Chapter 12 Alouatta Sur Peru

Book Title: Phylogeny, Molecular Population Genetics, Evolutionary Biology
and Conservation of the Neotropical Primates. Edited by M. Ruiz-García and J.

M. Shostell (2016). Nova Science Publisher Inc., New York, USA. Book ID: -
5975- ISBN: Hardcover 978-1-63485-165-7; E-book 978-1-63485-204-3. Pp. 395-434.
Chapter 12
WHICH HOWLER MONKEY (ALOUATTA, ATELIDAE,

PRIMATES) TAXA IS LIVING IN THE PERUVIAN
MADRE DE DIOS RIVER BASIN (SOUTHERN PERU)?
RESULTS FROM MITOCHONDRIAL GENE ANALYSES
AND SOME INSIGHTS IN THE PHYLOGENY
OF ALOUATTA
Manuel Ruiz-García1,*, Angela Cerón1,

Myreya Pinedo-Castro1 and Gustavo Gutierrez-Espeleta2
1
Laboratorio de Genética de Poblaciones-Biología Evolutiva. Unidad de Genética.
Departamento de Biología. Facultad de Ciencias. Pontificia Universidad Javeriana.
Bogotá DC., Colombia.
2
Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica
ABSTRACT
We sequenced mitochondrial genes (COI, COII and Cyt-b) from 138 howler monkeys
(Alouatta) representing six “a priori” species (A. pigra, A. palliata, A. coibensis trabeata,
A. seniculus, A. sara and A. caraya). These data were used to address two main issues: 1-
to determine which red howler monkey taxa were distributed in Southern Peru (four of
the sequenced were from there) and 2- to provide new insights about the systematics of
howler monkeys. Our data support six main findings. 1-There is strong molecular gene
heterogeneity between A. seniculus and A. sara. This verifies that A. sara (a taxon
currently living in Southern Peru) is a full species; 2- A. seniculus seniculus is present in
Colombia as well as across the northern and middle Peruvian Amazon. The alleged full
*
Corresponding author: mruizgar@yahoo.es, mruiz@javeriana.edu.co.
2 Manuel Ruiz-García, Angela Cerón, Myreya Pinedo-Castro et al.
species A. juara and A. puruensis seem to be molecularly un-differentiable from A. s.

seniculus. Therefore, until these potential taxa are studied from a karyological point of
view, only two red howler species are considered to be living in Peru: A. seniculus and A.
sara; 3- The cis and trans-Andean howler monkeys are clearly differentiated agreeing
with the results of Cortés-Ortiz et al., (2003). We obtained two temporal split estimates
(7.21 Millions of years ago, MYA, and 6.73 MYA) for the separation of the cis and trans-
Andean howler monkey species; 4- The evolution of the current mitochondrial
haplotypes for the cis-Andean taxa began earlier than the mitochondrial diversification
within the trans-Andean howler monkey taxa. This disagrees with the point of view of
several authors claiming that A. palliata was the ancestral taxon in the Alouatta genus.
Indeed, the haplotypes of A. palliata were derived from those of A. pigra, which agrees
with a unique migration of howler monkeys from South America to Central America. The
differentiation of both taxa occurred around 3 MYA; 5- No subspecies within A. palliata
were detected. Even A. coibensis trabeata seems to be an extension of A. palliata. 6- We
analyzed three species for possible population expansions. A. seniculus did not show any
evidence of population expansion but there was some evidence of population expansion
in A. sara. In contrast, A. palliata had a strikingly positive population expansion that is
correlated with its relatively more recent origin.
Keywords: Alouatta, mitochondrial genes (COI, COII and Cyt-b), population expansion,
molecular phylogeny, Southern Peru.
INTRODUCTION
The howler monkey (Alouatta) is the unique living genus within the subfamily
Alouattinae (or Mycetinae) (Rylands et al., 2000; Groves, 2001) and family Atelidae. Howler
monkeys are among the largest Neotropical primates and are similar in size to individuals
within the Ateles, Brachyteles and Lagothrix genera. Alouatta is also the most widely
distributed 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 woodlands (Crockett
and Eisenberg, 1987; Pope, 1992).
The systematics of Alouatta is complex due to the phenotypic similarity among many
taxa of this genus. The taxa also have extensive chromosomal rearrangements, probably a
result of chromosomic speciation (parapatric and stasipatric; Lewis, 1966; White, 1978; Reig,
1980; King, 1993). Within the A. seniculus complex (red howler monkeys), many
classifications have been registered. Cabrera (1958) determined four 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 nine (the four previously
cited plus A. s. amazonica [in a small area near to the mouth of the Purus River within the
Amazon River], A. s. insularis [in the Caribbean island of Trinidad], A. s. juara [in the Juara
River, mainly in the Brazilian Amazon], A. s. macconnelli [mainly in the Guiana area] and A.
s. puruensis [in the Purus River, mainly in the Brazilian Amazon]). This classification was
accepted by Stanyon et al., (1995). However, four of the forms Hill presented are misleading
Which Howler Monkey (Alouatta, Atelidae, Primates) Taxa ... 3
since he considered A. s. macconnelli and A. s. amazonica to be synonymous to A. s.

stramineus and A. s. juara and A. s. puruensis to be synonymous of A. s. seniculus. Rylands et
al., (1995 and 1996/1997) recognized seven of these forms as valid subspecies but included
another subspecies as A. s. sp following Bodini and Pérez-Hernández (1987), who claimed an
undescribed subspecies in the Venezuelan Llanos, north of the Orinoco River. 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) demonstrated
that this Bolivian taxon (46 chromosomes) has a very different karyotype from A. seniculus
(44 chromosomes) found in Colombia. They proposed that A. sara had a unique
X1X1X2X2/X1X2Y sex chromosome pattern. As we will discuss in this work, the unusual
chromosome mechanisms to determine sex in the howler monkeys have also been seen in
other Neotropical primates such as the Aotus (Ma el al., 1976, 1980; Pieczarka and
Nagamachi, 1988) and Cacajao (Dutrillaux el al., 1981). Other mammals including rodents
such as Lemnus (Bull and Bulmer, 1981) and insectivores like Sorex araneus (Ford et al.,
1957) also can have unusual sexual chromosomes.
Stanyon et al., (1995) and Consigliere et al., (1996) used G banding chromosomal
analyses to compare the Bolivian animals to the red howlers from Venezuela (A. s.
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 and A. s. stramineus as we will explain. A. sara possessed microchromosomes as
did A. s. seniculus and other red howler monkeys in contrast to that observed in other
Alouatta taxa. The first comprehensive molecular genetics work with Alouatta included the
majority of Alouatta taxa and determined A. sara to be a full species (Cortés-Ortiz et al.,
2003). Villalobos et al., (2004) also accepted A. sara as a full species. Groves’s seminal book
on primate taxonomy only recognized three subspecies (A. s. arctoidea, A. s. juara and A. s.
seniculus) with A. macconnelli and A. sara as full species (Groves, 2001). A. macconnelli was
elevated to the full species category because Lima and Seúanez (1989) 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. These chromosomes are similar to that
found by Lima and Seúanez (1989) for A. belzebul, but with differences regarding this taxon
in the extra number of nuclear organizing regions (NORs). The karyotype of A. macconnelli
has a 2N = 47 to 49 chromosomes with three microchromosomes (Bonvicino et al., 1995).
