FPR480502 Alouattapalliata Published

Original Research Article
Folia Primatol Received: April 26, 2017

DOI: 10.1159/000480502 Accepted after revision: August 20, 2017
Published online: ■■■
Phylogeography of the Mantled Howler Monkey

(Alouatta palliata; Atelidae, Primates) across Its
Geographical Range by Means of Mitochondrial
Genetic Analyses and New Insights about the
Phylogeny of Alouatta
Manuel Ruiz-García a Ángela Cerón a Sebastián Sánchez-Castillo a

Pilar Rueda-Zozaya b Myreya Pinedo-Castro a

Gustavo Gutierrez-Espeleta c Joseph Mark Shostell d

a Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética,

Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá,
Colombia; b Centro de Investigación en Ciencias Biológicas Aplicadas, Universidad

Autónoma del Estado de México, Toluca, Mexico; c Escuela de Biología, Universidad de

Costa Rica, San José, Costa Rica; d Department of Math Science and Technology, University

of Minnesota Crookston, Crookston, MN, USA
Keywords
Alouatta · Alouatta palliata · Mitochondrial gene sequences · Genetic diversity ·
Phylogenetics · Phylogeography
Abstract
We analyzed 156 specimens of diverse howler monkey taxa (Alouatta; Atelidae, Pri-
mates) for different mitochondrial genes (5,567 base pairs), with special emphasis on A.
palliata and related taxa. Our results showed no relevant differences among individuals
of different putative taxa, A. p. palliata, A. p. aequatorialis, A. coibensis coibensis, and A. c.
trabeata. We found no spatial differences in genetic structure of A. p. palliata throughout
Costa Rica, Nicaragua, and Honduras. A. p. mexicana (genetic distance: 1.6–2.1%) was the
most differentiated taxon within A. palliata. Therefore, we postulate the existence of
only 2 clearly defined subspecies within A. palliata (A. p. palliata and A. p. mexicana). A.
palliata and A. pigra (traditionally considered a subspecies of A. palliata) are 2 clearly dif-
ferentiated species as was demonstrated by Cortés-Ortiz et al. [Molecular Phylogenetics
and Evolution 2003;26:64–81], with a temporal split between both species around 3.6–
3.7 million years ago (MYA). Our results with the Median Joining Network procedure
showed that the ancestors of the cis-Andean Alouatta gave origin to the ancestors of the
trans-Andean Alouatta around 6.0–6.9 MYA. As Cortés-Ortiz et al. showed, A. sara and A.
© 2017 S. Karger AG, Basel Manuel Ruiz-García, Unidad de Genética

Departamento de Biología, Facultad de Ciencias
E-Mail karger@karger.com Pontificia Universidad Javeriana, Cra 7A, No. 43-82,
www.karger.com/fpr Bogotà, DC 110231 (Colombia)
E-Mail mruizgar @ yahoo.es
macconnelli are differentiable species from A. seniculus, although the first 2 taxa were
traditionally considered subspecies of A. seniculus. Our findings agree with the possibil-
ity that the ancestor of A. sara gave rise to the ancestor of A. pigra in northern South
America. In turn, the ancestor of A. pigra originated the ancestor of A. palliata. Two of our
results strongly support that the South American A. palliata (the putative A. p. aequato-
rialis) was the original population of this species. There is high genetic diversity and no
evidence of population expansion. The Central America A. palliata is the derived popula-
tion. It has low genetic diversity and there is clear evidence of population expansion.
However, both A. palliata and A. pigra probably migrated into Central America by 2 dif-
ferent routes: the Isthmus of Panama (A. palliata) and Caribbean island arch (A. pigra).
Finally, the red howler monkeys from the Trinidad Island in the Caribbean Sea were not
A. macconnelli (= A. s. stramineus) as Groves [Primate Taxonomy, 2001] maintained. This
taxon is more related to A. s. seniculus, although it formed a monophyletic clade. Future
molecular and karyotypic studies will show if the Trinidad red howler monkeys should
be considered as an extension of the Venezuelan taxon, A. arctoidea, as a subspecies of
A. seniculus (A. s. seniculus), or, in the case of extensive chromosomal rearrangements,
even a new species. © 2017 S. Karger AG, Basel
Introduction
The howler monkey (Alouatta) is the only living genus within the atelid subfam-
ily Alouattinae [Rylands et al., 2000; Groves, 2001]. Howler monkeys are among the
largest Neotropical primates and are similar in size to individuals of other genera,
such as Ateles, Brachyteles, and Lagothrix. Alouatta is also the most widely distrib-
uted Neotropical primate genus, ranging from Veracruz, Mexico [Estrada and
Coates-Estrada, 1984] to Corrientes, Argentina [Cabrera, 1939].
Howler monkeys are an ecologically flexible pioneer species that can live in many
types of diversified habitats, from primary rain forests to scrub and savanna wood-
lands [Crockett and Eisenberg, 1987; Pope, 1992]. Among the different recognized
Alouatta species (Table 1), the mantled howler monkey (A. palliata) extends from
southern Mexico (Veracruz, Oaxaca, Tabasco, Chiapas) throughout Central America
to the Pacific west coast of Colombia, Ecuador, and the area of Tumbes in northern
Peru [Hurtado et al., 2016].
Traditionally, 3 subspecies have been mentioned (A. p. mexicana, A. p. palliata,
and A. p. aequatorialis). A. p. mexicana was first described by Merriam in 1902 from
Minatitlan, Veracruz State in Mexico. This taxon has a more diffuse distribution of
light-banded hairs over the dorsum and a paler more silvery coat on the flanks. The
silver color also occurs on parts of the dorsum. In contrast, the other forms of A. pal-
liata are a walnut color. Its geographical distribution extends from the Mexican states
of Veracruz, Tabasco, Chiapas, Oaxaca and southern Campeche [Oropeza-Hernán-
dez and Rendón-Hernández, 2012] to central, and southern Guatemala [Rodríguez-
Luna et al., 1996]. In the Mexican Tabasco and Chiapas states, this taxon is sympatric
with the black howler monkey (A. pigra) [García-Orduña et al., 1999; Rodríguez-
Luna et al., 2001; Serio-Silva and Rico-Gray, 2004; Del Valle et al., 2005]. It probably
occurs in certain areas of Guatemala as well. In 1849, Gray described A. p. palliata in
Caracas, Venezuela, and later, in 1972, Sclater described the taxon at Lake Nicaragua,
2 Folia Primatol Ruiz-García et al.

DOI: 10.1159/000480502
Table 1. Different classifications of the species and subspecies of the genus Alouatta following
different authors
Authors Species Subspecies
Rylands et al. A. palliata A. p. mexicana, A. p. palliata, A. p. aequatorialis

[1997] A. pigra
A. coibensis A. c. coibenis, A. c. trabeata
A. seniculus A. s. seniculus, A. s. insulanus, A. s. stramineus,
A. s. macconnelli, A. s. amazonica, A. s. juara,
A. s. puruensis, A. s. ssp. (Venezuela)
A. arctoidea
A. sara
A. belzebul A. b. belzebul, A. b. nigerrima, A. b. discolor, A. b. ululata
A. fusca A. f. fusca, A. f. clamitans
A. caraya
Groves [2001] A. palliata A. p. mexicana, A. p. palliata
A. pigra
A. coibensis A. c. coibensis, A. c. trabeata
A. seniculus A. s. seniculus, A. s. arctoidea, A. s. juara
A. macconnelli
A. sara
A. belzebul
A. nigerrima
A. guariba A. g. guariba, A. g. clamitans
A. caraya
Cortés-Ortíz A. palliata A. p. mexicana, A. p. palliata, A. p. coibensis, A. p. trabeata,
et al. [2015] A. p. aequatorialis
A. pigra A. p. pigra, A. p. luctuosa
A. seniculus A. s. seniculus, A. s. juara, A. s. puruensis
A. arctoidea
A. sara
A. macconnelli
A. guariba A. g. guariba, A. g. clamitans
A. belzebul
A. caraya
Possible Alouatta species:
A. nigerrima
A. ululata
A. discolor
Nicaragua. This taxon has light yellowish hairs restricted to the flanks, whereas other
A. palliata forms have the lighter color extending into the dorsum. Its geographical
distribution is throughout southeastern Guatemala, Honduras, Nicaragua, Costa
Rica, and part of northern Panama. A. p. aequatorialis was described by Festa in 1903,
in Vinces, Guayas Province, Ecuador. Elliot [1913] claimed that the pelage was simi-
lar to that of A. p. palliata but that the general color was chocolate-brown instead of
black. Also, Lawrence [1933] found the mantle hairs to be golden-ochraceous in A. p.
aequatorialis, and more abundant in the posterior and slightly shorter than in A. p.
palliata. The geographical distribution extends through Panama and the Pacific west
Phylogeography of the Mantled Howler Monkey Folia Primatol 3

