one that orig-
inated L. l. tschudii and L. l. cana. Recall that both L. flavicauda and the L. l. tschudii
are found in mountain Andean areas. This could be evidence that the current Peru-
vian Andes were the original area where diversification began within Lagothrix and
L. lagothricha. However, this was not the case with BI.
The BI temporal split determined the split between the ancestors of L. flavicauda
and L. lagothricha to have occurred around 2.68 MYA (95% confidence interval:
2.34–2.97 MYA). The split between the ancestors of L. l. poeppigii and L. l. cana + L.
l. tschudii was around 2.33 MYA, while the split between the ancestors of cana and
tschudii was estimated to have occurred around 1.8 MYA (1.63–2.63 MYA). Later,
the mitochondrial diversification within L. l. tschudii started no later than 0.96 MYA
(0.81–2.19 MYA). Some of the MJN temporal splits were similar to those obtained
with BI. The split between the ancestors of L. flavicauda and L. lagothricha was esti-
mated at 2.45 ± 0.16 MYA, similar to the previous case. The splits between the ances-
tor of L. l. tschudii and the ancestors of L. l. cana, L. l. poeppigii, L. l. lugens, and L. l.
lagothricha were 0.90 ± 0.36, 1.25 ± 0.18, 1.00 ± 0.17, and 1.29 ± 0.18 MYA, respec-
tively. The mitochondrial diversification within L. l. tschudii started no later than 0.88
± 0.23 MYA, similar to that reported in the previous analysis. For instance, L. l. poep-
pigii showed an older temporal mitochondrial diversification compared to L. l.
tschudii (1.49 ± 0.13 MYA). Both of these were older than within L. l. cana (0.55 ±
0.08 MYA) or within L. l. lagothricha (0.32 ± 0.05 MYA). In general, the BI estimates
186.84.20.212 - 5/15/2019 7:07:03 PM

DOI: 10.1159/000497251
were higher than the MJN ones, as stated in other studies [Ruiz-García et al., 2017].
Nevertheless, both procedures revealed Pleistocene mitochondrial diversification for
all the Lagothrix taxa.
Genetic Distances and Genetic Heterogeneity

Table 2 shows the Kimura 2P genetic distance between the subspecies of L.
lagothricha, L. flavicauda, and Alouatta seniculus (outgroup). The smallest genetic
distances of L. l. tschudii were from L. l. cana (1.6%) and from L. l. lagothricha (1.7%),
whilst the greatest genetic distances were from L. l. lugens (3.3%) and from L. l. poep-
pigii (2.5%). It is remarkable to note that L. l. tschudii was the taxon which had the
smallest average genetic distance from all the subspecies of L. lagothricha as well as
the smallest from L. flavicauda (2.9%). This ratified (as did the MLT and the MJN
analyses) that L. l. tschudii was closely related with the initial diversification of L.
lagothricha after the split with the ancestor of L. flavicauda. It demonstrates the im-
portance of this area of the Peruvian Andes in the evolution of the Lagothrix genus.
Simultaneously taking together all 5 subspecies of L. lagothricha, the overall het-
erogeneity was highly significant for all the statistics (Table 3). The overall gene flow
indicated by γST, NST, and FST was substantially low (Nm = 0.92, 0.71, and 0.69, re-
spectively). This is expected with well-developed subspecies. However, cases of ge-
netic introgression and recent hybridization are common among L. lagothricha sub-
species as was shown by Ruiz-García et al. [2014].
Subspecies pair comparisons, using the FST and NST statistics (Table 4), exact
probability values with Markov chains, and gene flow (Table 5, 6), showed differ-
ences from some of the previous results. All of the pair comparisons were significant.
The less differentiated subspecies of L. lagothricha relative to the tschudii taxon were
L. l. lugens (FST = 0.322) and L. l. poeppigii (FST = 0.404). In contrast, L. l. cana (FST =
0.529) and L. l. lagothricha (FST = 0.652) were the most differentiated. The gene flow
estimates for subspecies pair comparisons showed values higher than 1 for the com-
parisons between tschudii-lugens (Nm = 1.65), tschudii-poeppigii (Nm = 1.10), lugens-
poeppigii (Nm = 1.20), and lugens-lagothricha (Nm = 1.08). All of the other pair com-
parisons showed very restrictive gene flow values.
The genetic heterogeneity analyses clearly revealed that tschudii is a well differ-
entiated taxon from the other Lagothrix lagothricha taxa and not a simple pelage vari-
ant from L. l. cana as it was traditionally considered [Fooden, 1963].
Genetic Diversity
The genetic diversity of L. l. tschudii in reference to other traditional taxa of Lago-
thrix can be seen in Table 7. L. l. tschudii showed medium to high levels of genetic di-
versity (Hd = 0.956 and π = 0.0075), similar to that estimated in L. l. cana (Hd = 0.822
and π = 0.0076) but higher than in L. l. lagothricha (Hd = 0.789 and π = 0.0057) and in
Fig. 1. Maximum likelihood tree based on 2 concatenated mitochondrial genes (COI and COII)
of 166 woolly monkeys (Lagothrix). For the first time, 10 exemplars of the tschudii taxon were
molecularly analyzed. The numbers in the nodes are bootstrap percentages higher than 50%. Se-
quences of Brachyteles hypoxanthus, Ateles chamek, Alouatta seniculus, and Aotus azarae bolivi-
ensis were used as outgroups.
(For figure see next pages.)
186.84.20.212 - 5/15/2019 7:07:03 PM