Thirty years earlier, 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 to 45 chromosomes and from three to five microchromosomes (four being the most
frequent number). There were pericentric inversions in chromosome 13 with an XX/XY
pattern. Torres and Leibocini (2001) analyzed 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 chromosome 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
microchromosomes) to those animals classified as A. macconnelli from the Jari River (Brazil)
by Lima et al., (1990). One individual even showed 50 chromosomes, with trisomy in
chromosome 11. The sex chromosome pattern in A. macconelli was also found in A. s.
stramineus (Uatama River, Brazil; Lima and Seuánez, 1991). However, these two taxa
showed differences and their karyotypes can be derived from each other by a reciprocal
translocation on chromosome 2 (Lima and Seúanez, 1991). Two molecular genetics studies
also ratified A. macconnelli as a full species (Cortés-Ortiz et al., 2003; Ruiz-García et al.,
2007). Groves (2001) considered A. s. amazonica and A. s. puruensis to be the same as A. s.
juara and A. s. insularis 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.
The author also classified A. nigerrima, a traditional subspecies classified within A. belzebul
within the A. seniculus complex (Cabrera, 1958; Hill, 1962; Rylands et al., 1995; Groves,
2001). However, Lima and Seuánez (1991) did not find any relevant chromosomal
differences between A. s. seniculus and A. juara (following the Gregorin’s terminology).
This variety of classifications should be observed in foundational books about
Neotropical primates. Here we list a few examples. For example, Mittermeier et al., (1988)
affirmed that almost certainly, within A. seniculus, a number of subspecies were included but
they preferred to not list the subspecies until further studies had been conducted. Auricchio
(1995) recognized two A. seniculus taxa in Brazil, A. s. seniculus (including within it, A. s.
juara and A. s. puruensis) and A. s. stramineus. Dos Reis et al., (2008) recognized four
species of red howler monkeys in Brazil: A. juara, A. macconnelli, A. puruensis and A.
seniculus. This last one was not recognized by Gregorin (2006) in Brazil. In the book edited
by Garber et al., (2009), Rylands and Mittermeier (2009) accepted six full species out of the
previously unique species, A. seniculus. They are: A. seniculus, A. arctoidea, A. macconnelli,
A. juara, A. puruensis and A. sara.
This confusing taxonomic situation regarding red howler monkeys also carries over into
Peru. Soini et al., (1989) and Aquino and Encarnación (1994) reported the existence of red
howler monkeys through many Peruvian Amazon areas, such as Manu National Park
(Southern Peruvian Amazon) as well as in the Pacaya-Samiria National Perk and in the
Tamshiyacu-Tahuayo Reserve (Northern Peruvian Amazon). They classified all of these
animals as A. s. seniculus. Groves (2001) on the other hand claimed the presence of two red
howler monkeys, A. seniculus juara and A. sara, in Peru.
Pacheco et al., (2009) accepted the classification of Gregorin (2006) and affirmed that A.
juara’s and A. puruensis’s distributions reached the mid and Southeastern section of Peru
(Purús area). As they accepted A. juara for Peru, then the presence of A. seniculus should be
rejected. Also, they commented on the possible existence of A. sara in Peru. Nevertheless,
Aquino et al., (2009) cited A. seniculus (not A. juara) as being in the central area of the
Peruvian Amazon (forests of the Contamana Sierra). In 2011 Pacheco and others claimed that
A. juara, A. puruensis and A. sara were the three possible red howler monkey species in Peru
(MINAM, 2011). However, they doubted the validity of the alleged new species of Alouatta
and concluded it better to consider that the unique red howler species in Peru was A. seniculus
sensu Hill (1962). For example, Voss and Fleck (2011) claimed, that due to the absence of
determinant information, it would be preferable to not use juara as a specific name. Also,
they commented that the presence of A. sara in Southern Peru is only assumed because this
taxon has been cited in Bolivia very near the Peruvian frontier by Wallace and Rumiz (2010)
and Mercado and Wallace (2010). The presence of A. sara is well documented in La Paz
Department (Apolobamba and Madidi National Park). Indeed, these authors commented that
in the eastern limit of Bolivia (Noel Kempff Mercado National Park), A. puruensis could be
present following Rylands and Brandon-Jones (1999). However, MINAM (2011) claimed
that since there is no voucher specimen of this taxon from Peru, the presence of A. sara is not
sustained for this country. In contrast, Gregory et al., (2012) cited A. sara in the Lower
Urubamba Region of Peru. The web page Primate Info Net (2014) (pin.primate.wisc.edu)
cited the two following red howler monkey taxa for Peru: A. s. seniculus and A. s. juara.
Boubli et al., (2008) speculated about the geographical distribution of A. puruensis in Peru
and Bolivia and whether it was the form in Northwestern Bolivia and extreme Southeastern
Peru in the basins of the upper Purus, Madre de Dios and Tampopata rivers. They also
speculated that the Inuya River may be the northern limit to A. puruensis in Peru.
Due to this uncertainty, we analyzed the sequences for three mitochondrial genes
(cytochrome b, Cyt-b; sub-unity cytochrome oxidase I, COI; sub-unity cytochrome oxidase II,
COII) for four red howler monkeys from the southern area of Peru (Madre de Dios and
Tambopata rivers) and six exemplars from the northern and middle Peruvian Amazon (Javari,
Napo, Ucayali, Tapiche and Pachitea rivers).
The mitochondrial genes are interesting markers for phylogenetic tasks because they lack
introns and include a rapid accumulation of mutations, rapid coalescence time, a negligible
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
species than other molecular markers (Moore, 1995). Supporting this, Cummings et al.,
(1995) showed that mitochondrial genomes have higher information content per base than
nuclear DNA. The Cyt-b gene is commonly used among the molecular markers relevant for
phylogeography, biosystematics, and genetic structure studies in mammalian populations
(Lavergne et al., 2010; Ruiz-García et al., 2015a). The COI gene has emerged as the standard
barcode region for animals, including mammals (www.mammaliabol.org) (Hebert et al.,
2003, 2004) and the COII gene has been employed extensively in the phylogenetics of
different mammalian groups, including primates (Ashley and Vaughn, 1995; Ruiz-García et
al., 2010, 2014).
Therefore, the main aims of the current work are to clarify the systematics of the red
howler monkeys in Peru, to describe several molecular population genetics statistics
(including possible demographic changes) within and among different Alouatta taxa, as well
as to obtain insights about the phylogeny of the howler monkeys.
MATERIAL AND METHODS

A total of 138 howler monkeys of different taxa were sampled across several countries
of Latin-America. Here we provide a breakdown of number and taxonomic type per sampled
location. There was one A. caraya (from Santa Ana de Yacumo, Mamore River, Bolivia),
three A. pigra (from San Miguel, Peten area in Guatemala) and five A. sara. One of the five
was from the Mamore River, Beni Department, Bolivia. The other four individuals, later
classified as A. sara, were from Chonta and La Torre, Tambopata River and Taricaya and
Madre de Dios River in Peru. There were also 11 A. seniculus (two from Santa Rita, Javari
River at the Peruvian side; one from Vencedores del Zapote, Napo River, Peru; one from
Puerto Inca, Pachitea River, Peru; two from Requena, Ucayali-Tapiche rivers, Peru; three
from the Antioquia Department in Colombia and two from the Valle del Cauca Department in
Colombia). Finally, there were 115 A. palliata. Of these, 103 were sampled from across Costa
Rica. One was from Los Katios National Park in the Choco Department and two were from
the Cordoba Department in Colombia. Other nine were from four different locations within
the Pacific area in Ecuador. Three Panamanian individuals belonged to the A. coibensis
trabeata taxon.