DOI: 10.1159/000480502
coast of Colombia and Ecuador to the Tumbes region of northern Peru [Aquino and
Encarnación, 1994; Encarnación and Cook, 1998]. In Colombia, this taxon is found
north of the Sinu and Atrato rivers to the Caribbean Coast [Hernández-Camacho and
Cooper, 1976], where it is sympatric with the Colombian red howler monkey, A.
seniculus.
Two other A. palliata forms were recognized in the past. A. p. coibensis was first
described by Thomas in 1902 from Coiba Island, Panama. Also, A. p. trabeata was
described by Lawrence in 1933 in Capina, Herrera Province, Azuero Peninsula, Pan-
ama. The first form has a veil more restricted to the flanks, a duller pelage than the
closely related form from the Azuero Peninsula, and it is smaller in size than other
Central American howler monkeys. The second form is distinguished principally by
its golden flanks and loins (golden-ochraceous tips of hairs), together with a browner
appearance over the rest of the body [Hill 1962]. Froehlich and Froehlich [1987] con-
cluded that both howler forms were very close (very few differences detected via a
multivariate analysis), but quite distinct from A. p. palliata, based on high frequencies
of complex dermatoglyphic patterns. Nevertheless, there is no single dermatoglyphic
pattern in these Panamanian forms relative to other A. palliata taxa. In spite of this,
and taking into account that the islands of Coiba and Jicarón were last connected to
the mainland about 24,000 and 15,000 years ago, respectively, Froehlich and Froehlich
[1987] concluded that these forms were 2 subspecies of a different species: A. coiben-
sis (A. c. coibensis and A. c. trabeata, respectively).
Only a few molecular studies have been conducted with A. palliata. Malmgren
and Brush [1978] studied the levels of genetic variation among 132 individuals from
the population of La Pacifica (Costa Rica) using 2-dimensional electrophoresis. They
only found variations in 2 out of 15 isoenzymatic loci. Froehlich and Thorington
[1982] studied 80 individuals from the population on Barro Colorado Island (Pana-
ma) and only reported variation for 2 serum proteins and 1 red cell enzyme. Later,
Zaldívar et al. [2003] analyzed 137 specimens for 8 different populations throughout
Costa Rica for total plasma proteins and for 14 red cell isoenzyme loci. They did not
detect any genetic variation for these markers. Their findings contrasted with the re-
sults for 2 South American Alouatta species (A. seniculus and A. belzebul), which had
genetic variability in 4 of these 14 loci.
Ellsworth and Hoelzer [1998] and Ellsworth [2000] analyzed 8 microsatellite
loci of samples from Panama, Costa Rica, and Mexico. A. palliata showed little ge-
netic diversity, and there was a northern decline in this diversity. The population
from Panama (southern area) showed the greatest genetic diversity, whereas the
population from Mexico (northern area) showed the least. Winkler et al. [2004]
analyzed 29 exemplars of A. p. palliata in 2 localities on Ometepe Island (Nicara-
gua) and 10 individuals of A. pigra, the black howler, for 12 microsatellites. The
sample of A. p. palliata showed less genetic variability than the sample of A. pigra.
Later Ellsworth and Hoelzer [2006], using DNA microsatellite loci, were surprised
that A. palliata and A. pigra (the 2 Central American Alouatta species clearly rec-
ognized) were the most genetically distinctive species in their study, suggesting that
they were not sister species as they previously believed. They suggested that A. pig-
ra may have descended from a different invasion of Central America that predated
the one leading to A. palliata. Additionally, A. pigra may have arrived in Central
America via a different route than that taken by the ancestors of A. palliata, using
the islands of the Caribbean archipelago as stepping stones to reach their current

DOI: 10.1159/000480502
location without moving up the Isthmus of Panama. Cortés-Ortiz et al. [2007, 2010]
detected hybrids between both A. palliata and A. pigra in a narrow region of south-
ern Mexico.
Milton et al. [2009] analyzed 10 polymorphic microsatellites in 76 A. p. palliata
collected from the Barro Colorado Island in Panama. This population was established
in 1914 from an unknown number of individuals when the island was created from
the surrounding mainland by the damming of the Chagres River to create the Panama
Channel. This sample exhibited medium-high levels of genetic diversity (expected
heterozygosity, H = 0.584 ± 0.063) for species of Alouatta. Furthermore, the authors
found that it did not have a strong genetic structure. Tests for genetic bottlenecks
based on departures from equilibrium expectations failed to reveal evidence of a re-
cent reduction in population size. However, they indicated a marked decline in pop-
ulation size in the last century.
Argüello-Sánchez [2012] analyzed hairs of 46 individuals of A. p. mexicana for
331 base pairs (bp) of the mitochondrial Cyt-b gene in 13 localities of the Mexican
Veracruz State. She estimated a medium to high elevated genetic diversity (haplo-
type diversity, Hd = 0.76; nucleotide diversity, π = 0.012), the existence of 2 differen-
tiated mitochondrial lineages living sympatrically in the Veracruz State, and no dif-
ferences in the amounts of genetic variability between continuous and fragmented
forests.
However, Dunn et al. [2014] estimated a different perspective for the population
of A. p. mexicana in the Selva Zoque (Veracruz State, Mexico). They amplified full-
length mitochondrial control region sequences (1,100 bp) from 45 individuals and
found 7 very similar haplotypes. The estimates of Hd = 0.49 and π = 0.0007 were very
low. Historical demographic analyses were consistent with a recent and mild popula-
tion expansion. Genetic diversity appears to be historically low in this taxon. Recent-
ly, Jasso-del Toro et al. [2016] determined the genetic diversity in 7 groups of A. p.
mexicana, 3 groups inhabiting continuous forests and 3 in fragmented forests, at Los
Tuxtlas Biosphere Reserve. They used fecal samples and analyzed 13 microsatellite
loci, with 8 of them being polymorphic. They obtained low levels of genetic diversity
and low genetic differentiation (Fst = 0.043) between continuous and fragmented
habitats of these howler monkeys.
Only 1 study [Cortés-Ortiz et al., 2003] tried to clarify the possible existence and
relationships of subspecies within A. palliata. They analyzed 9 A. p. mexicana, 2 A. p.
palliata (Costa Rica), 4 putative A. p. aequatorialis (although they were from Panama
and not from the South American distribution of Colombia, Ecuador, and Peru), 3 A.
c. trabeata, and 1 A. c. coibensis. Each was analyzed for 3 mitochondrial genes (Cyt-b,
ATP-6 and ATP-8) and 2 nuclear genes (CAL and RAG). The mitochondrial genes
showed minimal differences among individuals of A. p. mexicana, A. p. palliate, and
A. c. coibensis. They, in turn, were slightly differentiated from the Panamanian indi-
viduals of A. p. aequatorialis and A. c. trabeata, which were closely related. Thus, this
mitochondrial analysis did not differentiate between A. coibensis and A. palliata. Both
nuclear genes did not adequately discriminate different Alouatta species. Compara-
tively, the mitochondrial sequences were more helpful in resolving phylogenetic re-
lationships among Alouatta taxa.
Nevertheless, only a small number of individuals were analyzed in the previous
study. There were 2 unique individuals of A. p. palliata from a locality in Costa Rica
and no individuals of A. p. aequatorialis from South America. Also, only a small frac-

DOI: 10.1159/000480502
tion of the mitochondrial DNA was sequenced (1,642 bp). Therefore, our first aim is
to provide additional molecular data to resolve how many valid taxa are within A.
palliata. We provide a large sample size (124 individuals), including exemplars of A.
p. palliata from diverse areas of Costa Rica, Nicaragua, and Honduras. The data set
also includes individuals of A. p. aequatorialis from the Pacific area of Colombia and
Ecuador. Furthermore, we sequenced a larger fraction of mitochondrial DNA (5,567
bp). 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]. For all of these
reasons, mitochondrial gene trees are more precise in reconstructing the divergence
history among related taxa than other molecular markers [Moore, 1995]. Supporting
this, Cummings et al. [1995] showed that mitochondrial genomes have a higher in-
formation content per base than nuclear DNA. However, the mitochondrial genes
poses several problems, including heterogeneity in base composition at each codon
position, and third-codon position saturation. Additionally, extreme 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]. Species can diverge simultaneously with a pair of mitochondrial haplotypes or
they can diverge after a pair of haplotypes have diverged. However, it is possible that
some time after a population divides, a new haplotype may appear in the gene tree. A
migrant could carry the new haplotype to the other population and, finally, the new
haplotype is lost in one population and the ancestral haplotype is lost in the other.
Therefore, if we use the gene tree to estimate genetic heterogeneity and the divergence
time for the species tree, the species will appear to have diverged more recently than
they really did [Freeman and Herron, 1998]. Furthermore, mitochondrial data show
only the evolution of the female lineages, and this could miss hybridization events
between close species when males are the gene flow vectors (“mitochondrial capture”)
[Burrell et al., 2009].
On the other hand, the systematics of Alouatta is complex due to the phenotyp-
ic similarity among the taxa of this genus. They have extensive chromosomal rear-
rangements, probably as a result of chromosomal speciation hardly affecting this ge-
nus (parapatric and stasipatric) [Lewis, 1966; White, 1978; Reig, 1980; King, 1993].
As species differentiation has traditionally been completed through morphological
recognition, it makes it difficult to assign the real number of species in those genera
where morphological evolutionary stasis occurs (probably what has occurred in the
cases of Alouatta and Aotus in the Neotropical Primates) [Ruiz-García et al., 2016a,
c]. Therefore, our second aim is to provide new molecular data (a larger mitochon-
drial DNA fraction sequenced) and to analyze new Alouatta individuals (156 indi-
viduals) from new geographical areas to better understand the phylogenetic relation-
ships among Alouatta taxa.
Materials and Methods

A total of 156 howler monkeys of different taxa were sampled across several countries of
Latin-America (Fig. 1). Here, we provide a breakdown of number and taxonomic type per sample
location. We sequenced 124 individuals of A. palliata and related taxa. Of these, 103 were A. p.
palliata from across Costa Rica, Nicaragua, and Honduras. Another 14 were A. p. aequatorialis
(3 from Colombia, [1 from Los Katios National Park in the Choco Department and 2 from the

DOI: 10.1159/000480502
Color version available online
Cuba
Mexico Cayman Is. Haiti Dominican Rep.