Study DOI: 10.1159/000497251
Color version available online
poeppigii - Santa Rosa - Javari - Peru
poeppigii - Sarayaku 64 - Pastaza - Ecuador
poeppigii - Montalvo - Pastaza1 - Ecuador
poeppigii - Sarayaku 61- Pastaza - Ecuador
poeppigii - S. Jose Payamino 168 - Orellana - Ecuador
74 poeppigii - Sarayaku 62 - Pastaza - Ecuador
poeppigii - Shepagua 11 - Ucayali - Peru
poeppigii - Cerros de Orellana 5 - Loreto - Peru
poeppigii - Pto Portillo - Ucayali - Peru
poeppigii - Yarimaguas 9 - Peru
lugens - Southern Antioquia 809 - Colombia
poeppigii - Requena 54 - Tapiche River - Peru
poeppigii - Caballococha - Amazonas - Peru
76 poeppigii - Yaupi 123 - Morona Santiago - Ecuador
poeppigii - Gualaquiza 129 - Morona Santiago - Ecuador
poeppigii - Canelos 122 - Pastaza - Ecuador
poeppigii - Andoas Nuevo 115 - Pastaza - Ecuador
poeppigii - Pto. Amelia 124 - Peru
poeppigii - Andoas Nuevo 114 - Pastaza - Ecuador
poeppigii - Arajuno 118 - Pastaza - Ecuador
poeppigii - Contamana 144 - Peru
poeppigii - Tigre River 117 - Pastaza - Ecuador
50
poeppigii - Tigre River 119 - Pastaza - Ecuador