DNA were obtained from hair (all samples with the exception of A. palliata from Costa
Rica) and blood (the samples of A. palliata from Costa Rica) sampled from living howler
monkeys within Central and South America.
Molecular Procedures
The DNA from blood was extracted using the phenol-chloroform procedure (Sambrook
et al., 1989), while DNA samples from hairs were extracted with 10% Chelex resin (Walsh et
al., 1991). The 138 howler monkey individuals sampled were sequenced for three mt genes
(COI, COII, and Cyt-b). For the mt COI amplification (polymerase chain reaction, PCR), we
used the forward primer LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’), and
the reverse primer HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (657 base
pairs, bp) (Folmer et al., 1994) under the following PCR profile: 94°C for 5 min, followed by
39 cycles at 94°C for 30 s, 44°C for 45 s, 72°C for 45 s, and a final cycle of 72°C for 5 min.
For the amplification of the mt COII gene (located in the lysine and asparagine tRNAs) we
used the forward primer L6955 (5’ -AACCATTTCATAACTTTGTCAA-3’) and the reverse
primer H7766 (5’ -CTCTTAATCTTTAACTTAAAAG-3’) (720 bp) (Ashley and Vaughn,
1995; Collins and Dubach, 2000; Ruiz-Garcia et al., 2010, 2012a,b). We used the following
temperatures: 95° C for 5 min, 35 cycles for 45 s at 95° C, 30 s at 50° C and 30 s at 72° C and
a final extension time for 5 min at 72° C. For both genes, the PCRs were performed in a 25 l
volume with reaction mixtures including 4 l of 10 x buffer, 6 l of 3 mM MgCl2, 2 l of 5
mM dNTPs, 2l (8 mM) of each primer, 2 units of Taq DNA polymerase, 5 l of ddH2O and
2 l (20–80 ng/l) of DNA. The mt Cyt-b was amplified by PCR using the procedure of
Montgelard et al., (1997) (1,140 bp). The conditions for Cyt-b amplification were performed
in 25 l reactions including: 2 l of DNA, 2 l of 10 x buffer, 13 l ddH20, 2 l (25 mM)
MgCl2, 1 l (10 mM) each of forward and reverse primers, 2l (10 mM) dNTPs, and 2 units
of Taq DNA polymerase. The standard thermal cycling program consisted of 10 min at 95°C,
35 cycles of 35 s at 94°C, 35 s at 55 °C and 30 s at 70°C and a final extension time for 10 min
at 72°C. The total length of the sequences studied for the three genes was 2,517 base pairs
(bp).
All amplifications, including positive and negative controls, were checked in 2% agarose
gels. The gels were visualized in a Hoefer UV Transilluminator. Both mtDNA strands were
sequenced directly using BigDye Terminator v3.1 (Applied Biosystems, Inc.). We used a
377A (ABI) automated DNA sequencer. The samples were sequenced in both directions to
ensure sequence accuracy.
Data Analysis
Molecular Population Analyses

Two kinds of procedures were carried out to estimate genetic heterogeneity, and
theoretical gene flow estimates, among the diverse Alouatta species detected by the
phylogenetic analyses. We applied procedures to haplotypic frequencies (GST statistic) and to

nucleotide sequences (ST, NST and FST statistics, Hudson et al., 1992). To determine possible
differences among the Alouatta species pairs considered, we used the FST statistic (Weir and
Hill, 2002). The statistics used to determine the genetic diversity within the diverse Alouatta
species considered were: the number of polymorphic sites (S), the haplotypic diversity (Hd),
the nucleotide diversity (), the average number of nucleotide differences (k) and the 
statistic by sequence. These genetic heterogeneity and gene diversity statistics were
undertaken in the DNAsp 5.10 program (Librado and Rozas, 2009).
We used two procedure sets to determine possible historical population changes for the
different Alouatta species analyzed. 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 of Ramos-Onsins and Rozas (2002) to determine
possible population size changes in the Alouatta species we analyzed (Simonsen et al., 1995;
Ramos-Onsins and Rozas, 2002).
Phylogenetic Analyses
The sequence alignments were carried out manually and with the DNA alignment
program (Fluxus Technology Ltd.). The Modeltest Software (Posada and Crandall, 1998) and
the Mega 6.05 Software (Tamura et al., 2013) were applied to determine the best evolutionary
mutation model for the sequences analyzed of the three gene sequences concatenated. Akaike
information criterion (AIC; Akaike, 1974) and the Bayesian information criterion (BIC;
Schwarz, 1978) were used to determine the best evolutionary nucleotide model. Additionally,
we obtained maximum likelihood estimates of transition/transversion bias as well as
maximum likelihood estimates of the gamma parameter for site rates for the best evolutionary
nucleotide model obtained (Tamura et al., 2013).
The phylogenetic trees were obtained using three different procedures for the
concatenated genes: Neighbor-joining tree, Maximum likelihood tree, and Bayesian analysis
(NJ; Saitou and Nei, 1987; ML; Felsenstein, 1981). The Neighbor-joining tree utilized the
Kimura 2P genetic distance (Kimura, 1980). The Maximum likelihood tree used the HKY +
G model (Hasegawa-Kishino-Yano, 1985) and a discrete Gamma distribution (+ G) with five
rate categories. Both trees were constructed with the PAUP*4.0b8 program (Swofford, 2002)
and MEGA 6.05. The Bayesian analysis was performed using a HYK + G model with the
gamma distributed rate varying among sites, because it was determined to be the better model
using the FindModel program. This Bayesian analysis was completed with the BEAST v.
1.8.1 program (Drummond et al., 2012). Two separate sets of analyses were run, assuming a
Yule speciation model and a relaxed molecular clock with an uncorrelated log-normal rate of
distribution (Drummond et al., 2006). Results from the three independent runs (30,000,000
generations with the first 3,000,000 discarded, 10%, as burn-in and parameter values sampled
every 1000 generations) were combined with LogCombiner v1.8.0 Software (Rambaut and
Drummond, 2013a). Posterior probability values provide an assessment of the degree of
support of each node on the tree. The effective sample size (ESS) for the parameter estimates
and convergence were checked using the Tracer Program (version 1.6) (Rambaut et al.,
2013). The lower and upper 95% highest posterior densities (HPD) of these parameters as
well as the means, geometric means, medians, marginal densities and traces were estimated
with the same program. To determine the reality of the values of these parameters, we
obtained the autocorrelation tree (ACT) and ESS for parameter estimates. Sampled trees were
summarized with TreeAnnotator v1.8.0 (Rambaut and Drummond, 2013b) and visualized in
the FigTree Program (version. 1.4) (Rambaut, 2012). This program was run to estimate the
time to most recent common ancestor (TMRCA) for the different Alouatta species analyzed.