Anguilla
Belize Jamaica Antiqua and Barb.
Guatemala Dominica
Honduras
El Salvador Saint Lucia
Nicaragua
Curaçao Grenada
Costa Rica Trinidad and Tobago
Panama
Venezuela
A. c. coibensis French Guiana
Guyana
Colombia Suriname
Species distribution
A. belzebul belzebul (n = 1)
Ecuador
A. caraya (n = 1)
A. coibensis coibensis (n = 1)
A. coibensis trabeata (n = 2)
A. insulanus (n = 2)
A. macconnelli (n = 2) Brazil
A. palliata aequatorialis (n = 14) Peru
A. palliata mexicana (n = 4)
A. palliata palliata (n = 103)
A. pigra (n = 7)
A. sara (n = 8) Bolivia
A. seniculus seniculus (n = 11)
Fig. 1. Map with the geographical origins and sample sizes (n) of the A. palliata taxa and other
Alouatta species analyzed for 5,744 bp of mitochondrial DNA.
Cordoba Department], and 11 from Ecuador [2 from Esmeraldas Province and 9 from Manabí
Province]). Four were A. p. mexicana (hair samples donated by the Dirección General de Zoológi-
cos y Vida Silvestre in Mexico City). Two were Panamanian individuals (El Montuoso, Azuero
Peninsula) of the A. c. trabeata taxon. There was also 1 individual of A. c. coibensis (Coiba Island).
Another 32 specimens of different Alouatta taxa were sampled. There was 1 A. caraya from San-
ta Ana de Yacumo, Mamore River, Bolivia. Seven were A. pigra (3 from San Miguel, Peten area
in Guatemala; 3 animals from Mexico; [2 from Tabasco State, Tenosique and Macuspana, and 1
from Palenque, Chiapas State], and 1 from the Community Baboon Sanctuary, Bermudian Land-
ing, Belize). There were 8 A. sara; 4 of the 8 were from Bolivia (1 in Mayo-Mayo, Mamore River,
Beni Department, 2 from Puerto Mamoré, Cochabamba Department, and 1 from Santa Ana de
Madidi, La Paz Department). The remaining 4 were from Peru (2 were from Chonta and La
Torre, Tambopata River, 1 from the Taricaya River, and another from Infierno, Madre de Dios
River). There were 11 A. seniculus; 6 of these were from Peru (2 from Santa Rita, Javari River, 1
from Vencedores del Zapote, Napo River, 1 from Puerto Inca, Pachitea River, and 2 from Re-
quena, Ucayali-Tapiche rivers). The remaining 5 were from Colombia (3 from the Antioquia
Department and 2 from the Valle del Cauca Department). Two samples came from Trinidad and
Tobago (both individuals from the Mayaro area). There were also 2 samples of A. macconnelli
(both individuals from Carnopi River on French Guiana) and 1 of A. belzebul belzebul (Tocantins
River, Para State, Brazil; sample donated by Dr. H. Schneider). For outgroups, we used 10 indi-
viduals of L. lagotricha cana from different localities of the Madeira River (Brazil) and 19 indi-
viduals of A. geoffroyi from different locations within Costa Rica. DNA was obtained from hair
for all of the samples with the exception of A. palliata from Costa Rica, from which DNA was
obtained from blood.
Molecular Procedures
The DNA from blood was extracted using the phenol-chloroform procedure [Sambrock et
al., 1989], while DNA samples from hairs was extracted with 10% Chelex resin [Walsh et al.,

DOI: 10.1159/000480502
1991]. The samples were sequenced for 6 mitochondrial codifying genes: COI (657 bp), COII (720
bp), Cyt-b (1, 140 bp), ND4 (710 bp), ND5 (1, 806 bp) and ND6 (534 bp). These genes, especially
COI, COII, and Cyt-b, have shown a very good phylogenetic signal at the intraspecific level in the
Neotropical primate species [Andrews and Easteal, 2000; Ascunce et al., 2003; Lavergne et al.,
2010]. They are genes without indels and they are easy to align. The gene sequences employed
summed up to 5,567 bp. For the amplification conditions (polymerase chain reaction) and prim-
ers used, see Ashley and Vaughn [1995], Collins and Dubach [2000a], Folmer et al. [1994], Mont-
gelard et al. [1997], Morales-Jiménez et al. [2015], Pastorini et al. [1998], and Ruiz-García et al.
[2010, 2012a, b].
All amplifications, including the positive and negative controls, were checked in 2% agarose
gels. The gels were visualized in a Hoefer UV Transilluminator. Both mitochondrial DNA strands
were sequenced directly using a BigDye Terminator v3.1 (Applied Biosystems Inc.). We used a
377A (ABI) automated DNA sequencer and sequenced in both directions to ensure sequence ac-
curacy.
Data Analysis
Phylogenetics Procedures
MrModeltest v2.3 [Nylander, 2004] and Mega 6.05 software [Tamura et al., 2013] were ap-
plied to determine the best evolutionary mutation model for the sequences analyzed for each
individual gene, for different partitions, and for all the concatenated sequences. The Akaike in-
formation criterion (AIC) [Akaike, 1974; Posada and Buckley, 2004] and the Bayesian informa-
tion criterion (BIC) [Schwarz, 1978] were used to determine the best evolutionary nucleotide
model.
Phylogenetic trees were constructed using 2 procedures: maximum likelihood tree (MLT)
and Bayesian analysis (BI). (1) An MLT was obtained using RAxML v.7.2.6 software [Stamataki,
2006] for the 6 mitochondrial genes. To select the best fitting model, 50 independent iterations
were run using 3 data partitions (codon 1, codon 2, codon 3). Additionally, 50 iterations were run
using 2 data partitions (codons 1 + 2 combined, codon 3). For each analysis, the HKY + G mod-
el [Hasegawa et al., 1985] was used to search for the MLT and topological support was estimated
with 500 bootstrap replicates [Stamatakis, 2006]. (2) BI was performed using a HYK + G model
with the gamma distribution rate varying among sites, because it was determined to be the better
model using MrModeltest v2.3 software. BI 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, codon 3) with 6 MCMC chains sampled every 1,000 generations for 40 million gen-
erations after a burn-in period of 4 million generations. We checked for convergence using Trac-
er 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. The results from
different runs were combined using LogCombiner v1.8.0 software [Rambaut and Drummond,
2013a] and TreeAnnotator v1.8.0 software [Rambaut and Drummond, 2013b]. A Yule speciation
model and a relaxed molecular clock with an uncorrelated log-normal rate of distribution [Drum-
mond et al., 2006] was used. Posterior probability values provide an assessment of the degree of
support of each node on the tree. Values higher than 0.95 were considered strong support for
monophyletic clades [Erixon et al., 2003; Huelsenbeck and Rannala, 2004]. Trees were visualized
in the FigTree v1.4 software [Rambaut, 2012]. This program was run to estimate the time to most
recent common ancestor for different nodes of BI. We used a prior of 16 ± 1 million years ago
(MYA; 95% confidence interval 14.36–17.64) for the split of the ancestor of Alouatta and the an-
cestor of Ateles-Lagothrix, and a prior of 11 ± 1 MYA (95% confidence interval 9.35–12.64) for
the split between the ancestors of Ateles and Lagothrix, following Opazo et al. [2006], Perelman
et al. [2011], Springer et al. [2012], and Jameson Kiesling et al. [2015], which in turn are estimates
that agree quite well with the paleontological record [MacFadden, 1990; Rosenberger, 2002; Kay,
2015].
To estimate possible divergence times among the haplotypes found in Alouatta for the 6
mitochondrial genes studied, we constructed a Median Joining Network (MJN) [Bandelt et al.,
1999] using Network 4.6.0.1 software (Fluxus Technology Ltd). Additionally, the ρ statistic [Mor-
ral et al., 1994] and its standard deviation [Saillard et al., 2000] were estimated and transformed

DOI: 10.1159/000480502
into years. The ρ statistic is unbiased and highly independent of past demographic events. This
approach is named “borrowed molecular clocks” and uses direct nucleotide substitution rates
inferred from other taxa [Pennington and Dick, 2010]. Thus, we used 1e mutation each 195,000
years for the howler monkeys following an average value for different mitochondrial genes ob-
tained for different primate taxa [Ashley and Vaughn, 1995; Ruiz-García and Pinedo-Castro,
2010; Ruiz-García et al., 2016a]. One advantage of the MJN procedures compared to traditional
trees is that they explicitly allow for the coexistence 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 taxa began to evolve first and also to
identify the more recently derived taxa.
Genetic Diversity and Heterogeneity Analyses

We used the following statistics to determine the genetic diversity, for different Alouatta
taxa: number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), average num-
ber of nucleotide differences (k), and θ statistic by sequence. These genetic diversity statistics were
calculated with the DNAsp 5.1 [Librado and Rozas, 2009] and Arlequin 3.5.1.2 programs [Excof-
fier and Lischer, 2010].
We used the γST, NST, and FST statistical tests to measure genetic heterogeneity and possible
indirect gene flow estimates among different taxa within A. palliata and among different species
of Alouatta [Hudson et al., 1992]. Significance was estimated with permutation tests using 10,000
replicates.
We used the Kimura 2P genetic distance [Kimura, 1980] to determine the percentage of
genetic differences within different A. palliata taxa and among the different species of Alouatta
studied. The Kimura 2P genetic distance is a standard measurement for barcoding tasks [Hebert
et al., 2003, 2004]. In the current work, we only show these genetic distances with the mitochon-
drial COII gene. For Neotropical primates and Hominoidea, there is an average genetic distance
at the COII gene of 15.68% ± 1.73% among genera, 5.82% ± 1.64% among species within a genus,
and 2–4% for subspecies within species [Collins and Dubach, 2000b; Ascunce et al., 2003]. We
take these values as orientative ones in an identical way as some barcoding markers are used for
other taxonomic groups by other authors (for instance, Kartavtsev [2011] at the mitochondrial
COI gene from 20,731 vertebrate and invertebrate animal species; 3.78 ± 1.18% for subspecies or
semispecies, and 11.06 ± 0.53% for species within a genus).
Demographic Evolutionary Changes

To determine possible historical female population changes in 2 A. palliata populations
(Central American and Colombian-Ecuadorian), we relied on 3 procedures. First, 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 [Harpending et
al., 1993; Harpending, 1994] to determine the similarity between the observed and the theoretical
curves. Second, 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 permutations.
Third, a Bayesian skyline plot was obtained for the concatenated mitochondrial sequences 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 40,000,000
generations (the first 4,000,000 were 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 mar-
ginal 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 (HPD) and the trace of the root height
as the treeModel.rootHeight. However, Bayesian demographic analyses based on a single marker
are often misleading and inaccurate [Heller et al., 2013; Grant, 2015]. Thus, those results should
be taken with caution.