92 poeppigii - Pisiqui 4 - Ucayali - Peru
98 poeppigii - Pisiqui 6 - Ucayali - Peru
poeppigii - Pisiqui 7 - Ucayali - Peru
98 poeppigii - Requena 53 - Tapiche River - Peru
60
poeppigii - Nanay River - Loreto - Peru
94 poeppigii - Barranca 10 - Loreto Peru
100 poeppigii - Yurimaguas - Loreto - Peru
100 poeppigii - Barranca 9 - Loreto - Peru
lugens - Caguan - PNN Los Picachos - Caqueta - Colombia
poeppigii - Pano River 1 - Napo - Ecuador
poeppigii - Pantoja - Napo River - Peru
poeppigii - Benjamin Constans - Javari River Brazil
92 poeppigii - Tecohavi - Javari River - Brazil
poeppigii - Pano River 2 - Napo - Ecuador
poeppigii - Jeberos - Loreto - Peru
poeppigii - Yarinacocha - Ucayali - Peru
52
poeppigii -Yurimaguas 4 - Loreto - Peru
74
poeppigii - Contamana 3 - Loreto - Peru
62
poeppigii - Contamana 142 - Loreto - Peru
poeppigii - Cerros de Orellana - Loreto - Peru
60
poeppigii - Apaga - Alto Amazonas - Peru
poeppigii - Santa Maria - Napo River - Peru
poeppigii - Cononaco 2 - Pastaza - Ecuador
poeppigii - Chimbote - Amazon River - Peru
poeppigii - PN Yasuni 108 - Ecuador
poeppigii - Andoas Nuevo - Pastaza - Ecuador
lugens - Huila - Tolima 2 - Colombia
84 lagothricha - Putumayo 7 - Amazonas - Colombia
lagothricha - Pto lbacaba - Vaupes - Colombia
lagothricha - Pebas River - Vaupes - Colombia
lagothricha - Negro River 1 - Brazil
lagothricha - El Porvenir - Amazonas - Colombia
lagothricha - El Progreso - Amazonas - Colombia
76 lagothricha - Negro River 2 - Brazil
lagothricha - Negro River 3 - Brazil
lagothricha - Cotube River 30 - Amazonas - Colombia
lagothricha - MiritiParana River - Amazonas Colombia
lagothricha - Putumayo 6 - Amazonas - Colombia
lagothricha - La Libertad - Amazonas Colombia
lagothricha - Cuyabeno Sucumbios - Ecuador
lagothricha - Nueva Union - Napo River - Peru
lagothricha - Apaporis River - Colombia
lagothricha - Cotube River 34 - Amazonas - Colombia
lagothricha - Negro River C1 - Brazil
lagothricha - El Vergel 1 - Amazonas-Colombia
lagothricha - 20 de Julio - Amazonas - Colombia
lagothricha - Santa Clotilde - Napo River - Peru
lagothricha - Nuevo Jordan - Amazonas - Colombia
lagothricha - La Pedrera - Amazonas - Colombia
lagothricha - Calderon 61 - Amazonas - Colombia
lagothricha - Calderon 63 - Amazonas - Colombia
lagothricha - El Vergel 2 - Amazonas - Colombia
lagothricha - Nariño - Putumayo - Colombia
lagothricha - Araracuara - Caqueta - Colombia
92 lagothricha - Amacayacu - Amazonas - Colombia
1
(Figure continued on next page.)
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DOI: 10.1159/000497251
lagothricha - PtoRastrojo - Miriti Parana River - Colombia
lugens - North - eastern - Antioquia - Colombia
lugens - Cundinamarca 5 - Colombia
lugens - Tolima 1 - Colombia
62 lugens - Cundinamarca 1 - Colombia
94 lugens - Huila - Tolima 4 - Colombia
50 lugens - Southern Antioquia 11 - Colombia
74 82
poeppigii - Negro Urco -Napo River - Peru
78
54 lugens - Tolima - Colombia
poeppigii - Requena 5 -Tapiche River - Peru
lugens - Bolivar - Colombia
lugens - La Macarena - Meta - Colombia
lagothricha - Pto. Inirida 107 - Guainia - Colombia
lugens - Chinauta - Cundinamarca - Colombia
lugens - Cauca - Colombia
lugens - Southern Magdalena Valley - Colombia
lugens - Neiva - Huila - Colombia
lugens - Meta 25 - Colombia
86 tschudii - Cuzco 14 - Peru
tschudii - Cuzco 15 - Peru
50 tschudii - Cuzco 11 - Peru
tschudii - La Convencion - Cuzco 8 - Peru
tschudii - Apolobamba 8 - La Paz - Bolivia
tschudii - Madidi 1 - La Paz Bolivia
54
tschudii - Madidi 2 - La Paz Bolivia
66
tschudii - Manu River - Madre de Dios - Peru
64 cana - Madeira 1 - Brazil
cana - Branco 2 - Acre - Brazil
100
52
cana - Tefe 18 - Brazil
cana - Tapajos - Brazil
cana - Roosevelt - Brazil
cana - Purus2 - Brazil
cana - Humaita 33 - Brazil
cana - Manicore 39 - Brazil
cana - Manicore 44-Brazil
cana - Purus 4 - Brazil
cana - Madeira 2 - Brazil
74
cana - Novo Aripuana 35 - Brazil
72 cana - Manicore 38 - Brazil
cana - Manicore c41 - Brazil
L. flavicauda - Ulcabamba - Bagua Grande - Peru
100
L. flavicauda - Abra Patricia - Upper Mayo River - Peru
L. flavicauda - Colca - Upper Mayo River - Peru
L. flavicauda - Comunidad LaHiguera - Ulcabamba - Peru
L. flavicauda - Shipasbamba Amazonas - Peru
L flavicauda - Paitoa - San Martin Department - Peru
Brachyteles hypoxanthus - Brazil
Ateles chamek - Santa Cruz - Bolivia
100 Alouatta seniculus - Nanay River 50 - Peru
70
Alouatta seniculus - Tapiche River 48 - Peru
100 Alouatta seniculus - Tapiche River 45 - Peru
88 Alouatta seniculus - Cauca - Colombia
Aotus azarae boliviensis - Santa Cruz - Bolivia
1
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Study DOI: 10.1159/000497251
Ateles chamek - Santa Cruz - Bolivia
Brachvteles hypoxanthus - Brazil
L. flavicauda - Abra Patricia - Upper Mayo River - Peru
1,(0.78), <0.2;2.02> L. flavicauda - Ulcabamba - Bagua Grande - Peru
L. flavicauda - Colca - Upper Mayo River - Peru
L. flavicauda - Comunidad La Higuera - Ulcabamba - Peru
L. flavicauda - Santa Rosa - Amazonas Department - Peru
L. flavicauda - Shipasbamba - Amazonas Department - Peru
0.72,(1.01), <0.35;2.22> poeppigii - Santa Maria - Napo River - Peru
poeppigii - Chimbote - Amazon River - Peru
1,(1.64), <0.08;1.69>
poeppigii - Tecohavi - Javari River - Brazil
1,(10.26), <9.99;11.79> poeppigii - Benjamin Constans - Javari River - Brazil
0.78,(0.57), <0.05;0.88> poeppigii - Apaga - Alto Amazonas - Peru
0.64,(0.51), <0;0.48> poeppigii - Requena 6 - Tapiche River - Peru
1,(2.2), <1.86;2.68> poeppigii - Contamana 3 - Peru
0.87,(0.86), <0.42;1.65> poeppigii - Contamana 142 - Peru
poeppigii - Yarinacocha - Ucayali - Peru
poeppigii - Cerros de Orellana - Loreto - Peru
poeppigii - Moyobamba 143 - Peru