We used two priors. One was around 16.13 + 2.0 MYA (95% HPD: 12.21-19.42 MYA) for
Alouatta and the Atelinae (Ateles, Brachyteles and Lagothrix). The second was around 11.25
+ 2.0 MYA (95 % HPD: 7.96-14.54 MYA) for the split between Ateles and Lagothrix
(Meireles et al., 1999; Opazo et al., 2006; Perelman et al., 2011; Springer et al., 2012; and
Von Dornum and Ruvolo, 1999).
We constructed a Median Joining Network (MJ) (Bandelt et al., 1999) using Network
4.2.0.1 Software (Fluxus Technology Ltd) to estimate possible divergence times among the
haplotypes found in Alouatta. Additionally, the  statistic (Morral et al., 1994) and its
standard deviation (Saillard et al., 2004) were estimated and transformed into years. The 
statistic is unbiased and highly independent of past demographic events. We took the
mutation rate of the mt COII gene as an average for the three mitochondrial genes used.
Ruvolo et al., (1991) determined a mutation rate of 0.85% per million years per lineage for
Hominoidea at the mt COII gene. This represents one mutation each 199,402 years. This
mutation rate was almost identical to that determined by Ruiz-García and Pinedo-Castro
(2010) for Lagothrix at the same gene (one mutation every 191,000 years). Ashley and
Vaughn (1995) and Ruiz-García et al., (2011) determined one mutation every 199,000 years
for Aotus. Herein we used a rate of one mutation every 195,000 years for Alouatta.
RESULTS
Molecular Population Genetics
Clearly, the genetic heterogeneity among the different Alouatta taxa studied was very
significant (Table 1). This agrees with the fact that they are different species. Similarly, the
theoretical gene flow estimates among the taxa were extremely small (Nm = 0.09-0.13),
which supports with the existence of different species. The highest degree of genetic
heterogeneity was between A. seniculus-A. palliata (FST = 0.938) and between A. seniculus-A.
pigra (FST = 0.936). These values were highly significant especially between one cis-Andean
taxon and the two trans-Andean taxa (Table 2). The A. seniculus-A. sara pair was also highly
significant (FST = 0.641), which indicates that they are different species and both live in Peru.
Table 3 lists the gene statistics of the 11 A. seniculus and the five A. sara individuals.
Gene diversity was considerably higher for A. sara ( = 0.0322 and k = 23) than for A.
seniculus ( = 0.0057 and k = 4.15).
Table 1. Overall genetic heterogeneity and gene flow (Nm) statistics for different
Alouatta species for three concatenated mitochondrial genes. *Significant Probability (P
< 0.05)
Genetic differentiation
P Gene flow
estimated
 = 411.000 df = 105 0.0000*
HST = 0.0825 0.0000* γST = 0.7895 Nm = 0.13
KST = 0.7758 0.0000* NST = 0.8429 Nm = 0.09
KST* = 0.4524 0.0000* FST = 0.8387 Nm = 0.10
ZS = 3,674.6439 0.0000*
ZS* = 8.0124 0.0000*
Snn = 1.0000 0.0000*
Table 2. Population FST (below) and NST (above) pairs among the different Alouatta
species studied for three concatenated mitochondrial genes
Alouatta species 1 2 3 4
1 - 0.9293 0.9414 0.7992
2 0.9275 - 0.9388 0.7782
3 0.9387 0.9359 - 0.6449
4 0.7928 0.7720 0.6407 -
1 = A. palliata; 2 = A. pigra; 3 = A. seniculus; 4 = A. sara. All the values
were significant
Table 3. Gene diversity statistics for the different Alouatta species studied for three
concatenated mitochondrial genes. The statistics estimated were the number of
haplotypes (NH), the haplotypic diversity (Hd), the nucleotide diversity (), the average
number of nucleotide differences (K) and the  statistic (= 2Ne; Ne = effective female
population size;  = mutation rate per generation) by sequence
Alouatta taxa NH Hd  K 
A. palliata 22 0.662 + 0.045 0.0036 + 2.573 + 17.78 +
0.0011 1.389 4.503
A. pigra 2 0.667 + 0.114 0.0037 + 2.667 + 2.667 +
0.0024 1.919 1.919
A. seniculus 7 0.909 + 0.066 0.0058 + 4.145 + 4.097 +
0.0014 2.232 1.937
A. sara 5 1.000 + 0.126 0.0321 + 23.00 + 27.36 +
0.0093 12.26 0.999
Table 4. Demographic statistics applied to three different Alouatta species (A. palliata, A.
seniculus, A. sara)
Fu & Li Fu & Li
Tajima D Fu’s Fs raggedness rg R2
D* F*
A. palliata P[D < - P[D* < - P[F* < - P[Fs < - P[rg < 0.0527] P[R2 <
2.789] = 8.811] = 7.438] = 8.928 ] = = 0.296 0.038] =
0.001** 0.001** 0.001** 0.004** 0.039*
A. seniculus P[D < P[D* < - P[F* < - P[Fs < - P[rg < 0.103] P[R2 <
0.051] = 0.071] = 0.122] = 0.815] = = 0.460 0.146] =
0.553 0.426 0.444 0.327 0.332
A. sara P[D < - P[D* < - P[F* < - P[Fs < P[rg < 0.140] P[R2 <
1.205] = 1.432] = 1.306] = 0.683] = = 0.229 0.218] =
0.026* 0.038* 0.036* 0.386 0.521
* P < 0.05; ** P < 0.01, significant population expansions
We analyzed for possible historical demographic changes in A. seniculus, A. sara and A.

palliata and analyzed for possible historical demographic changes (Table 4 and Figure 1). For
A. seniculus, neither test nor the mismatch distribution clearly showed any demographic
change during the evolutionary history of this species. In the case of A. sara, three of the tests
employed showed significant evidence of population expansion (Tajima D test, Fu and Li D*
and Fu and Li F*). All the tests we used for A. palliata were statistically significant indicating
population expansion. Thus, the two cis-Andean howler species showed less evidence of
population expansions than the trans-Andean howler species, which showed a very striking
population expansion.
Phylogenetic Inferences
The BIC and the Akaike information criterion showed that HKY + G (9,749.833 and
6,902.338, respectively) was the nucleotide substitution model which best fit with the three
concatenated genes. The maximum likelihood estimate of transition/transversion bias was
2.07 (maximum log likelihood was -3,160.23).
The Kimura 2P genetic distances (Table 5) showed values ranging from around 6% to 9%
among the different Alouatta taxa studied. These are typical values of different species
(Ascunce et al., 2003).
(A)
(B)
(C)
Figure 1. Mismatch distributions (pairwise sequence differences) at the three concatenated

mitochondrial DNA genes (COI, COII and Cyt-b) for the different Alouatta taxa considered: A. palliata
(A), A. seniculus (B) and A. sara (C).
Table 5. Kimura (1980) 2P genetic distances among the different Alouatta taxa
considered. Below, genetic distance values in percentages (%); above, standard errors in
percentages (%)
Alouatta
1 2 3 4 5 6
pecies
1 - 0.8 1.1 1.0 1.2 1.7
2 5.3 - 1.3 1.0 1.2 1.9
3 8.4 8.0 - 0.8 0.9 1.6
4 9.3 8.4 5.5 - 1.7 1.6
5 8.0 7.2 5.5 7.6 - 1.5
6 15.1 16.0 14.3 16.4 13.6 -
1= A. palliata; 2 = A. pigra; 3 = A. seniculus; 4 = A. sara; 5 = A. caraya; 6 = Lagothrix lagotricha cana
Figure 2. (Continued)
Figure 2. Neighbor-Joining tree with the Kimura (1980) 2P genetic distance with the 138 howler
monkeys (Alouatta) studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The
number in the nodes are bootstrap percentages.