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Results
Phylogeography of A. palliata and Phylogenetic Inferences for Different Alouatta

Species
BIC and AICc showed that the HKY + G (122,119.6 and 84,481.401, respective-
ly) was the best model explaining the nucleotide substitution evolution for the 6 mi-
tochondrial genes we analyzed. The 2 phylogenetic trees (MLT, BI; Fig. 2a, b) showed
similar patterns with very few differences. The 2 trees showed a clear differentiation
between the trans- and cis-Andean Alouatta taxa (99%, and p = 1, respectively). In
the trans-Andean Alouatta clade, the 2 trees showed the split between A. palliata and
A. pigra (86%, and p = 1). It is noteworthy to mention that of the 4 A. p. mexicana
analyzed, 3 showed haplotypes of A. pigra and only 1 showed a haplotype of A. pal-
liata. One tree (MLT) showed that the nonhybridized specimen of A. p. mexicana was
the most differentiated specimen within the A. palliata clade (only 27% of bootstrap).
The finding is in agreement with the genetic distances (see below) because the hap-
lotype of this specimen was the most differentiated within A. palliata. However, in
BI, as well as the MJN analysis, this specimen of A. p. mexicana was intermixed with
other A. palliata with other geographical origins. Another interesting trait in this
analysis is that the 2 individuals of A. c. trabeata sampled from the Azuero Peninsula
and the individual of A. c. coibensis (Coiba Island) in Panama were undifferentiated
from the specimens of A. palliata. They intermixed among them.
The individuals of A. palliata from Colombia and Ecuador (putatively A. p. ae-
quatorialis) in the MLT formed little clusters within the big cluster of A. p. palliata
from Costa Rica, Nicaragua, and Honduras. The 3 Colombian specimens showed a
cluster with 63% bootstrap and 7 Ecuadorian A. palliata specimens formed another
cluster with a very low bootstrap (9%). Nevertheless, 4 other Ecuadorian specimens
were intermixed with animals from Costa Rica. In BI, the Colombian cluster (p =
0.94) and the Ecuadorian cluster (6 individuals, p = 0.91) were also present but inter-
mixed with individuals of A. p. palliata from Costa Rica, Nicaragua, and Honduras.
Thus, we detected no clear differentiation between the putative A. p. palliata and A.
p. aequatorialis subspecies with mitochondrial genes. Additionally, no spatial struc-
ture among the individuals of A. p. palliata in Costa Rica, Nicaragua, and Honduras
were detected in either of the phylogenetic trees.
A. belzebul (74% and p = 1) was the first ancestor to diverge in the cis-Andean
Alouatta clade. The ancestor of A. caraya diverged next (MLT; for BI the ancestor of
A. caraya + A. macconnelli). A. macconnelli was the first of the red howler monkeys
to diverge, followed by the ancestor of A. sara (Madre de Dios River Basin, southern
Peru and Bolivia). These results support the findings of Cortés-Ortiz et al. [2003] and
Ruiz-García et al. [2016a]. From a molecular point of view these 2 taxa clearly dif
ferentiated from A. seniculus, a taxon of which they were sometimes considered sub-
species.
A relevant result of this work is the relationship of 2 individuals of red howler
monkey from Trinidad Island. It is the first time that this red howler monkey taxon
was genetically analyzed. The most related taxon was A. s. seniculus from Colombia
and Peru, but it was clearly differentiated from it forming a monophyletic clade (99%
and p = 1), which could have interesting systematic consequences. No significant spa-
tial structure seems to be present within A. s. seniculus throughout Colombia and in
the northern and central Peruvian Amazon.

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Fig. 2. MLT with 156 howler

monkeys (Alouatta) studied
for 5,744 bp of mitochondrial
DNA. The numbers in the
nodes are bootstrap percent-
ages.
(Figure continued on next pages.)

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Fig. 3. MJN with haplotypes found at 5,744 bp of mitochondrial DNA for the 156 howler monkeys
(Alouatta) analyzed. Green circles, L. l. cana; light gray, A. geoffroyi; dark blue circles, A. senicu-
lus; pink circle, A. insulanus; black circles, A. macconnelli; light brown circle, A. belzebul; light
blue circle, A. caraya; white circles, A. sara; orange cicles, A. pigra; yellow circles, A. p. palliata;
dark brown circles, A. p. aequatorialis; gray circles, A. c. coibensis and A. c. trabeata; blueish green
circle, A. p. mexicana. Red circles indicate missing intermediate haplotypes.
The MJN analysis (Fig. 3) showed the possible evolution of the haplotypes of the
Alouatta taxa analyzed. Following this analysis, the cis-Andean Alouatta taxa first ap-
peared and gave origin to the trans-Andean Alouatta taxa. The haplotypes of the current
A. s. seniculus were the first to appear in Alouatta, followed by those of A. insulanus, A.
macconnelli, A. belzebul, A. caraya, and A. sara. This last taxon has cis-Andean Alouat-
ta haplotypes more related to those of the trans-Andean Alouatta taxa. The genetic dis-
tance analysis (see below) also showed A. sara as the cis-Andean Alouatta taxon with
the smallest genetic distances in regard to the trans-Andean Alouatta species. Of both
trans-Andean Alouatta species, the haplotypes of A. pigra seem to have appeared first.
The haplotypes of A. palliata (the youngest Alouatta taxon analyzed) seem to have aris-
en out of these A. pigra haplotypes. Haplotype 9 of A. palliata was shared by individuals
of A. p. palliata, A. p. aequatorialis, and A. coibensis. Therefore, these 3 putative taxa are
probably a unique taxon. Several different haplotypes, both of A. p. palliata and A. p.
aequatorialis, were derived from haplotype 9. Also, haplotype 10 is highly represented
in many individuals sampled in Costa Rica, Nicaragua, and Honduras. The haplotype
of A. p. mexicana is derived from this highly represented Central American haplotype.

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Table 2. Genetic diversity in the different A. palliata taxa studied and in the different Alouatta
species analyzed for mitochondrial sequences with a length of 5,744 bp
NH Hd π K θ
A. palliata subspecies
A. p. palliata 14 0.567 ± 0.054 0.0025 ± 0.0010 1.806 ± 1.050 11.907 ± 3.197
A. p. aequatorialis 9 0.912 ± 0.059 0.0094 ± 0.0036 6.736 ± 3.379 11.949 ± 4.738
A. p. trabeata 1 0 0 0 0
Alouatta species
A. palliata 23 0.674 ± 0.043 0.0038 ± 0.0010 2.694 ± 1.443 19.18 ± 4.812
A. pigra 6 0.844 ± 0.103 0.0072 ± 0.0016 5.178 ± 2.738 5.302 ± 2.471
A. seniculus 7 0.909 ± 0.066 0.0058 ± 0.0009 4.145 ± 2.232 4.097 ± 1.937
A. sara 5 0.786 ± 0.151 0.0202 ± 0.0088 14.43 ± 7.246 21.98 ± 9.779
The statistics estimated were the number of haplotypes (NH), the haplotypic diversity (Hd),
the nucleotide diversity (π), the average number of nucleotide differences (K) and the θ statistic
(Neμ; Ne = effective female population size; μ = mutation rate per generation) by sequence.
Temporal Splits among Alouatta Taxa

The BI estimation for the split of the ancestor of the cis- and trans-Andean Al-
ouatta taxa was around 6.9 MYA (95% HPD: 7.85–6.02 MYA), which is very similar
to that obtained with the MJN procedure (between A. sara and A. pigra: 6.045 ± 0.585
MYA; between A. s. seniculus and A. pigra: 6.268 ± 0.418 MYA).
The mitochondrial radiation within the cis-Andean Alouatta clade was around
4.27 MYA (95% HPD: 7.2–3.78 MYA; split of the ancestor of A. belzebul from the
remaining cis-Andean Alouatta taxa) with the BI estimate. This was considerably
lower in the MJN estimate (2.86 ± 0.37 MYA; split between the A. s. seniculus and A.
sara haplotypes). The mitochondrial differentiation between the ancestors of A. pigra
and A. palliata was 3.64 MYA (95% HPD: 4.69–3.31 MYA) for the BI estimation, re-
markably similar to that obtained with the MJN procedure (3.705 ± 0.850 MYA).
The mitochondrial diversification within the detected haplotypes of A. palliata
was 3.56 MYA (95% HPD: 3.06–4.48 MYA) with the BI estimation. It was consider-
ably lower with the MJN estimate (0.375 ± 0.149 MYA). The same occurred for the
diversification of the current haplotypes of A. pigra. The BI estimate showed 1.95
MYA (95% HPD: 3.72–1.08 MYA), whereas the MJN estimate offered 0.585 ± 0.163
MYA. The BI estimates showed that the mitochondrial diversification first began in
A. palliata. In contrast, the MJN estimates supported mitochondrial diversification
first occurring in A. pigra.
The temporal split between A. s. seniculus and A. s. insulanus occurred around
2.46 MYA (95% HPD: 4.08–1.62 MYA) with the BI estimate, and considerably more
recently with MJN (0.829 ± 0.162 MYA). The mitochondrial diversification within A.
s. seniculus was 1.93 MYA for BI and 0.656 ± 0.237 MYA for MNJ. In A. sara, the BI
and MJN values were 1.75 MYA (95% HPD: 3.42–0.71 MYA) and 1.389 ± 0.187 MYA,
respectively.
The temporal splits between the cis- and trans-Andean Alouatta groups, as well
as the split between the ancestors of A. pigra and A. palliata, were remarkably similar

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Table 3. Genetic heterogeneity between pairs
1 2 3 4 5 6
A. palliata subspecies
1 – 0.1708 0.4489*
2 0.1725 – 0.0733
3 0.4529* 0.0754 –
Alouatta species
1 – 0.9404** 0.8576** 0.8880** 0.9631** 0.9680**
2 0.9376** – 0.7275** 0.9125** 0.9238** 0.8432**
3 0.8530** 0.7242** – 0.8071** 0.8134** 0.7891**
4 0.8855** 0.9086** 0.8019** – 0.9420** 0.9445**
5 0.9617** 0.9217** 0.8109** 0.9395** – 0.9697**
6 0.9666** 0.8415** 0.7869** 0.9419** 0.9687** –
Population FST (below the main diagonal) and NST (above the main diagonal) pairs among
the different A. palliata taxa and Alouatta species analyzed (1, A. palliata; 2, A. seniculus; 3, A. sara;
4, A. pigra; 5, A. macconnelli; 6, A. insulanus) for mitochondrial sequences with a length of 5,744
bp. * p < 0.05, ** p < 0.001.
for both procedures used. However, for other temporal splits within the Alouatta ge-
nus, the MJN estimates were systematically lower than the BI estimates.
Genetic Diversity in A. palliata and in Other Alouatta Species