0.87,(0.99), <1.6;2.64> poeppigii - Pano River 2 - Napo - Ecuador
poeppigii - Caballococha - Amazonas - Peru
1,(9.45), <9.3;9.69> poeppigii - Laguna Venecia - San Martin - Peru
1,(0.37), <0.16;1.24> poeppigii - Yurimaguas 9 - Peru
1,(0.63), <0.57;1.73> poeppigii - Laguna Venecia - San Martin - Peru
0.96,(0.41), <0.01;1> poeppigii - Nanay River - Loreto - Peru
1,(0.44), <0.09;0.94> poeppigii - Pisiqui 6 - Ucayali -Peru
poeppiqii - Pisiqui 4 - Ucayali - Peru
1,(0.99), <0.23;1.53>
poeppigii - Pisiqui 7 - Ucayali - Peru
1,(1.14), <0.36;1.53> poeppigii - Andoas Nuevo 114 - Pastaza - Ecuador
poeppigii - Rio Tigre 117 - Pastaza - Ecuador
0.54,(1.82), <1.48;2.53>
0.95,(0.1), <0;0.58> poeppigii - Rio Tigre 119 - Pastaza - Ecuador
0.65,(1.43), <0.89;2.17> poeppigii - Arajuno 118 - Pastaza - Ecuador
poeppigii - Pto Amelia 124 - Peru
0.56,(0.51), <0.02;1.15>
0.99,(0.92), <0.46;1.74> poeppigii - Yaupi 123 - Morona Santiago- Ecuador
0.98,(0.55),<0.15;1.12> poeppigii - Gualaquiza 129 - Morona Santiago - Ecuador
0.6,(0.83), <0.25;1.33> poeppigii - Andoas Nuevo 115 - Pastaza - Ecuador
lugens - Caguan - PNN Los Picachos - Caqueta - Colombia
0.5,(0.68), <0.13;1.26> poeppigii - Pano River 1 - Napo - Ecuador
poeppigii - Pantoja - Napo River - Peru
poeppiqii - Puyo 111 - Ecuador
poeppigii - in9 - Moyobamba - Peru
1,(2.68), <2.34;2.97>
poeppigii - Santa Rosa - Javari - Peru
poeppigii - Reauena 5 - Tapiche River - Peru
poeppigii - Pucallpa - Peru
poeppigii - Shepagua 11 - Ucayali - Peru
poeppigii - Montalvo - Pastaza1 - Ecuador
poeppigii - Cerros de Orellana 5 - Loreto - Peru
0.94,(0.9), <0.01;1.04> poeppigii - Sarayaku 62 - Pastaza - Ecuador
poeppigii - S. Jose Pavamino 168 - Orellana - Ecuador
cana - Tapajos - Brazil
cana - Roosevelt - Brazil
cana - Branco 1- Acre - Brazil
cana - Tefe 22 - Bazil
0.93,(0.74), <0.07;1.06> cana - Humaita132 - Brazil
0.6,(0.34), <0.0;5.9>
0.94,(0.91), <0.22;1.15>
cana - Humaita131 - Brazil
0.99,(0.31), <0.04;1.82>
cana - Manicore c41 - Brazil
0.9,(0.77), <0.15;1.3>
2
(Figure continued on next page.)
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DOI: 10.1159/000497251
0.98,(0.67), <0.07;1.22> cana - Purus 4 - Brazil
1,(1.39), <1.19;2.34> cana - Tefe 29 - Brazil
1 cana - Madeira 1 - Brazil
0.9,(1.18), <1.63;2.63> cana - Manicore 42 - Brazil
1,(2.4), <2.19;2.89>
1,(0.37), <0.08;0.81> tschudii - Cuzco 15 - Peru
0.97,(0.62), <0.25;1.39> tschudii - Cuzco 11 - Peru
tschudii - Apolobamba 8 - La Paz - Bolivia
tschudii - La Convencion - Cuzco 8 - Peru
1,(0.91), <0.58;1.9>
tschudii - Madidi 1 La Paz - Bolivia
tschudii- Cuzco 1- Peru
1,(0.96), <0.81;2.19> tschudii - Madidi 2 - La Paz - Bolívia
tschudii- Manu River - Madre de Dios - Peru
lugens - North - eastern - Antioquia - Colombia
lugens - Huila -Tolima 1 - Colombia
lugens - Cauca - Colombia
lugens - Southern Magdalena Valley - Colombia
0.91,(0.95), <0.7;1.97> lugens - Cundinamarca 1 - Colombia
lugens - Neiva - Huila - Colombia
lugens - Meta 25 - Colombia
lugens - Chinauta - Cundinamarca - Colombia
lugens - La Macarena - Meta - Colombia
1,(2.07), <1.52;2.49>
lagotricha - Pto Inirida 107 - Guainia - Colombia
poeppigii - Negro Urco - Napo River - Peru
0.94,(1.12), <0.7;1.68> lugens - Tolima - Colombia
0.6,(0.71), <0.36;1.28> lugens - Southern Antioquia 47 - Colombia
1,(1.64), <0.92;1.95> lugens - Cundinamarca 5 - Colombia
1,(0.53), <0.24;0.85>
0.85,(2.4), <1.88;2.73>
lagotricha - Pto Rastroio - Miriti - Parana River - Colombia
lagotricha - Pto lbacaba - Vaupes - Colombia
1,(0.01), <0;0.95>
lagotricha - Putumayo 6 - Amazonas - Colombia
lagotricha - Santa Clotilde - Napo River - Peru
0.79,(0.72), <0.04;0.89> lagotricha - Negro River 1 - Brazil
1,(2.26), <1.39;2.45> poeppigii - PN Yasuni 108 - Ecuador
0.57,(0.78), <0.12;1.21> lagotricha - El Porvenir - Amazonas - Colombia
lagotricha - Nariño - Putumayo - Colombia
lagotricha - La Libertad - Amazonas - Colombia
lagotricha - Pebas River - Vaupes - Colombia
lagotricha - La Pedrera - Amazonas - Colombia
0.84,(1.94), <1.1;2.17> lagotricha - Putumayo 7 - Amazonas - Colombia
lagotricha - El Vergel 1 - Amazonas - Colombia
lagotricha - Cotube River 30 - Amazonas - Colombia
lagotricha - Calderon 61 - Amazonas - Colombia
lagotricha - Negro River C1 - Brazil
lagotricha - 20 de Julio - Amazonas - Colombia
lagotricha - Nuevo Jordan - Amazonas - Colombia
lagotricha - Nueva Union - Napo River - Peru
0.5,(1.87), <1;2.03> lagotricha - Cotube River 34 - Amazonas - Colombia
lagotricha - Cuyabeno - Sucumbios - Ecuador
lagotricha - Miriti-Parana River - Amazonas - Colombia
lagotricha - Apaporis River - Colombia
lagotricha - El Vergel 2 - Amazonas - Colombia
0.54,(0.22), <0.01;0.95> lagotricha - Negro River 2 - Brazil
lagotricha - Negro River 3 - Brazil
lagotricha - Calderon 63 - Amazonas - Colombia
0.96,(0.38), <0.02;0.92> lagotricha - Araracuara - Caqueta - Colombia
lagotricha - Amacayacu - Amazonas - Colombia
lagotricha - El Progreso - Amazonas - Colombia
0.76,(0.65), <0.02;1.46> Alouatta seniculus - Nanay River 50 - Peru
1,(1), <0.16;2.52> Alouatta seniculus - Tapiche River 48 - Peru
1,(4.36), <1.2;5.84> Alouatta seniculus - Tapiche River 45 - Peru
1,(1.56), <0.15;2.35> Alouatta seniculus - Cauca - Colombia
2.0
2
Fig. 2. Bayesian tree with 166 woolly monkeys (Lagothrix) studied for 2 concatenated mitochondrial genes (COI
and COII). For the first time, 10 exemplars of the tschudii taxon were molecularly analyzed. The numbers in the
nodes are posterior probabilities higher than 0.5 (first number), the median temporal split (second number), and
the interval of 95% high posterior density (in parentheses). As outgroups, sequences of Brachyteles hypoxanthus,
Ateles chamek, Alouatta seniculus, and Aotus azarae boliviensis were employed.
186.84.20.212 - 5/15/2019 7:07:03 PM