The Kimura 2P genetic NJ tree clearly showed two main groups of howler monkeys for
the three mitochondrial genes (Figure 2). One main clade consisted of three cis-Andean
howler monkeys taxa (A. caraya, A. seniculus and A. sara). The other main clade contained
the two trans-Andean howler taxa we analyzed (A. pigra and A. palliata). The splits of these
five Alouatta taxa were correlated with high bootstrap percentages. The four Peruvian howler
monkeys sampled in the Madre de Dios River Basin belonged to A. sara. On the other hand,
the other six Peruvian howler monkey sampled at the Javari River (frontier with Brazil), at the
Napo River, at the Ucayali-Tapiche rivers and at the Pachitea River were closely related to
the howler monkeys sampled in different areas of Colombia, which traditionally have been
designated as A. s. seniculus. Therefore, the molecular data detected at least two different
Alouatta species in the Peruvian Amazon: one (A. seniculus) in the northern and middle
Peruvian Amazon (from the Napo River until the middle Ucayali River), and another (A.
sara) in the Southern Peruvian Amazon (Madre de Dios River basin). This tree also showed
that no differentiated subspecies within A. palliata were detected in Costa Rica, Panama,
Colombia or Ecuador (A. palliata palliata and A. p. aequatorialis).
Figure 3. Maximum likelihood tree with the 138 howler monkeys (Alouatta) studied for three
concatenated mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are bootstrap
percentages.
The ML tree (Figure 3) showed the same phylogenetic relationships as the previous tree
with one exception: A. caraya was the most differentiated of the Alouatta taxa employed in
this study.
Figure 4. Bayesian tree with the 138 howler monkeys (Alouatta) studied for three concatenated
mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are posterior probabilities higher
than 0.5, the median temporal split and the interval of 95 % High Posterior Density (HPD).
The Bayesian tree (BI tree; Figure 4) showed a split of the ancestor of Alouatta from the
other Atelidae around 15.93 MYA (95% HPD: 14.93-16.85 MYA; P = 1). The split of the
ancestors of Lagothrix and Ateles occurred around 11.18 MYA (95% HPD: 10.3-12.23 MYA;
P = 1). The diversification within Alouatta began around 7.21 MYA (95% HPD: 6.20-8.01
MYA; P = 1), with the ancestor of A. seniculus being the first to diverge. Within A. seniculus,
the mitochondrial diversification began around 3.94 MYA (95% HPD: 1.93-5.86 MYA; P =
1). The next ancestral branch to diverge within the remaining Alouatta taxa was the ancestor
of A. sara around 6.61 MYA (P = 0.28). The mitochondrial diversification in A. sara began
around 2.27 MYA (95% HPD: 1.2-4.72 MYA; P = 1). The split of the ancestors of the two
trans-Andean howler monkey species (A. pigra and A. palliata) was around 6.06 MYA (P =
0.47) with the diversification within A. pigra around 2.12 MYA (95% HPD: 1.7-3.89 MYA;
P = 1) and within A. palliata around 4.58 MYA (95% HPD: 4.04-7.21 MYA; P = 0.82).
Remarkably different to that obtained in the two previous trees, A. caraya was more related to
A. pigra than to the other cis-Andean howler monkey species. The mitochondrial
diversification within Alouatta obtained with this procedure clearly overestimates the
temporal splits obtained by Cortes-Ortiz et al., (2013). Nevertheless, in an identical fashion to
the other two trees, six Peruvian howler monkeys were clearly identified as A. seniculus and
the four animals from the Madre de Dios River basin were clearly related to A. sara. This
ratifies the existence, at least, of two different species of Alouatta in the Peruvian Amazon.
Figure 5. Median Joining Network (MJN) with haplotypes found at the three concatenated
mitochondrial genes (COI, COII and Cyt-b) gene for the 138 howler monkeys (Alouatta) analyzed.
Black circles = Lagothrix lagotricha cana; pink circle = A. caraya; blue circles = A. seniculus; green
circles = A. sara; yellow circles = A. pigra; brown circles = A. palliata. Red circles indicate missing
intermediate haplotypes.
The Median Joining Network (MJN) procedure (Figure 5) clearly differentiated the
haplotypes of Lagothrix and Alouatta with an estimated temporal split around 18.33 + 1.69
MYA ( = 94 + 8.72), which agrees quite well with the temporal splits estimated by many
authors between Alouatta and Atelinae (Meireles et al., 1999; Opazo et al., 2006; Perelman et
al., 2011; Springer et al., 2012; Von Dornum and Ruvolo, 1999). Of all the haplotypes, A.
caraya was the one most related to the original mitochondrial diversification of this genus.
This is also supported by the ML tree. The temporal split between this more ancestral cis-
Andean howler monkey haplotype and the first differentiated trans-Andean howler monkey
haplotype (one of A. pigra) was around 6.73 + 0.35 MYA ( = 34.5 + 1.80). This agrees with
the temporal split between both Alouatta groups estimated by Cortes-Ortiz et al., (2003). The
temporal splits between A. caraya-A. seniculus and A. caraya-A. sara were around 3.41 +
0.28 MYA ( = 17.5 + 1.41) and 3.99 + 0. 28 MYA ( = 20.5 + 1.41), respectively. Clearly,
this analysis also differentiated the haplotypes of A. seniculus and A. sara with a time division
of around 4.51 + 0.84 MYA ( = 23.12 + 4.32). The temporal splits between A. seniculus and
A. sara with reference to the two trans-Andean species (A. pigra and A. palliata) were 4.88 +
0.35 MYA (A. seniculus-A. pigra:  = 25 + 1.80), 5.36 + 0.41 MYA (A. seniculus-A. palliata:
 = 27.5 + 2.12), 4.29 + 0.35 MYA (A. sara-A. pigra:  = 22 + 1.80) and 4.78 + 0.41 MYA
(A. sara-A. palliata:  = 24.5 + 2.12), respectively. The mitochondrial diversification within
A. sara (around 2.22 + 0.29 MYA;  = 11.4 + 1.54) was older than in A. seniculus (around
0.86 + 0.30 MYA;  = 4.29 + 1.53) for this procedure. This disagrees with that obtained for
the BI tree, which showed an older mitochondrial diversification for A. seniculus than for A.
sara. On the other hand, the ancestor of A. pigra seems to be older than that of A. palliata.
The time split between both trans-Andean Alouatta species was around 3.12 + 0.41 MYA (
= 16 + 2.12) and the mitochondrial diversification within A. palliata was around 1.29 + 0.23
MYA ( = 6.59 + 1.18).
DISCUSSION
How Many Taxa of Red Howler Monkeys are in Peru?
Clearly our study reveals a striking and significant genetic heterogeneity among the five
“a priori” Alouatta taxa we analyzed, which is related to the relevant and noteworthy
karyological differences found by many studies for this Neotropical primate genus.