The genetic diversity estimates for the overall sample of A. palliata analyzed
showed that this taxon has lower values in reference to other Alouatta species studied
(Table 2). A. palliata showed a value of Hd = 0.674 ± 0.043 and π = 0.0038 ± 0.001,
whereas A. seniculus (Hd = 0.909 ± 0.066, π = 0.0058 ± 0.0009), A. sara (Hd = 0.786 ±
0.151 and π = 0.0202 ± 0.0088), and A. pigra (Hd = 0.844 ± 0.103 and π = 0.0072 ±
0.0016) showed higher values.
Within A. palliata, the putative taxon A. p. aequatorialis in Colombia and Ecua-
dor (Hd = 0.912 ± 0.059 and π = 0.0094 ± 0.0036) yielded considerably higher genet-
ic diversity levels than the putative taxon A. p. palliata in Central America (Hd = 0.567
± 0.054 and π = 0.0025 ± 0.001).
Genetic Heterogeneity and Genetic Distances among A. palliata Taxa and

among Alouatta Species
The relative genetic heterogeneity statistics revealed comparatively little differ-
entiation among the 3 taxa enclosed within A. palliata (aequatorialis, coibensis tra-
beata, and palliata; γST = 0.1043, p = 0.274; FST = 0.183, p = 0.042, and NST = 0.1806,
p = 0.048). These results agree quite well with the phylogenetic analyses previously
mentioned. Although some of these values are slightly significant, they are relatively
small and their respective theoretical gene flow estimates are higher than 1 (Nm =
4.29, 2.27, and 2.23, respectively). Therefore, important historical gene flow has oc-
curred among these taxa. Supportive of previous results, this finding agrees quite well
with the existence of a unique A. palliata taxon. The genetic heterogeneity between

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Table 4. Kimura 2P genetic distances among the different A. palliata subspecies and Alouatta
species considered at the mitochondrial COII gene
1 2 3 4 5 6 7 8 9 10
A. palliata taxaa
1 – 0.2 0.2 0.1 0.4
2 0.8 – 0.2 0.1 0.5
3 0.5 0.8 – 0.2 0.5
4 0.2 0.6 0.2 – 0.5
5 1.6 2.1 1.8 1.6 –
Alouatta species and outgroupsb
1 – 1.1 0.9 0.9 0.9 1.0 1.0 1.0 1.4 1.4
2 8.0 – 1.2 1.0 0.7 1.2 1.0 1.1 1.5 1.5
3 5.5 8.4 – 0.7 1.1 0.6 0.8 1.0 1.3 1.5
4 7.0 8.8 4.9 – 0.9 0.8 0.7 0.9 1.4 1.6
5 6.7 4.9 7.6 7.3 – 1.0 0.9 1.1 1.5 1.4
6 6.3 8.5 2.3 5.3 7.8 – 0.8 1.0 1.4 1.6
7 5.4 7.3 4.8 6.0 7.4 4.7 – 0.8 1.5 1.6
8 7.1 7.9 6.8 7.6 6.9 7.0 6.5 – 1.4 1.6
9 14.3 12.6 12.0 14.1 15.3 13.8 13.8 13.8 – 1.3
10 13.6 15.1 14.3 15.8 15.8 16.2 15.8 15.2 12.2 –
Below, genetic distance values in percentages (%); above, standard errors in percentages (%).
a
Subspecies considered: 1, A. p. palliata; 2, A. p. aequatorialis (Ecuador); 3, A. p. aequatorialis
(Colombia); 4, A. c. trabeata; 5, A. p. mexicana.
b Alouatta species considered: 1, A. caraya; 2, A. palliata; 3, A. seniculus; 4, A. sara; 5, A. pigra;
6, A. insulanus; 7, A. macconnelli; 8, A. belzebuth; 9, A. geoffroyi; 10, L. l. cana.
taxa pairs (Table 3) showed relatively small values in all cases for the 3 comparisons.
Disappointingly, we only had 1 sequence of a pure sample of A. p. mexicana, which
prevented a similar analysis of this last taxon.
The Kimura 2P genetic distance matrix based only on the mitochondrial COII
gene (Table 4) among 5 different A. palliata populations (aequatorialis in Ecuador,
aequatorialis in Colombia, coibensis in Panama, palliata in Costa Rica, Honduras,
and Nicaragua, and mexicana in Mexico) showed very small values among the first 4
populations (0.2–0.8%). The genetic distances between these 4 populations and the
mexicana are slightly higher (1.6–2.1%), but they are still relatively small.
The relative genetic heterogeneity statistics among the different Alouatta species
showed very high values (γST = 0.8299, p = 0.001; FST = 0.8904, p = 0.0005; and NST =
0.8933; p = 0.0005) and their respective gene flow values were very low (Nm = 0.10,
0.06 and 0.06, respectively). These findings agree quite well with the fact that these
mitochondrial sequences have great power to differentiate Alouatta species. The
genetic heterogeneity between species pairs (Table 3) showed extremely high values
for all comparison pairs. Several facts are remarkable. First, A. sara was the species
which simultaneously showed the most reduced genetic heterogeneity with both cis-
Andean Alouatta species (seniculus, macconnelli, and insulanus) as well as with trans-

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1.E3
1.E2
Effective population size of females
1.E1
1.E0
1.E–2
1.E–3
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
a Time, millions of years
1.E1
Effective population size of females
1.E0
1.E–2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
b Time, millions of years
Fig. 4. Bayesian skyline plot analysis to determine possible demographic changes across the nat-
ural history of 2 A. palliata populations: A. p. palliata from Central America (a) and A. p. aequa-
torialis from Colombia and Ecuador (b).
Andean Alouatta species (palliata and pigra). This could mean that the ancestor of A.
sara had some role in the origin of these 2 cis-Andean and trans-Andean groups.
Second, the 4 red coat Alouatta taxa (seniculus, sara, macconnelli, and insulanus) were
highly differentiated from a molecular point of view, even though scientists have re-
cently considered them as subspecies of A. seniculus. Third, insulanus is highly dif-

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ferentiated from both A. s. seniculus and A. macconnelli. Some authors [e.g., Groves,
2001] considered that the red howler monkey from Trinidad Island could be A. mac-
connelli. Our results indicate that none of these species are likely to be the red howler
monkey inhabiting Trinidad Island.
The Kimura 2P genetic distance matrix with just the mitochondrial COII gene
among 8 Alouatta taxa is shown in Table 4. The lowest genetic distance for A. pallia-
ta was with A. pigra (4.9%), whereas the genetic distance range with the cis-Andean
Alouatta species was 7.3–8.8%. The lowest genetic distances for A. caraya with the
other cis-Andean Alouatta species were with A. maconnelli (5.4%) and with A. se-
niculus (5.5%). In this analysis, A. insulanus clearly showed a closer relationship with
A. seniculus (2.3%) than with A. macconnelli (4.7%). All the Alouatta species showed
a genetic distance with L. l. cana within a range of 13.6–16.2%. The range was 12.0–
15.3% for A. geoffroyi.
Demographic Changes in the A. palliata

When we only analyzed the Central American A. palliata sample, the evidence
of female demographic growth was high. Five out of 6 tests showed significant popu-
lation expansions, with the exception of the R2 test. The mismatch distribution (rg
statistic) also showed a significant population expansion for the Central American A.
palliata sample. Assuming that a generation in Alouatta is around 7 years, this expan-
sion began around 850,000 years ago. The Bayesian skyline plot showed female pop-
ulation growth in the last 500,000 years (Fig. 4). The growth was exceptionally strong
in the last 300,000 years. In contrast, the Colombian and Ecuadorian A. palliata sam-
ple showed less evidence of female population growth. Only 3 out of 6 tests were sig-
nificant for population expansions (Fu and Li D*, Fu and Li F*, and Tajima tests).
The mismatch distribution (rg statistic) did not detect any demographic change for
this population. The Bayesian skyline plot detected a unique change – a population
decrease in the last 10,000–15,000 years. Thus, we show clear evidence in favor of
more important female population growth for the Central American A. palliata pop-
ulation than for the Colombian and Ecuadorian one.
Discussion
Molecular Relationships among Different Taxa of the A. palliata Group and Its
Systematics
To date, this study contains the greatest sample size (124 individuals) and longest
mitochondrial gene sequences (5,567 bp) to analyze the phylogenetic relationships
of different taxa within A. palliata, including A. coibensis. There were no relevant
molecular differences among the individuals of putative taxa such as A. p. palliata,
A. p. aequatorialis, A. c. coibensis, and A. c. trabeata. We detected no spatial structure
within A. p. palliata throughout Costa Rica, Nicaragua, and Honduras. Indeed, some
haplotypes were widely distributed throughout a large part of the distribution range
of A. palliata. For instance, haplotype 9 was distributed in Honduras and Costa Rica,
in both endemic Panamanian putative taxa (A. c. coibensis and A. c. trabeata), and in
Ecuador. Therefore, the traditional morphological subspecies, A. p. palliata and A. p.
aequatorialis, were not molecularly differentiable throughout Honduras, Nicaragua,
Costa Rica, Colombia, and Ecuador. Although Cortés-Ortiz et al. [2003] conjectured