Study DOI: 10.1159/000497251
L. ﬂavicauda
L. l. tschudii
L. l. lugens L. l. cana
L. l. lagothricha
L. l. poeppigii
Fig. 3. Median Joining Network with haplotypes found at the 2 concatenated mitochondrial genes
(COI and COII) for the 166 woolly monkeys (Lagothrix) analyzed. Gray circle = Lagothrix flavicauda;
green circles = Lagothrix lagothricha tschudii; black circles = Lagothrix lagothricha cana; pink
circles = Lagothrix lagothricha lugens; blue circles = Lagothrix lagothricha lagothricha; yellow
circles = Lagothrix lagothricha poeppigii. Red circles indicate missing intermediate haplotypes.
L. flavicauda (Hd = 0 and π = 0). The genetic diversity level for tschudii was lower than
in L. l. lugens (Hd = 0.850 and π = 0.0357) and L. p. poeppigii (Hd = 0.974 and π = 0.0166).
Thus, although L. l. tschudii has the narrowest distribution range of all the taxa
of L. lagothricha, it has a considerable amount of genetic diversity, which could be
related to the original genetic diversification in Lagothrix.
Demographic Evolution in L. l. tschudii

The different statistics used to determine historical demographic change pro-
vided evidence of a slight female population expansion for L. l. tschudii. The Tajima
D, and the Ramos-Onsins and Rozas R2 statistics (p = 0.0482 and p = 0.0481, respec-
tively) showed moderately significant negative values, which agree with population
expansion. The other statistics and the mismatch distribution (Fig. 4) fell slightly shy
of the level of significance probably due to the low number of sequences analyzed.
The BSP analysis (Fig. 5) yielded a slight and constant population expansion dur-
ing the last 900,000 years, although there was some increase that began 100,000 YA
and a very slight population decrease in the last 10,000 YA (Holocene). Therefore,
there was no evidence of substantial demographic changes throughout the natural
history of L. l. tschudii.
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DOI: 10.1159/000497251
Table 2. Kimura 2P genetic distance [Kimura, 1980] in percentages (%) among 5 different taxa
of Lagothrix lagothricha, L. flavicauda, and Alouatta seniculus (Atelidae, Primates) (below the
main diagonal) and standard deviations in percentages (%) (above the main diagonal) at the
mtCOI and COII genes
L. l. L. l. L. l. L. l. L. l. L. A.
lugens poeppigii lagothricha cana tschudii flavicauda seniculus
L. l. lugens 0.4 0.3 0.4 0.3 0.6 1.3

L. l. poeppigii 4.2 0.4 0.3 0.4 0.6 1.3
L. l. lagothricha 3.1 2.5 0.4 0.4 0.7 1.4
L. l. cana 3.7 2.8 2.0 0.3 0.6 1.4
L. l. tschudii 3.3 2.5 1.7 1.6 0.6 1.3
L. flavicauda 4.7 3.8 3.1 3.0 2.9 1.4
A. seniculus 15.8 14.9 14.4 14.8 14.1 14.2
Table 3. Overall genetic heterogeneity and gene flow (Nm) statistics for the 5 subspecies of Lago-
thrix lagothricha analyzed at the mtCOI and COII genes
Genetic differentiation p Gene flow

estimated
χ2 = 613.128 df = 372 0.0001** GST = 0.1045 Nm = 4.28

HST = 0.1027 0.0001** γST = 0.3516 Nm = 0.92
KST = 0.3368 0.0001** NST = 0.4129 Nm = 0.71
KST* = 0.2865 0.0001** FST = 0.4188 Nm = 0.69
ZS = 3646.757 0.0001**
ZS* = 7.4219 0.0001**
Snn = 0.9614 0.0001**
* p < 0.05; ** p < 0.01.
Discussion
Systematics and Phylogeny of L. l. tschudii

This is the first molecular work which analyzed samples of the tschudii taxon
to elucidate its phylogenetic relationship with the other taxa of Lagothrix. Puch-
eran [1857] made the first reference to tschudii. It was of Peruvian or Bolivian
origin and referred to as Lagothrix humboldtii by Geoffroyi [1851]. Later, other
specimens from the Peruvian area of Cusco were also designated as L. humboldtii
[Thomas, 1899, 1901, 1920]. However, the name of L. humboldtii was also used for
different specimens of L. l. lagothricha in Brazil (Vaupes River) and Venezuela
[Geoffroyi, 1812]. It was also used for specimens of L. l. poeppigii near the Napo
River in Ecuador [Slack, 1863] and those of L. l. lugens in Bogota and Tolima, Co-
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Study DOI: 10.1159/000497251
Table 4. FST pair statistics (below the main diagonal) and NST pair statistics (above the main
diagonal) among 6 different Lagothrix taxa by means of the mtCOI and COII genes
L. l. L. l. L. l. L. l. L. l. L.
lugens poeppigii lagothricha cana tschudii flavicauda
L. l. lugens 0.2756* 0.3099* 0.3687* 0.3138* 0.5900**

L. l. poeppigii 0.2783* 0.4614* 0.4641* 0.4038* 0.7079**
L. l. lagothricha 0.3223* 0.4627* 0.7029** 0.6535** 0.9328**
L. l. cana 0.3771* 0.4641* 0.7016** 0.5306** 0.8704**
L. l. tschudii 0.3219* 0.4039* 0.6516** 0.5291** 0.8729**
L. flavicauda 0.6006** 0.7082** 0.9318** 0.8694** 0.8714**
* p < 0.01, ** p < 0.001.
Table 5. Exact probability tests (p) to estimate genetic heterogeneity (below the main diagonal)
and standard deviations (above the main diagonal) among 6 different Lagothrix taxa by means of
the mtCOI and COII genes
L. l. L. l. L. l. L. l. L. l. L.
tschudii lugens poeppigii lagothricha cana flavicauda
L. l. tschudii 0.0000 0.0145 0.0000 0.0000 0.0023