We begin this section with a discussion about the number of red howler monkey species
in Peru. It’s clear that at least two different species of red howler monkeys are present in
Peru: A. seniculus and A. sara. The main subspecies of A. seniculus is A. s. seniculus, but
other subspecies could be conserved within this species, distributed in the Northern Peruvian
Amazon (Napo, Javari, Tapiche and lower Ucayali rivers) and also in the middle Peruvian
Amazon (Pachitea River). A. sara was previously referred to as A. s. sara in the Southern
Peruvian Amazon along the Tambopata and Madre de Dios rivers. This is the first study
which unquestionably shows the existence of A. sara in Peru. Our results also show
indisputable arguments in favor of A. sara as a different species from A. seniculus, coinciding
with Cortés-Ortiz et al., (2003), using several different mitochondrial gene sequences. It’s
interesting to note that mitochondrial genes are extremely more useful to discriminate
different Alouatta species (and also in many other organisms) than nuclear gene sequences.
For instance, Cortés-Ortiz et al., (2003) showed that neither RAG1 nor CAL (nuclear genes)
data were capable of resolving the phylogenetic relationships among different Alouatta
species. These nuclear genes could only discriminate between Alouatta and Ateles species.
The same was found for other Neotropical primate phylogenetic studies (Collins and Dubach,
2001; Cropp and Boinski, 2000).
Minezawa et al., (1985) showed 2N = 50 for females (with X1X1X2X2) and 2N = 49 for
males (X1X2Y), with a range of 2-6 microchromosomes (six microchromosomes were more
frequent) for A. sara. 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
chromosomically differentiated from A. seniculus in Colombia (2N = 44 also with X1X1X2X2
and X1X2Y1Y2, 26 acrocentric chromosomes with 3-4 microchromosomes following Torres
and Leibocini, 2001, contrasting with the erroneously XX and XY system determined by
Yunis et al., 1976). Consinglieri et al., (1998) showed numerous interchromosomal
rearrangements in Alouatta. They demonstrated that A. belzebul had six unique apomorphic
associations and that A. sara and A. seniculus arctoidea shared seven derived associations.
Additionally A. sara had four apomorphic associations and A. seniculus arctoidea had seven.
Indeed, Consiglieri et al. (1996) showed, at least, 16 chromosome rearrangements including
numerous Robertsonian and tandem translocations and intra-chromosomal rearrangements
between A. sara and A. s. arctoidea (same karyological characteristics as A. s. seniculus;
Consiglieri et al., 1996; Torres and Ramírez, 2003). These karyotypic differences can in fact
be considered high enough to ensure reproductive isolation of A. sara from other red howler
monkeys (Stanyon et al., 1995; Consigliere et al., 1996).
Within A. s. seniculus, there was also internal polymorphism. Torres and Leibocini
(2001) showed that chromosomes 1, 2, 3, 6 and X showed heteromorphism with variability in
size and placement of the constitutive heterochromatin. In chromosome 1, there was a
polymorphism not having centromeric heterochromatin, which was presented as a
heterozygote condition in four of the eleven animals.
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 peripherally
isolated in the Yungus 2 refugia. Brown (1982) proposed this refugia on the basis of butterfly
endemism data presented in the context of the general forest refugia model (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). For this reason, Cortés-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, 2008) explained that the dry-wet
cycles were not exclusive to the Pleistocene and therefore these cycles should also be
important during the Pliocene. Cortés-Ortiz et al., (2003) speculated that the appearance of
the Madeira River could be essential to the split between these two red howler monkey
species, because the river forms the eastern boundary of A. seniculus and A. sara. We
estimated a temporal split somewhat higher than Cortés-Ortiz et al., (2003) (4.5 MYA with
the MNJ procedure). This age agrees quite well with a very important geological change in
the Amazon that had impacts on the rivers of the region. Espurt et al., (2007, 2010)
demonstrated that the Nazca Ridge subduction imprint had a significant influence on the
eastern side of the Andes by means of the Fitzcarrald Arch. This uplift is responsible for the
atypical three-dimensional shape of the Amazon’s foreland basin. Related with the Nazca
Ridge subduction, 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 divided A. sara and A. seniculus. This
event could also isolate the Bolivian dolphins from other Amazonian pink river dolphins
(Ruiz-García et al., 2016).
Another question is which of the two taxa could be first. The levels of gene diversity
were considerably higher in A. sara than in A. seniculus (3.22% vs. 0.57%), although the
geographical range extension of the second is considerably higher than that of the first.
Dobzhansky (1971) showed that original populations have the greatest levels of gene
diversity. However, if there were different molecular lineages of A. sara (from other Alouatta
taxa), they should artificially increase the gene diversity of A. sara. The MNJ procedure also
showed an older mitochondrial diversification in A. sara (around 2.22 MYA) than in A.
seniculus (0.86 MYA), which also coincides with the ancestors of A. sara as older than the
ancestors of A. seniculus. Some karyotypic results also agree with this possibility. Consiglieri
et al., (1998), starting from the hypothetical ancestral karyotype, estimated the number of
derived associations for each of three howler monkeys taxa. There were six for A. belzebul,
11 for A. sara, and 14 for A. seniculus arctoidea. It is clear then from the hybridization data
alone that the two red howler taxa have more derived karyotypes than A. belzebul and that A.
s. arctoidea is more derived than A. sara. Therefore, A. sara is more primitive than one A.
seniculus subspecies. The phylogenetic tree presented by Villalobos et al., (2004) also yielded
the ancestors of A. sara as more ancestral than that of A. seniculus. In contrast, other proofs
agree better with A. seniculus as an older taxon than A. sara. Our BI tree showed that the
ancestors of A. seniculus diverged before the ancestors of A. sara (thus both MNJ and BI
procedures did not agree in this aspect). Generally, the most ancestral populations show less
evidences of population expansions than the most modern populations. No tests, for A.
seniculus, showed population expansions, whilst three tests showed population expansions in
A. sara, which could be in agreement with a young origin for this last taxon. Although
Bovicino et al., (2001), did not enclose sequences of A. sara, they showed neighbor joining
and parsimony trees with mt Cyt-b gene sequences. They grouped A. belzebul and A. fusca as
sister branches in one clade. In a separate clade, A. seniculus was the most basal offshoot,
followed by A. nigerrima as a sister lineage of the most derived A. macconnelli and A.
stramineus. The grouping of A. belzebul and A. fusca as sister branches was also reported
with γ1-globin pseudogene sequence data, with A. caraya as the most basal offshoot followed
by A. seniculus and, subsequently, by the most derived A. belzebul/A. fusca clade (Meireles et
al., 1999). Thus, this question required further analyses.
What is clear is that the red howler monkeys are more related among themselves than
with other Alouatta taxa. A. sara, A. seniculus artoidea (Consigliere et al., 1996, 1998;
Minezawa et al., 1985), A. seniculus seniculus (Yunis et al., 1976; Torres and Levobivi,
2001), A. macconelli (Lima et al., 1990; Vassart et al., 1996) 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. fusca
(Koiffman, 1982; Koiffman and Saldanha, 1974; De Oliveira, 1996; De Oliveira et al., 1995,
1998, 2000, 2002).