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that a minor phylogeographic break separating northern and southern A. palliata
could be located near Panama’s Sona Peninsula [Bermingham and Martin, 1998], we
did not find any such geographical point break. In the MLT, only the 3 Colombian
specimens formed a small significant cluster. In the BI, this Colombian cluster and
another small Ecuadorian cluster only showed a limited, but significant, importance.
However, these clusters were inside a large cluster of A. p. palliata. Even the 3 Pana-
manian exemplars sampled (A. c. coibensis and A. c. trabeata) could not be molecu-
larly differentiated from A. palliata. Thus, molecular results, as well as those of Cor-
tés-Ortiz et al., [2003], do not validate A. coibensis as a separate species from the for-
merly known A. palliata panamensis. This disagrees with the claim of Froehlich and
Froehlich [1987], who analyzed dermal ridge patterns in hands and feet. They de-
tected a different frequency in dermatoglyphic patterns between A. palliata and the
putative A. coibensis. However, this could be the result of founder effect or genetic
drift on the constitution of the howler monkeys on the Azuero Peninsula and on
Coiba Island. Therefore, neither the Biological Species Concept [Mayr, 1942, 1963]
nor the Phylogenetic Species Concept [Cracraft, 1983] help to differentiate A. coiben-
sis from A. palliata. The minor differences of the mitochondrial gene sequences be-
tween A. coibensis and A. palliata support a very recent split between the taxa. These
results favor the existence of only 1 species, not 2.
A. p. mexicana (genetic distance at mitochondrial COII gene: 1.6–2.1%) was the
most genetically differentiated taxon within A. palliata as well as that most morpho-
logically differentiated. This genetic distance is within the range (2–4%) of different
subspecies for Neotropical primates [Collins and Dubach, 2000b; Ascunce et al.,
2003]. Cortés-Ortiz et al. [2015] recognized 5 subspecies in A. palliata (A. p. mexi-
cana, A. p. palliata, A. p. coibensis, A. p. trabeata, and A. p. aequatorialis). Neverthe-
less, we postulate the existence of only 2 subspecies within A. palliata (A. p. palliata
and A. p. mexicana).
Molecular Relationships between A. palliata and A. pigra and the Origins of the
Central American Howler Monkeys
A. palliata and A. pigra are molecularly differentiated species, first demonstrated
by Cortés-Ortiz et al. [2003]. However, A. pigra was traditionally considered a sub-
species of A. palliata (A. p. pigra, Lawrence in 1933, from Uaxactún, Petén, Guate-
mala). In the current work, we estimated that a temporal split between these 2 species
occurred around 3.6–3.7 MYA. This estimate is very similar to the value of 3 MYA
calculated by Cortés-Ortiz et al. [2003] and the estimate of 3.12 MYA determined by
Ruiz-García et al. [2016a]. These results stress the importance of the Panama Isthmus
in the speciation of Central American howler monkeys [Fleagle, 1988]. The final clo-
sure of the Isthmus of Panama is estimated to have occurred about 3 MYA [Coates et
al., 1992], which agrees quite well with these temporal splits.
Also, the karyotypes of A. palliata and A. pigra are considerably different. The
most frequent A. palliata karyotype has 2N = 53 with an X1X2Y trivalent in males [Ma
et al., 1975, Solari and Rahn, 2005], whereas A. pigra has 2N = 58 and an X1X2Y1Y2
quadrivalent in males [Steinberg et al., 2008].
The split between the trans- and cis-Andean howler monkeys showed that the
ancestors of the cis-Andean Alouatta gave origin to the ancestors of the trans-Ande-
an Alouatta around 6.0–6.9 MYA. The BI estimate of Ruiz-García et al. [2016a] was
around 7.21 MYA, whilst the estimate of Cortés-Ortiz et al. [2003] was 6.8 MYA, and

DOI: 10.1159/000480502
the MJN estimate of Ruiz-García et al. [2016a] was 6.73 MYA. This temporal split
coincides with the formation of the Northern Andes [Lundberg et al., 1998].
It seems clear that the South American howler monkeys began their mitochon-
drial evolution much earlier than the trans-Andean howler monkeys. The data of
Cortés-Ortiz et al. [2003] correlated very well with our affirmation. These authors
estimated that the diversification of the South American howler monkey species oc-
curred around 4.8–5.1 MYA (4.27 MYA in our BI estimation), near the end of the
Miocene, while the diversification of the cis-Andean howler monkeys, as we showed,
was around 3–3.7 MYA.
This is opposite to the ideas of Villalobos et al. [2004]. These authors claimed that
A. palliata was the most basal taxon for the genus and the sister taxon to all other
Alouatta species. Furthermore, they specified that this agreed with previously report-
ed topologies [Meireles et al., 1999; Bonvicino et al., 2001]. However, none of these
cited works included sequences of A. palliata, and therefore their conclusions are mis-
leading. To support their hypothesis, they claimed that A. palliata had been described
as having an XY system for males and XX for females, a claim based on a population
studied on Barro Colorado Island in Panama [Ma et al., 1975]. This is the original
sexual chromosome system in the vast majority of mammals and primates, but the
sexual chromosome system is more complex in many Alouatta taxa. Therefore, any
“normal” XX/XY system could be considered the original one. However, the findings
of Ma et al. [1975] indicated a different sex chromosome system for the Panamanian
animals. The system is not XX/XY as claimed by Villalobos et al. [2004]. Instead, fe-
males have 2N = 54 and X1X1X2X2 and males have 2N = 53 and X1X2Y. Thus, there is
no doubt that both Central American howler monkey taxa are differentiated species.
This aligns with the work of Smith [1970]. Cortés-Ortiz et al. [2003] detected a 5.7%
difference between A. palliata and A. pigra. Similarly, our calculated value was around
5%. Our molecular results also permited some evaluation of the 2 hypotheses present-
ed by Smith [1970] to account for the origins of A. palliata and A. pigra. His first
model posits a single colonization of Central America from South America. This is
followed by the separation of A. palliata in the Talamancan region of Costa Rica from
A. pigra in the north. His second hypothesis recognizes 2 sequential invasions of Cen-
tral America by the trans-Andean Alouatta ancestor into South America. Also, Cortés-
Ortiz et al. [2003] claimed 2 possible scenarios. (1) Both trans-Andean howler monkey
species would have been separated by forest reduction in Central America during the
last Pliocene-Pleistocene dry-wet glacial periods [Haffer, 1969, 1982, 1997, 2008].
They would have had isolated populations persisting in the highlands of Guatemala,
Mexico, and Belize, and in Costa Rica’s Talamanca Mountains. (2) Parapatric specia-
tion could also be important across the ecotone that separates the drier and lower for-
est of the Mexican Yucatan peninsula occupied by A. pigra, from the moist, tall forests
inhabited by A. palliata. The A. pigra-A. palliata contact zone is placed between the
Grijalva and Usumacinta rivers, with the possibility that either or both of these rivers
might have acted as barriers to gene flow between the 2 howler monkey species.
Our results, however, could give support to other hypotheses. We agree with the
possibility that the ancestor of A. sara (MJN) gave rise to the ancestor of A. pigra in
northern South America. In turn, in northwestern South America, the ancestor of A.
pigra gave rise to the ancestor of A. palliata. Two results strongly support the South
American A. palliata (the putative A. p. aequatorialis) as the original population of
this species: (1) considerable higher genetic diversity than the Central American A.

DOI: 10.1159/000480502
palliata population, and (2) no evidence of population expansion. The Central Amer-
ican A. palliata is the derived population (low genetic diversity and clear evidence of
population expansion). In this sense, our results correlated well with the second hy-
pothesis of Smith [1970] as well as with Ellsworth and Hoelzer [2006] with microsat-
ellites. If the first hypothesis of Smith [1970] and the 2 hypotheses of Cortés-Ortiz et
al. [2003] of 1 unique colonization process of howler monkeys in Central America
[Fleagle, 1988; Rowe, 1996]) were true, we would expect these species to be geneti-
cally similar. A. pigra should have been particularly linked to the northern A. palliata
populations in phylogenetic analyses, which was not the case. For instance, the MJN
procedure showed that the ancestor of A. sara gave origin to the ancestor of A. pigra
and in turn this gave origin to the ancestor of A. palliata. As the A. palliata popula-
tion with the highest levels of genetic diversity is the north-western South American
one (Colombia and Ecuador; the putative A. p. aequatorialis), this should be inter-
preted as the origin of both ancestors of A. pigra and A. palliata being in north-west-
ern South America, and not “in situ” in Central America. Therefore, our results
suggest that both species probably represent independent invasions into Central
America.
It can be considered that both A. palliata and A. pigra migrated into Central
America if we take into consideration the following 2 scenarios. One scenario is mi-
gration through the Isthmus of Panama. Until relatively recently, the scientific com-
munity considered that the Great American Biotic Interchange began around 3 MYA
when the Isthmus was completed [Coates et al., 1992]. However, we now know that
the Isthmus of Panama formation began earlier and seems to be associated with the
Northern Andean uplift, around 24 MYA [Farris et al., 2011]. Additionally, recent
research suggests that the rise of the Isthmus was not as much an event as a process
[Knowlton and Weigt, 1998], which has probably resulted in intermittent periods of
divided and connected lands, possibly over the past 18 MYA. This would mostly be a
Miocene process [Montes et al., 2012, 2015]. If this was the case, then A. pigra arrived
first. If this taxon was able to colonize the region before the final formation of the
Isthmus, A. pigra may have become isolated after crossing into Central America dur-
ing an earlier period of land connection [Knowlton and Weigt, 1998]. The ancestor
of A. pigra could have occupied a larger range throughout Central America with sub-
sequent range reduction following by a second colonization by the ancestor of the
current A. palliata. There are examples with Panamanian and Costa Rican fresh water
fishes that suggest reduced sea levels in that area around 5–7 MYA (Miocene) [Ber-
mingham and Martin, 1998], which could have created land connections used by the
ancestor of the current A. pigra. The fact that the levels of genetic diversity are clear-
ly higher in A. pigra than in A. palliata, although its current geographical range is
considerably smaller, agrees quite well with an older colonization process involving
large reproductive populations of A. pigra.
A second scenario considers migration via the Caribbean island arch. Another
possibility is that the ancestor of A. pigra could have reached Central America via is-
land hopping from northeastern South America through the Caribbean island ar
chipelago. Different fossils (Antillothrix, Insulacebus, Xenothrix, and Paralouatta)
[Cooke et al., 2016] show that monkeys existed on at least some of the islands (i.e.,
Cuba, Haiti, and Jamaica) in the past. Two species from Cuba, P. varonai and P. ma
rianae [MacPhee et al., 2003; MacPhee and Meldrum, 2006] have been related to
Alouattinae [Rosenberger et al., 2015], although no consensus exists among paleopri-