L. l. lugens 0.00000* 0.0000 0.0000 0.0000 0.0000
L. l. poeppigii 0.02845* 0.00000* 0.0000 0.0000 0.0002
L. l. lagothricha 0.00000* 0.00000* 0.00000* 0.0000 0.0021
L. l. cana 0.00000* 0.00000* 0.00000* 0.00000* 0.0013
L. flavicauda 0.00715* 0.00000* 0.00791* 0.00300* 0.00115*
* Significant probability.
lombia [Geoffroyi, 1851; Pucheran, 1857; Thomas, 1880]. This information pre-
cludes our use of humboldtii for the 10 individuals we analyzed from the Cusco
Department (Peru) and from northern Bolivia (Apolobamba, Madidi). Further-
more, Allen [1900] designated an individual from Juliaca (= Inca Mines) in Puno
(Peru) as Alouatta nigra, and Elliot [1909] named 1 specimen Lagothrix thomasi
from the Comberciato River in Cusco. Since tschudii was used prior to either nig-
ra or thomasi, it seems that the correct name for the taxon we studied should be L.
l. tschudii.
We agree with the view of Fooden [1963] that cana, poeppigii, lagothricha, and
lugens are subspecies within L. lagothricha [molecular arguments in Ruiz-García et
al., 2014] and not full species as it was claimed by Groves [2001]. We disagree with
Fooden [1963] concerning the fact that he did not recognize tschudii as a fifth sub-
species in L. lagothricha and different from L. l. cana. However, he did morpho-
logically differentiate the Cusco animals from those of the upper and middle Ma-
deira River, upper Ucayali River, and upper Pachitea River. He described the ani-
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DOI: 10.1159/000497251
Table 6. Gene flow (Nm) estimations (below the main diagonal) among 6 different taxa of Lago-
thrix by means of the mtCOI and COII genes
L. l. L. l. L. l. L. l. L. l. L.
tschudii lugens poeppigii lagothricha cana flavicauda
L. l. tschudii
L. l. lugens 1.6552
L. l. poeppigii 1.1049 1.1963
L. l. lagothricha 0.2259 1.0773 0.7386
L. l. cana 0.4719 0.8547 0.6977 0.2189
L. flavicauda 0.0933 0.6204 0.3941 0.0600 0.1296
Table 7. Genetic diversity of 6 taxa of Lagothrix at the mtCOI and COII genes using the number
of haplotypes (NH), the haplotype diversity (Hd), the nucleotide diversity (π), and the θ statistic
(= Neμ) by sequence
NH Hd π θ
L. flavicauda 1 0.000±0.000 0.0000±0.0000 0.000±0.000

L. l. tschudii 8 0.956±0.059 0.0075±0.0024 8.130±3.616
L. l. lugens 14 0.850±0.053 0.0357±0.0127 57.039±17.91
L. l. poeppigii 46 0.974±0.014 0.0166±0.0019 30.676±8.519
L. l. lagothricha 16 0.789±0.080 0.0057±0.0014 11.864±3.977
L. l. cana 14 0.822±0.063 0.0076±0.0026 5.439±1.234
Ne, effective female population size; μ, mutation rate per generation.
mals from the Comberciato River (Cusco) as those with the darkest pelage series
within L. l. cana. He wrote that an adult female has a wide and dark dorsal streak
and a greatly reduced olivaceous ticking. Overall, it appears as a blackish animal
with olivaceus flanks. A juvenile from the same river was generally blackish with
slight olivaceus ticking on the flanks. In contrast, the animals of the aforemen-
tioned areas in Brazil and in the other Peruvian areas were considerably paler. Dif-
ferent from Fooden [1963], Groves [2001] recognized tschudii as a differentiable
taxon but as a subspecies within L. cana. Groves [2001] described the individuals
of tschudii as much darker than the other specimens of cana, with a deep blackish
gray, with a tinge of red and with head, limbs, and tail completely black. Our mo-
lecular results emphasize that neither author is completely correct: tschudii should
be a real subspecies but within L. lagothricha.
We support the descriptions of Fooden [1963] and Groves [2001] because we
have seen live and dead animals in the Andean highlands from southern Peru (Cusco
and Puno Departments) and northern Bolivia (La Paz Department). They are very
dark and have very long hair. Furthermore, they are similar to some of the dark phe-
notypes seen in some areas of Colombian Andean for L. l. lugens. L. l. tschudii is ex-
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Study DOI: 10.1159/000497251
0.20 Exp
Obs
0.15
Fig. 4. Mismatch distri
butions (pairwise sequence 0.10
differences) at the 2 concate-
nated mtDNA genes (COI
and COII) for the sequence 0.05
set of Lagothrix lagothricha
tschudii. Exp, theoretical ex- 0
pected curve with a popula- 0 5 10 15 20 25 30
tion expansion; Obs, observ Pairwise differences
ed curve with the data ana-
lyzed.
tremely different from L. l. cana of the Amazonian lowlands in Peru and Brazil (for
instance along Madeira River), which have very short hair and a totally different coat
color. Therefore, there is a noteworthy correlation between the clearly recognizable
phenotype of tschudii and the molecular differences we found between the two taxa.
Indeed, we showed that the MLT only yielded a bootstrap of 24% for the monophyly
of L. l. tschudii + L. l. cana (42% for a Neighbor-Joining tree with the Kimura 2P dis-
tance). The MJN procedure showed that L. l. tschudii was closer than L. l. cana to the
common ancestor of L. flavicauda and L. lagothricha. Some of the genetic heteroge-
neity and gene flow analyses also showed that other taxa of L. lagothricha (such as L.
l. lugens, the other Andean taxon of L. lagothricha) could be more related to L. l.
tschudii than to L. l. cana. This could validate our previous comment that the Andean
mountains could have played an extremely important role in the initial divergence
within Lagothrix and within L. lagothricha. The BI (p = 0.9) was the only analysis
which showed a considerable relationship between L. l. cana and L. l. tschudii.
The temporal split between the ancestor of L. l. tschudii and L. l. cana for BI was
1.8 MYA. The split between L. l. tschudii and all of the other subspecies of L. lagothricha
ranged from 1.3 to 0.9 MYA for the MJN. The diversification within L. l. tschudii was
estimated to have occurred no later than 0.96 MYA (BI) or 0.88 MYA (MJN). The last
phase of the Gelasian occurred around 2.5–1.8 MYA. This was a period characterized
by the last stages of a global cooling trend that led to the Quaternary ice ages [Interna-
tional Commission on Stratigraphy, 2007]. The temperature was around 4 ± 2 ° C lower
than today, and overall precipitation was less (by 500–1,000 mm). At 2,500 masl, the
temperature was 10 ° C less than what it is today [Van der Hammen, 1992]. L. l. tschudii
nowadays lives at this altitude, although the taxon has probably increased its altitudinal
distribution since the last ice age [Colwell and Rangel, 2010]. This also coincides with
the last formation phase of the central Andes where L. l. tschudii lives. The entire An-
dean mountain chain between Cajamarca and Huancavelica in Peru was formed during
this period by volcanic activity. A divergence of 1.30–0.9 MYA for the split between the
ancestors of L. l. tschudii and the other subspecies of L. lagothricha, and the diversifica-
tion within L. l. tschudii, coincides with the time that the Buenos Aires fauna trans-
formed into a typical semi-arid Patagonian fauna, represented by the guanaco, Lesto-
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DOI: 10.1159/000497251
1.E2
1.E1
log female population size
1.E0
1.E–2
0 0.25 0.5 0.75 1
Time in millions of years
Fig. 5. Bayesian skyline plot analysis to determine possible demographic changes across the natu-
ral history of Lagothrix lagothricha tschudii at 2 mtDNA genes (COI and COII).
delphys, and Lyncodon. This period was a time for haplotype diversification for many
carnivores, including the Pampas cat [Cossíos et al., 2009], the foxes of the Lycalopex
genus [Ruiz-García et al., 2013], and the jaguarundi [Ruiz-García et al., 2018]. There-
fore, the climate was considerably colder and drier than today and could have influ-
enced the mitochondrial differentiation between L. l. tschudii and the other taxa within
L. lagothricha. This period also coincides with some of the glaciations detected in dif-
ferent areas of South America. Glaciations have been registered in the Argentinian Pa-
tagonia since 3.5 MYA [Mercer, 1984]; in La Paz (Bolivia) since 3.27 MYA [Clapperton,
1981], and in Chile, 3 glaciations have been recorded since 2 MYA [Caviedes and Pas-
koff, 1975].
Genetic Diversity in L. l. tschudii and in the other Lagothrix taxa