Following the typological nomenclature of Gregorín (1996, 2006) and based on
geographical distribution, the animals sampled at the Javari and Ucayali rivers should be A.
juara. Furthermore, the animal from the Pachitea River (middle Ucayali River) should be A.
puruensis. Nevertheless, they were clearly related (with very small genetic distances) with the
A. s. seniculus individuals from Colombia. Therefore, our molecular results don’t agree with
A. juara and A. puruensis as full species. They are taxa within A. seniculus. Only,
karyological analyses could show if these taxa are really different from A. seniculus because
from a molecular point of view they were undifferentiated from A. s. seniculus. The
morphological characteristics used by Gregorin (1996, 2006) are probably not good
characters for reconstructing evolutionary histories. Their genetic basis are unknown and
positive natural selection could affect them. Thus, everything seems to indicate that there are
two different species of red howler monkeys, A. seniculus and A. sara, in the Peruvian
Amazon.
Molecular Phylogenetic Insights into the Systematics of Alouatta
Our BI split estimate for the initial diversification of Alouatta was a litter higher to that
obtained by Cortés-Ortiz et al., (2003). Our estimate was around 7.21 MYA, whilst the
estimate of Cortés-Ortiz et al., (2003) was 6.8 MYA. Nevertheless, our MJN estimate was
6.73 MYA, which is nearly identical to that estimated by the quoted authors. This temporal
split coincides with the formation of the Northern Andes (Lundberg et al., 1998). Similarly,
our BI temporal estimate for the diversification within the trans-Andean (Central American;
A. palliata and A. pigra) howler monkeys was higher (6.06 MYA) than the 3 MYA estimate
of Cortés-Ortiz et al., (2003) and our own MNJ estimate of 3.12 MYA. It’s important to note
the similarity of these last two estimates.
In this work, as well as in previous studies we carried out regarding Neotropical
mammalian species the MNJ temporal splits are systematically lower than those obtained by
BI trees (Ruiz- García et al., 2010, 2012a,b, 2014, 2015b, 2016; Ruiz-García and Pinedo,
2010). We suspect that the MJN procedure (borrowed molecular clock) produces more
realistic temporal splits than do the BI estimations (fossil-calibrated DNA phylogeny). This is
especially true when the mutation rates are well estimated and substantiated as those in the
current work. Another advantage of the MJN procedures compared to traditional trees is that
they explicitly allow for the co-existence of ancestral and descendant haplotypes, whereas
traditional trees treat all sequences as terminal taxa (Posada and Crandall, 2001). Use of the
MJN procedures allows us to observe which current taxa began to evolve first and also to
identify the more recently derived taxa. From our MJN, it seems clear that the South
American (cis-Andean) howler monkeys began their mitochondrial evolution much earlier
than the trans-Andean howler monkeys. This conclusion is quite logical. The clear population
expansion detected for A. palliata (for all the statistics employed) revealed that this species
has a younger population than the two cis-Andean species (A. seniculus and A. sara). Recall,
there was scant evidence of any population expansions in the latter two species. Furthermore,
haplotypes of A. pigra, derived from cis-Andean haplotypes generated the haplotypes of A.
palliata. Indeed, the traditional morphological subspecies, A. p. palliata and A. p.
aequatorialis, were not molecularly differentiable throughout Costa Rica, Colombia and
Ecuador. Therefore, although Cortés-Ortiz et al., (2003) conjectured that the minor
phylogeographic break separating northern and southern A. palliata could be located near
Panama’s Sona Peninsula (Bermingham and Martin, 1998), we didn’t find any geographical
point break. Even the three Panamanian exemplars sampled (A. coibensis trabeata) could not
be molecularly differentiated from A. palliata.
Our own MJN estimate and that of Cortés-Ortiz et al., (2003) (both around 3 MYA) for
the differentiation of trans-Andean howler monkeys agree quite well with the completion date
of the Panama Isthmus. This stresses the importance of the Panama Isthmus in the speciation
of Central American howler monkeys. The data of Cortés-Ortiz et al., (2003) correlated very
well with our affirmation that the molecular evolution of the cis-Andean howler monkeys
preceded the evolution of the trans-Andean. These authors estimated that the diversification
of the South American howler monkey species occurred around 4.8–5.1 MYA, near the end
of the Miocene. They also estimated that the temporal split between A. belzebul and A.
guariba occurred approximately 3.9–4.0 MYA. This is opposite to that claimed by Villalobos
et al., (2004). These authors claimed that their results demonstrated 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 reported 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 misleading.
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 the 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. The findings of Torres and Ramírez (2003) did show some Colombian A.
palliata to have the highest diploid number for the genus (2N = 56 and XX /XY sex
chromosome system). However the findings of Ma et al., (1975) indicated a different sex
chromosome system for the Panamanian animals. The system is not the XX/XY one claimed
by Villalobos et al., (2004). Instead, females have 2N = 54 and X1X1X2X2 and males have 2N
= 53 and X1X2Y (30 acrocentric chromosomes versus 34 acrocentric chromosomes in the
Colombian animals). The karyotype comparisons of the Colombian and Panamanian
individuals showed that the A4 pair of the Panamanian individuals was replaced in the
Colombian individuals by two acrocentric pairs of different sizes (B15 and B24; the second
pair is the result of a Robertsonian fussion of pairs 15 and 24). Additionally, the B19 pair of
the Colombian male did not show the heteromorphism observed in the Panamanian males.
The remaining chromosomes were similar. However, the molecular results showed that no
important differences were found among Panamanian and the Colombian individuals. Our
molecular results, as well as those of Cortés-Ortiz et al., (2003), don’t validate A. coibensis
trabeata as a separate species from the formerly known A. palliata panamensis, as stipulated
by Froehlich and Froehlich (1987) who analyzed dermal ridge patterns in hands and feet.
Our results, as well as that of Cortés-Ortiz et al., (2003) clearly differentiated A. pigra
from A. palliata. This aligns with Smith’s earlier work (1970). Cortés-Ortiz et al., (2003)
detected a 5.7% difference between A. palliata and A. pigra. Similarly, our calculated percent
was 5.3. Our molecular results also permit some evaluation of the two hypotheses presented
by Smith (1970) to account for the origins of A. palliata and A. pigra. Smith’s 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 two sequential invasions of Central America by the trans-
Andean Alouatta ancestor in South America. Our results more strongly agree with the first
than with the second hypothesis. The explanations of Cortés-Ortiz et al., (2003) are highly
plausible: 1- Both of these 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- Also, parapatric speciation could be important here across the ecotone that
separates the drier and lower forest 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 two howler monkey
species. However, Cortés-Ortiz et al., (2007), in Tabasco (Mexico), reported hybridization of
individuals with a mosaic of morphological characteristics between A. palliata and A. pigra.
These included individuals living in various grades of disturbed vegetation and that had
characteristics of both species. However, this seems to be an exception rather than the rule.
Other cases of hybridization for different Alouatta taxa were those reported by Aguiar et al.,
(2007), who described hybridization between A. caraya and A. fusca clamitans in a group of
eight individuals observed near the Paraná River in Brazil, in the ecotone between rain forest
and the Cerrado, showing intermediate morphological variation. Also, hybridization has been
documented to occur in captivity between A. caraya and A. fusca (De Souza et al., 2010).