DOI: 10.1159/000480502
matologists [MacPhee et al., 1995; Kay, 2015]. The evolution of the pygmy sloth
(Bradypus pygmaeus) from El Escudo Island in the Panamanian Caribbean Sea could
parallel that of A. pigra. Ruiz-García et al. [2017a] proposed several hypotheses on the
origins of B. pygmaeus. Based on an analysis of BI and mitochondrial control region
sequences, they estimated the split between the lineages of B. pygmaeus and B. tridac-
tylus to have occurred approximately 4.5 MYA. This could be interpreted as occur-
ring within the last Miocene and early Pliocene through the Caribbean Sea island
arch. The sloths migrated from current Guianas and Northeastern Brazil to the Is-
lands of Central America. Based on MJN and factorial analyses of these mitochon-
drial control region sequences, the authors claimed that the ancestor of B. pygmaeus
could have originated from a certain B. variegatus haplotypes similar to what they
detected in the Negro River (Amazon). This split could have occurred around 4.3 ±
0.6 MYA during the Pliocene. For this hypothesis, an island arch in the Caribbean Sea
connecting the northern area of South America to Central America is essential.
Independently of what route was used by the ancestors of A. pigra, A. palliata
seems to have colonized Central America more recently compared to A. pigra. The
low genetic diversity found in the Central American A. palliata resulted from a
founder effect followed by a rapid population expansion within Central America. A
rapidly expanding small founder population of A. palliata would retain low levels of
genetic variation. This species has high fecundity, a flexible diet, and social system,
which enables them to rapidly colonize a wide variety of habitats and experience high
rates of population growth [Fendigan et al., 1998; Clarke et al., 2002]. Thus, the A.
palliata populations have not had sufficient time to regenerate high levels of genetic
variability. This explanation seems to be more parsimonious than the low genetic di-
versity due to multiple population crashes resulting from the yellow fever epidemics
or other recent catastrophic events within Central America.
Cortés-Ortiz et al. [2015] claimed the possibility of the existence of 2 subspecies
within A. pigra (A. p. pigra and A. p. luctuosa). However, our results showed that in-
dividuals from different parts of Mexico, Guatemala, and Belize did not show differ-
entiated clusters by geographical regions. Therefore, we did not find evidence of dif-
ferent subspecies within A. pigra.
Although A. palliata and A. pigra are clearly different species, Cortés-Ortiz et al.
[2007] reported hybridization of individuals with a mosaic of morphological charac-
teristics between A. palliata and A. pigra in Tabasco (Mexico). These included indi-
viduals living in various grades of disturbed vegetation that had characteristics of
both species. We detected 3 specimens of A. p. mexicana (there was no doubt that
morphologically these specimens were A. p. mexicana; therefore, there are no misla-
beled individuals in this case; photos of these exemplars can be requested from the
first author) with mitochondrial DNA of A. pigra. This could be a case of recent hy-
bridization, like that detected by Cortés-Ortiz et al. [2003, 2007]. While hybridization
is a possible explanation, other possible explanations include incomplete lineage sort-
ing, or historic introgression (to be differentiated from recent hybridization) [Ruiz-
García et al. 2016c]. Other cases of hybridization for different Alouatta taxa were
those reported by Aguiar et al. [2007], who described hybridization between A. ca-
raya and A. guariba clamitans (= fusca) in a group of 8 individuals observed near the
Paraná River in Brazil. This was in the ecotone between the rainforest and the Cer-
rado, showing intermediate morphological variation. The chromosome complement
of A. g. clamitans showed a wide variation in diploid number, with 2N = 45, 46, and

DOI: 10.1159/000480502
52 [De Oliveira et al., 2000], and the diploid number for A. caraya as 2N = 52 [Mudry
et al., 1984, 1998]. Thus, perhaps animals with 52 chromosomes should hybridize
without problems.
Some Phylogenetic Insights for the South American Howler Monkeys with
Comments on the Alouatta from Trinidad Island
Our BI and MLT analyses showed the ancestor of A. belzebul as the first to di-
verge in the cis-Andean howlers. This agrees quite well with the results of Cortés-
Ortiz et al. [2003]. These authors showed that the clade of A. belzebul-A. guariba was
the first to diverge in the cis-Andean Alouatta. Indeed, the most extreme karyological
diversity in Alouatta is found in A. guariba. De Oliveira et al. [1995, 1998, 2000] iden-
tified at least 4 different karyomorphic groups: 2N = 52 with XY/XX, 2N = 48 or 50
with XY/XX, 2N = 49 with X1X2Y/50 with X1X1X2X2, and 2N = 45 with X1X2Y/46 with
X1X1X2X2. A. belzebul also presented a similar sex chromosome system of 50 with
X1X1X2X2/49 with X1X2Y [Armada et al., 1987]. Nevertheless, our MJN analysis
showed A. s. seniculus as the taxon with haplotypes most closely related to other gen-
era of Atelidae (and then the most original within Alouatta). De Oliveira et al. [2002]
concluded that the ancestral sex chromosome system for the genus Alouatta should
consist of X1X2/Y1Y2 chromosomes. Four cases of XX/XY patterns were detected in
Alouatta: in 1 population of A. g. clamitans (Espiritu Santo state) [De Oliveira et al.,
2000], in Colombian A. palliata individuals [Torres and Ramírez, 2003] (56 with XY/
XX), in karyotypes of A. s. seniculus [Yunis et al. 1976], and in A. caraya [Mudry et
al., 1984, 1998]. We can discard A. palliata as the original Alouatta species, as we pre-
viously explained. However, any of the ancestors of A. guariba, A. caraya, and A. se-
niculus could be the original ones. Our data cannot resolve this question.
Many classifications have been registered within the red howler monkey com-
plex. Cabrera [1958] determined 4 subspecies: A. s. arctoidea (mainly in Venezuela),
A. s. sara (Bolivia), A. s. seniculus (in Colombia, northwestern Venezuela, and Peru),
and A. s. stramineus (south of the Orinoco River and between the Negro and Branco
rivers, mainly in the northern Brazilian Amazon). Hill [1962] elevated the number of
subspecies within A. seniculus to 9. These include the 4 previously cited plus A. s.
amazonica (in a small area near the mouth of the Purus River within the Amazon
River). Also included were A. s. insulanus (on the Caribbean island of Trinidad), A.
s. juara (on the Juara River, mainly in the Brazilian Amazon), A. s. macconnelli (main-
ly in the Guiana area), and A. s. puruensis (on the Purus River, mainly in the Brazilian
Amazon). This classification was accepted by Stanyon et al. [1995]. Rylands et al.
[1995, 1997] recognized 7 of these forms as valid subspecies. Two previous subspecies
were elevated to the species category by Rylands et al. [1995] – A. arctoidea and A.
sara. A. sara was elevated to full species because Minezawa et al. [1985] demonstrat-
ed that this Bolivian taxon (46 chromosomes) had a very different karyotype from A.
seniculus (44 chromosomes) found in Colombia. Stanyon et al. [1995] and Consi-
gliere et al. [1996] used G banding chromosomal analyses to compare the Bolivian
animals to the Venezuelan A. arctoidea. Both Alouatta taxa differed at least in 16
chromosomal rearrangements. However, these last authors found a sex-chromosome
pattern in A. sara identical to that found in A. macconnelli/A. s. stramineus. The sem-
inal book by Groves [2001] on primate taxonomy only recognized 3 subspecies of A.
seniculus (A. s. arctoidea, A. s. juara, and A. s. seniculus) with A. macconnelli and A. sa-
ra as full species. A. macconnelli was elevated to the full species category because Lima

DOI: 10.1159/000480502
and Seúanez [1991] and Lima et al. [1990] showed that this taxon has a X1X1X2X2/
X1X2Y1Y2 sex chromosome pattern, with the Y chromosome being translocated onto
an autosome. The karyotype of A. macconnelli has a 2N = 47 to 49 chromosomes with
3 microchromosomes [Bonvicino et al., 1995]. The first karyological study with A. s.
seniculus showed 44 chromosomes [Bender and Chu, 1963]. Later, Yunis et al. [1976]
studied 23 specimens and found a range of 43–45 chromosomes and from 3 to 5 mi-
crochromosomes (4 being the most frequent number). There were pericentric inver-
sions in chromosome 13 with an XX/XY pattern. Torres and Leibocini [2001] ana-
lyzed another 12 Colombian A. s. seniculus specimens. All of these animals had 44
chromosomes. Additionally, males in this study had a translocation from the Y chro-
mosome to autosomal chromosome 3 (X1X2Y1Y2). These results disagree with those
of Yunis et al. [1976]. Vassart et al. [1996] studied 42 exemplars from French Guiana
and determined that they were very similar (47–49 chromosomes, 1–3 microchromo-
somes) to those animals classified as A. macconnelli from the Jari River (Brazil) by
Lima et al. [1990]. The sex chromosome pattern in A. macconelli was also found in A.
s. stramineus (Uatama River, Brazil) [Lima and Seuánez, 1991]. Groves [2001] con-
sidered A. s. amazonica and A. s. puruensis to be the same as A. s. juara, and A. s. in-
sulanus to be the same as A. macconnelli. Gregorín [2006] accepted A. macconnelli as
a full species and elevated A. juara and A. puruensis to full species as well. However,
Lima and Seuánez [1991] did not find any relevant chromosomal differences between
A. s. seniculus and A. juara (following the terminology of Gregorín).
We show, as Cortés-Ortiz et al. [2003] did, that A. macconnelli and A. sara are
differentiable species from A. seniculus. Ruiz-García et al. [2007, 2016b] also showed
that the dynamics of DNA microsatellites were different between A. s. seniculus and
A. macconnelli. Also, De Oliveira et al. [2002] revealed that A. macconnelli and A. s.
arctoidea differ by multiple translocations, which may further contribute to repro-
ductive isolation between A. macconnelli and A. seniculus. For A. sara, Minezawa et
al. [1985] showed 2N = 50 for females (with X1X1X2X2) and 2N = 49 for males (X1X2Y),
with a range of 2–6 microchromosomes (6 microchromosomes were more frequent).
Later, Stanyon et al. [1995] determined 2N = 50 for both females and males (with
X1X1X2X2 and X1X2Y1Y2) and 28 acrocentric chromosomes. Clearly, A. sara is chro-
mosomically differentiated from the A. seniculus of Colombia (2N = 44 also with
X1X1X2X2 and X1X2Y1Y2, 26 acrocentric chromosomes with 3–4 microchromo-
somes) [Torres and Leibocini, 2001]. Indeed, Consiglieri et al. [1996] revealed at least
16 chromosome rearrangements, including numerous Robertsonian and tandem
translocations, and intrachromosomal rearrangements between A. sara and A. s. arc-
toidea (the same karyological characteristics as A. s. seniculus) [Consiglieri et al.,
1996; Torres and Ramírez, 2003]. These karyotypic differences can in fact be consid-
ered high enough to ensure the reproductive isolation of A. sara from other red howl-
er monkeys [Stanyon et al., 1995; Consigliere et al., 1996].
Several hypotheses should be proposed to explain the split between A. sara and
A. seniculus. Minezawa et al. [1985] claimed this to be the result of A. sara being pe-
ripherally isolated in the Yungus 2 forest refugia [Haffer, 1969, 1982, 1997, 2008].
However, Cortés-Ortiz et al. [2003] estimated a late Pliocene divergence between A.
seniculus and A. sara (2.4 MYA). We estimated a temporal split between the species
to have occurred around 3.03 MYA (BI) and 2.86 MYA (MJN). For this reason, Cor-
tés-Ortiz et al. [2003] argued against this split because it was too old to be explained
by the Pleistocene refugia hypothesis. However, we must not forget that Haffer [1997,