We have shown that the genetic diversity for L. l. tschudii is high, considering
that it is a taxon with a very limited geographical distribution (the smallest geograph-
ic distribution for a current taxon of L. lagothricha). This could be related to the orig-
inal genetic diversification within Lagothrix. Only L. l. poeppigii and L. l. lugens yield-
ed levels of genetic diversity higher than L. l. tschudii. The ancestor of L. l. poeppigii
was probably the first one to come from the Andean mountains to colonize the Am-
azonian lowlands. Thus, L. l. poeppigii spent most time evolving in the Amazon and,
for this reason, it is the Amazonian L. lagothricha taxon with the highest genetic di-
versity. In the case of L. l. lugens, the high levels of genetic diversity are easy to under-
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Study DOI: 10.1159/000497251
stand. Ruiz-García and Pinedo-Castro [2010] and Ruiz-García et al. [2014] analyzed
specimens from the Colombian Eastern Cordillera, the upper Magdalena River Val-
ley, and the almost extinct populations from the northern end of the Colombian Cen-
tral Cordillera (Antioquia, Córdoba, and Bolivar). The specimens of these last popu-
lations were genetically fixed by intensive gene drift. There were high levels of overall
genetic diversity when all of the sequences of lugens were together as a unique taxon.
However, authors who estimated a very limited genetic diversity in populations of L.
l. lugens may misunderstand our results [Botero and Stevenson, 2014; Botero et al.,
2015]. These authors sampled individuals of this subspecies in a relatively restricted
area of Colombia (Meta, Guaviare, Caqueta, and Huila Departments), whereas we
sampled L. l. lugens in all the areas of Colombia where this subspecies is found (Ca-
queta, Cauca, Huila, Tolima, Meta, Cundinamarca, Cordoba, Bolivar, and Antioquia
Departments). The populations on the left side of the Eastern Andean cordillera
(Magdalena Valley and northern Central Cordillera in the Cordoba, Bolivar, and An-
tioquia Departments) are extremely small and intensely affected by gene drift. These
populations were not studied by Botero and Stevenson [2014] and Botero et al. [2015].
When we unite all the individuals in 1 unique group, this greatly enhances the ge-
netic diversity of this subspecies. Thus, the claim of these authors that diversifying
natural selection affects the mtCOII gene in Lagothrix is not supported.
Fooden [1963] commented about possible intergradation of tschudii with L. l.
poeppigii in the areas of Hacienda Cadena (Cusco, 500 masl) and Puno (1,800 masl).
Both localities are on the Inambari River. The animals of these areas have a tawny-
olive color. Following Fooden [1963], the tawniness of these individuals is the result
of a reddish-brown sub-basal band. This band is whitish in the classical cana. Never-
theless, our molecular results did not provide evidence of mitochondrial introgres-
sion or recent hybridization between L. l. tschudii and L. l. poeppigii.
Demographic Changes
The demographic evolutionary history of L. l. tschudii with BSP is very similar to
that shown by L. l. lugens. L. l. lugens showed a very slight increase during the last 0.75
MY with some detectable decrease in the last 25,000 years [Ruiz-Garcia et al., 2014]. In
the case of L. l. tschudii, the increase was extremely slight during the last 0.9 MY, with
some decrease in the last 10,000 years. The decrease of both Andean forms could be in
tandem with climatic events that began around 35,000 YA, in the middle of the Pleni-
glacial period, when the environment was colder and drier. For instance, the Bogota la-
goon was dry in that period [Van der Hammen, 1992]. In fact, the maximum glaciation
extension in the Colombian Andes was around 35,000 YA, with ice from 3,000 ± 100
masl and covering around 17,108 km2 [Van der Hammen, 1985]. Afterwards, another
period of extreme cold occurred from 23,000 to 20,000 YA (Dryas I) as MacNeish [1979]
showed for Pikimachay Cove in Peru with his pollen and soil acidity study. The Last
Glacial Maximum was next, from 19,000 to 16,500 YA, when the snow cover in the Cen-
tral Andes reached its maximum. Finally, from 14,500 to 12,000 YA (Dryas II), the tem-
perature was extremely cold. Glaciation reached a maximum in the Manachaque Valley
(Cordillera Blanca) and in the Upismayo-Jalacocha at the Vilcanota Cordillera, both in
Peru, as well as in the Nevado of ChoqueYapu in Bolivia and in the Chimborazo in Ec-
uador. Rodbell and Seltzer [2000] showed that the glaciers in the Cordillera Blanca (San
Martin Department in Peru) reached their maximum 12,000 YA, and the ice limit then
was around 3,170–3,827 masl. Today, this limit is around 4,600 masl. Maslin and Burns
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DOI: 10.1159/000497251
[2000] showed that the Amazon River reached its maximum dry peak around 16,000–
15,000 YA, and this situation continued until 12,000 YA. The analysis with O18 isotopes
revealed that the mouth of the Amazon River only had 40% of the water compared to
today. In the tropical forest, the temperature descended around 2–6 ° C, and in some ar-
eas, such as the Fuquene lagoon near Bogota, the precipitation was only half of what it
is today [Van der Hammen, 1992]. The temperature of the Atlantic Ocean near the Bra-
zilian coasts descended 6 ° C [Clark, 2002]. This climate change might have produced the
last massive extinction event, which eliminated around 80% of the large vertebrates of
North America. For example, this eliminated the pumas in North America [Culver et al.,
2000] as well as 40 other species, including Smilodon, lions, cheetahs, etc. This could ex-
plain the female population decrease in both Andean taxa, L. l. tschudii and L. l. lugens.
In contrast, the Amazon taxa of L. l. poeppigii and L. l. lagothricha showed strong
population expansions (1 and 0.3 MYA, respectively) with no evidence of population
decrease. For L. l. cana, also an Amazonian taxon, the evidence of population expan-
sions was more limited than in these 2 taxa of L. lagothricha. Therefore, the 2 current
Andean taxa of L. lagothricha showed female populations decreasing in the last tens
of thousands of years, which is a different demographic trajectory relative to the oth-
er 3 Amazonian taxa.
The evidence compiled in this work is suggestive of tschudii being a distinct tax-
on within L. lagothricha, but not conclusive. We basically have 2 pieces of evidence,
that from examinations of pelage and from the molecular phylogeny with mt genes.
We need evidence from more loci and more localities before the evidence is strong
enough to be conclusive. Additionally, future studies should include more samples of
L. l. tschudii from transitional areas containing both L. l. cana and L. l. poeppigii. Such
samples could be analyzed to try to find possible introgression or recent hybridization
among these taxa. Nuclear gene sequences are desirable to determine if there is, or
not, some gene flow between tschudii and other taxa of Lagothrix.
Acknowledgments
Thanks to Dr. Diana Alvarez, Pablo Escobar-Armel, Nicolás Lichilín, Luisa Castellanos-
Mora, Kelly Luengas, Hugo Gálvez, and Alan Velarde for their help in obtaining Lagothrix sam-
ples over the last 20 years. Thanks to Instituto von Humboldt (Villa de Leyva in Colombia; Janeth
Muñoz), to the Peruvian Ministry of Environment, PRODUCE (Dirección Nacional de Extrac-
ción y Procesamiento Pesquero), Consejo Nacional del Ambiente and the Instituto Nacional de
Recursos Naturales from Peru, to the Colección Boliviana de Fauna (Dr. Julieta Vargas) and to
CITES Bolivia, to the Ministerio del Ambiente in Coca (Ecuador), and the Brazilian IBAMA for
their role in facilitating the acquisition of collection permits in Colombia, Peru, Bolivia, Ecuador,
and Brazil. We also thank the many people of diverse Indian tribes in Peru (Bora, Ocaina, Ship-
igo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana, and Alamas), in Colombia
(Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri, Yucuna, Curripacos, and Desano), in
Bolivia (Kallawaya, Uchipiamonas, and Tacana), and Ecuador (Kichwa, Huaorani, Shuar, and
Achuar).
Disclosure Statement
The authors report no conflicts of interest. The authors alone are responsible for the content
and writing of this article.
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Study DOI: 10.1159/000497251
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Folia Primatologica 2019 Lagothrix Lagotricha Tschudii