DOI: 10.1159/000480502
2008] explained that the dry-wet cycles were not exclusive to the Pleistocene and,
therefore, these cycles should also be considered important during the Pliocene. Cor-
tés-Ortiz et al. [2003] speculated that the appearance of the Madeira River could be
essential to the split between these 2 red howler monkey species, because the river
forms the eastern boundary of A. seniculus and A. sara. Based on the stated hypoth-
eses, the Madeira River could have formed around 4 MYA (Paleogeography hypoth-
esis) or around 1–2 MYA (Hydrological Recent Change hypothesis). The first hy-
pothesis is based on the research of Espurt et al. [2007, 2010], who demonstrated that
the Nazca Ridge subduction imprint had a significant influence on the eastern side of
the Andes by means of the Fitzcarrald Arch. Related with the Nazca Ridge subduc-
tion, arc volcanism in the Peruvian Andes ceased around 4 MYA [Rosenbaum et al.,
2005]. Thus, the older time estimate of the Fitzcarrald Arch uplift is around 4 MYA
(Pliocene). This could explain changes in the relationship of the Mamore-Beni Rivers
(or Beni Lake) with other drainage river systems, which could have split the ancestors
of A. sara and A. seniculus. This event could also have isolated the Bolivian dolphins
from other Amazonian pink river dolphins [Ruiz-García et al., 2017b]. The second
hypothesis is based on the works of Ribas et al. [2012] and Lynch-Alfaro et al. [2015].
Bonvicino et al. [2001] commented that the X1X2Y1Y2/X1X1X2X2 sex chromo-
somal system found in A. sara and A. s. arctoidea consists of shared chromosomes
that are identical Y1, Y2, and X2 chromosomes [Stanyon et al., 1995; Consiglieri et al.,
1998]. They did not know whether they were the same as the Y1, Y2, and X2 of A.
macconnelli/A. stramineus. They concluded that these rearrangements could have oc-
curred independently (and involving different autosomes) in 2 separate ancestors, 1
in A. macconnelli/A. stramineus and another in A. sara and A. arctoidea. If this is cor-
rect, it agrees with our finding that the ancestor of A. sara could be found in the ori-
gin of the trans-Andean Alouatta in northwestern South America. The current geo-
graphical distribution of A. sara is far from this area, as well as from Central America.
However, the closely related ancestor of A. arctoidea (not analyzed here or in Cortés-
Ortiz et al. [2003]) was probably living very close to the geographical area where the
ancestor of A. pigra could have appeared in northwestern South America. The ances-
tor of A. palliata originated later.
Finally, the red howler monkeys from the island of Trinidad in the Caribbean
Sea do not belong to A. macconnelli (= A. s. stramineus) [Stanyon et al., 1995; Sampaio
et al., 1996, Figueiredo et al., 1998; Bonvicino et al., 2001], as Groves [2001] main-
tained. This taxon is more related to A. s. seniculus, although it formed a monophy-
letic clade. As we did not analyze samples of the putative Venezuelan taxon A. arctoi-
dea, we cannot know the relationship between the Trinidad red howler monkeys and
this last taxon. Only future molecular and karyotypic studies could determine if the
Trinidad Alouatta corresponds to A. arctoidea, to a subspecies of A. seniculus (A. s.
insulanus; in our molecular study, a genetic distance of 2.3% from A. s. seniculus), or
even to a new species (for which it is necessary to detect clear chromosomal rear-
rangements). Anecdotally, we did observe some behavioral and physiological differ-
ences between the red howler monkeys from Trinidad and the red howler monkeys
from Colombia and Peru.
Molecular and karyotype data clearly support that the the red howler monkeys
are more closely related to each other than to other Alouatta taxa. A. sara, A. artoidea
[Minezawa et al., 1985; Consigliere et al., 1996, 1998], A. s. seniculus [Yunis et al.,
1976, Torres and Levobivi, 2001], A. macconelli [Lima et al., 1990, Vassart et al., 1996]

DOI: 10.1159/000480502
and A. stramineus [Lima and Seuánez, 1991] all showed microchromosomes (or B
chromosomes). However, microchromosomes have not been reported for A. palliata
[Ma et al., 1975, Torres and Ramírez, 2003], A. caraya [Egozcue and Egozcue, 1966;
Mudry et al., 1994, 1998], A. belzebul belzebul [Armada et al., 1987], A. nigerrima
[Armada et al., 1987], and A. guariba [Koiffmann, 1982; Koiffmann and Saldanha,
1974; De Oliveira, 1996; De Oliveira et al., 1995, 1998, 2000, 2002].
Therefore, the Alouatta genus should experience speciation with relative fre-
quency by means of chromosomal changes (parapatric and stasipatric speciation).
These striking karyotypical changes plus molecular genetics analyses should help to
establish an accurate account of the phylogeny and systematics of this Neotropical
primate genus more easily than in other organisms with greater chromosomal sta
bility.
Ackowledgments
Thanks to Dr. Diana Alvarez, Pablo Escobar-Armel, Nicolás Lichilín, Armando Castellanos,
Andrés Laguna, Fernando Nassar, Luz Mercedes Botero, Marcela Ramírez, Connie Stelle, and
Hugo Gálvez for their respective help in obtaining howler monkey samples during the last 20
years. Thanks to the Instituto von Humboldt (Villa de Leyva in Colombia; Janeth Muñoz, Clau-
dia Alejandra Medina, and Fernando Botero), to the Peruvian Ministry of Environment, PRO-
DUCE (Dirección Nacional de Extracción y Procesamiento Pesquero), Consejo Nacional del
Ambiente, and the Instituto Nacional de Recursos Naturales (INRENA) from Peru, to the Colec-
ción Boliviana de Fauna (Dr. Julieta Vargas), to CITES Bolivia and Ministerio del Ambiente in
Santo Domingo de Tsáchilas (Ecuador) for their role in facilitating the obtainment of the collec-
tion permits in Colombia, Ecuador, Peru, and Bolivia. The Costa Rican howler monkeys were
sampled with the collection permits provided by the Costa Rican government to Dr. Gustavo
Gutiérrez-Espeleta. We would also like to express our thanks to the Dirección General de Zoológi-
cos y Vida Silvestre in Mexico City (Mexico) for facilities to obtain some of the samples used in
this study. Thanks also goes to ARCAS (Guatemala) and the Jardín Botánico de Portoviejo de la
Universidad Técnica de Manabí (Ecuador) for providing hair samples of A. pigra and A. palliata,
respectively. The first author is also thankful for the help of many people of diverse Indian tribes
in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana,
and Alamas), Bolivia (Sirionó, Canichana, Cayubaba, and Chacobo), and Colombia (Jaguas, Ti-
cunas, Huitoto, Cocama, Tucano, Nonuya, Yuri, and Yucuna) for their assistance in obtaining
samples of howler monkeys.
Disclosure Statement
The authors report no conflicts of interest. The authors alone are responsible for the content
and writing of this article.

DOI: 10.1159/000480502
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DOI: 10.1159/000480502

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Original Research Article

Folia Primatol Received: April 26, 2017

Phylogeography of the Mantled Howler Monkey

a Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética,

Autónoma del Estado de México, Toluca, Mexico; c Escuela de Biología, Universidad de

of Minnesota Crookston, Crookston, MN, USA

© 2017 S. Karger AG, Basel Manuel Ruiz-García, Unidad de Genética

2 Folia Primatol Ruiz-García et al.

Authors Species Subspecies

Rylands et al. A. palliata A. p. mexicana, A. p. palliata, A. p. aequatorialis

Phylogeography of the Mantled Howler Monkey Folia Primatol 3

4 Folia Primatol Ruiz-García et al.

Phylogeography of the Mantled Howler Monkey Folia Primatol 5

Materials and Methods

6 Folia Primatol Ruiz-García et al.

Mexico Cayman Is. Haiti Dominican Rep.

Costa Rica Trinidad and Tobago

Phylogeography of the Mantled Howler Monkey Folia Primatol 7

8 Folia Primatol Ruiz-García et al.

Genetic Diversity and Heterogeneity Analyses

Demographic Evolutionary Changes

Phylogeography of the Mantled Howler Monkey Folia Primatol 9

Phylogeography of A. palliata and Phylogenetic Inferences for Different Alouatta

10 Folia Primatol Ruiz-García et al.

Fig. 2. MLT with 156 howler

Phylogeography of the Mantled Howler Monkey Folia Primatol 11

12 Folia Primatol Ruiz-García et al.

Phylogeography of the Mantled Howler Monkey Folia Primatol 13

14 Folia Primatol Ruiz-García et al.

Temporal Splits among Alouatta Taxa

Phylogeography of the Mantled Howler Monkey Folia Primatol 15

Genetic Diversity in A. palliata and in Other Alouatta Species

Genetic Heterogeneity and Genetic Distances among A. palliata Taxa and

16 Folia Primatol Ruiz-García et al.

6, A. insulanus; 7, A. macconnelli; 8, A. belzebuth; 9, A. geoffroyi; 10, L. l. cana.

Phylogeography of the Mantled Howler Monkey Folia Primatol 17

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Demographic Changes in the A. palliata

Phylogeography of the Mantled Howler Monkey Folia Primatol 19

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Phylogeography of the Mantled Howler Monkey Folia Primatol 21

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Phylogeography of the Mantled Howler Monkey Folia Primatol 23

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Phylogeography of the Mantled Howler Monkey Folia Primatol 25

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Phylogeography of the Mantled Howler Monkey Folia Primatol 27

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Phylogeography of the Mantled Howler Monkey Folia Primatol 29

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Phylogeography of the Mantled Howler Monkey Folia Primatol 31

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Phylogeography of the Mantled Howler Monkey Folia Primatol 33

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