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Original Research Article

Folia Primatol Received: April 19, 2018

First Molecular Phylogenetic Analysis of the

© 2019 S. Karger AG, Basel Manuel Ruiz-García

2 Folia Primatol Ruiz-García et al.

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 3

4 Folia Primatol Ruiz-García et al.

Taxa of Lagothrix Geographic origin n

Materials and Methods

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 5

6 Folia Primatol Ruiz-García et al.

Genetic Diversity and Heterogeneity Analyses

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 7

8 Folia Primatol Ruiz-García et al.

Genetic Distances and Genetic Heterogeneity

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 9

poeppigii - Tigre River 119 - Pastaza - Ecuador

10 Folia Primatol Ruiz-García et al.

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 11

12 Folia Primatol Ruiz-García et al.

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 13

Demographic Evolution in L. l. tschudii

14 Folia Primatol Ruiz-García et al.

L. l. lugens 0.4 0.3 0.4 0.3 0.6 1.3

Genetic differentiation p Gene flow

χ2 = 613.128 df = 372 0.0001** GST = 0.1045 Nm = 4.28

* p < 0.05; ** p < 0.01.

Systematics and Phylogeny of L. l. tschudii

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 15

L. l. lugens 0.2756* 0.3099* 0.3687* 0.3138* 0.5900**

* p < 0.01, ** p < 0.001.

L. l. tschudii 0.0000 0.0145 0.0000 0.0000 0.0023

16 Folia Primatol Ruiz-García et al.

L. flavicauda 1 0.000±0.000 0.0000±0.0000 0.000±0.000

Ne, effective female population size; μ, mutation rate per generation.

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 17

18 Folia Primatol Ruiz-García et al.

Genetic Diversity in L. l. tschudii and in the other Lagothrix taxa

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 19

20 Folia Primatol Ruiz-García et al.

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 21

22 Folia Primatol Ruiz-García et al.

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 23

24 Folia Primatol Ruiz-García et al.

Downloaded by: M. Ruiz-García - 442282

Lagothrix lagothricha tschudii, First Molecular Folia Primatol 25

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