Nevertheless, the chromosome complement of Alouatta fusca damitans showed a wide
variation in the diploid number, with 2N = 45, 46 and 52 (De Oliveira et al., 2000) and the
diploid number for A. caraya is 2N = 52 (Mudry et al., 1984, 1998). Thus, perhaps animals
with 52 chromosomes should hybridize without a problem. Indeed, Zúñiga-Leal and Defler
(2013) confirmed sympatry between A. seniculus and A. palliata along the west bank of the
Atrato River (Chocó Department in Colombia) but they did not find hybridization between
the two species. According to local information, sympatry between the two species continues
to an undetermined point up the Atrato River, but no hybrids have been discovered between
these two taxa. This could be related with the fact of the important chromosome differences
between the two species (Torres and Leibocini, 2001; Torres and Ramírez, 2003).
Molecularly and karyotypically speaking, there is clear evidence that A. macconnelli is a
full species. From a molecular perspective, Cortés-Ortiz et al., (2003) showed that this taxon,
A. sara and A. seniculus formed monophyletic groups (with 100% of bootstrap) and had a
level of gene divergence among them similar to that observed between A. palliata and A.
pigra. Ruiz-García et al., (2007) also showed that the dynamics of DNA microsatellites were
different between A. s. seniculus and A. macconnelli. Although, we don’t enclose
mitochondrial sequences of A. macconnelli in this study, in a parallel study, we also show the
differentiation of both red howler taxa. From a chromosomal perspective, A. macconnelli was
recognized as a full species by Lima and Seuánez (1991) and Vassart et al., (1996). Also, De
Oliveira et al., (2002) revealed that A. macconnelli and A. s. arctoidea differ by multiple
translocations which may further contribute to reproductive isolation between A. macconnelli
and A. seniculus.
However, the degree of differentiation between A. macconnelli and A. stramineus is not
very clear. Bonvicino et al., (1995) proposed that A. stramineus (west Trombetas River) and
A. macconnelli (east Trombetas River) should be considered as separate species basing their
results in the analysis of quantitative cranial traits. Armada et al., (1987) and Lima and
Seuánez (1991) showed that A. macconnelli and A. stramineus, although sharing the same
diploid number (47, 48 or 49) and sex chromosome system (X1X2Y1Y2/X1X1X2X2), differed
in two chromosome pairs. It was a result of a translocation event (reciprocal homozygote
translocation between chromosomes 2 and 20). This is why genetic introgression between
them was considered unlikely and they were considered valid species by Bonvicino et al.,
(1995). But other authors have revealed minor differences between the two red howler taxa.
Figueiredo et al., (1998), using the mitochondrial COII gene (one of the genes that we
analyzed here), presented a strong genetic homogeneity among the three populations studied.
There was a low number of nucleotide differences, phylogenetic continuity and the complete
absence of any geographic partitioning of the sequences. Sampaio et al., (1996) using
biochemical data (20 protein loci; genetic divergence of 0 to 0.2%) and cytogenetics showed
that A. macconelli and A. stramineus were related closely enough to be considered a single
species. Bonvicino et al., (2001) used karyotypic data to confirm the molecular topology of
the tree that they obtained from the mitochondrial Cyt-b gene. Their finding showed a closer
relationship between A. macconnelli and A. stramineus than either to A. nigerrima. They
showed 2N = 50 in the female, with nine pairs of biarmed autosomes against 11 pairs in A.
macconnelli and A. stramineus.
Indeed, very low genetic distance values (average 0.3%; around a divergence of 300,000
years) were observed between A. macconnelli and A. stramineus. These values were lower
than divergence estimates within other species such as A. caraya (temporal diverge between
haplotypes of this species is around 0.6 MYA) or A. belzebul (temporal diverge between
haplotypes of this species is around 0.8 MYA). Other studies of G-banding data from A.
macconnelli individuals collected from the Uatuma and Jari rivers revealed two distinct
karyotypes due to two independent Robertsonian translocations or a centric fission-fusion
event (De Oliveira et al., 2002; Lima and Seuánez, 1991).
The Uatuma River karyotype showed chromosome forms 2p/2q and 16 and the Jari
karyotype 2q and 2p/16. For these authors, however, it is unclear whether one chromosomal
difference has the potential to act as a profound post-mating isolating mechanism. Therefore,
it is highly probably that both of these red howler forms belong to the same species that
contains two subspecies: A. stramineus stramineus and A. stramineus macconnelli. However,
these red howler monkeys should be studied in more detail to determine the real status of
these forms.
The most external cis-Andean howler monkey that we found was A. caraya, with a
temporal split of around 3.4-4 MYA with reference to the two red howler taxa we studied.
Bonvicino et al., (2001) estimated this divergence to have occurred around 4.2 MYA.
Another estimate by Cortés-Ortiz et al., (2003) of 4.0 MYA is almost the same. Therefore,
there is clear similarity among these three estimates for the split between A. caraya and the
red howler monkey species. Also, the three works showed A. caraya as the sister clade of the
red howler monkeys. However, other authors claimed a different relationship status for A.
caraya. Hershkovitz (1949) divided the five species that were accepted at that time into three
different Alouatta groups, based on morphological studies of the hyoid bone. The three
groups are: Alouatta seniculus (A. seniculus, A. belzebul and A. fusca); A. palliata (A.
palliata; two further species, A. pigra and A. coibensis, were recognized and included in the
A. palliata group by Mittermeier et al., 1981); and A. caraya (A. caraya). Mittermeier et al.,
(1981) and Gregorín (1996) placed A. palliata as the most basal genus. We previously
discussed the uncertainty of this claim. This genus was followed by A. caraya and next the
clade formed by A. fusca and the Amazonian species, A. seniculus and A. belzebul. Also,
Meireles et al., (1999) used globin pseudogene phylogeny to determine A. caraya as a
monotypic clade separated from the rest of the South American Alouatta species. Other
authors, however, showed that the position of several Alouatta taxa did not correspond with
Hershkovitz’s (1949) scheme. For instance, De Oliveira (1996) used cytogenetical data to
propose the placement of A. belzebul nigerrima within the A. seniculus group. Sampaio et al.,
(1996), using biochemical and chromosomal data, found that A. seniculus and A. belzebul
were not the closest species to the A. seniculus group. Their compiled chromosomal data
supported A. belzebul and A. fusca to have the closest relationship. De Oliveira et al., (2002)
used chromosomal data to propose another classification. Alouatta can be divided into two
distinct species groups, the first with A. caraya and A. belzebul and the second with A.
macconnelli, A. sara, A. seniculus arctoidea and A. fusca. This classification agrees with the
fact that eight out of 28 ancestral Neotropical primate chromosomes are conserved in A.
macconnelli, 11 in A. fusca clamitans, 14 in A. fusca fusca and 15 in A. caraya. Thus, taking
this data into account, A. macconnelli was the most derived taxa and A. caraya was the most
ancestral one. In addition, De Oliveira et al., (2002) observed high level of species-specific
(autapomorphic) rearrangements, reflecting the extensive karyological variation within this
genus. The number varies between two in A. caraya to 10 in A. macconnelli. The variation in
number should stress A. macconnelli as the most derived taxa and A. caraya as the most
ancestral one. This coincides with Mudry et al., (1998), who showed a very stable karyotype
for A. caraya (data from 25 males and 22 females from different geographical origins; Mudry
et al., 1994). Thus, while chromosomal speciation mechanisms should be very important in
the red howler monkeys, there is no evidence that these mechanisms are present in A. caraya.
But, as we mentioned, the three mitochondrial genes support A. caraya as the sister clade of
the red howler monkey clade. Bonvicini et al., (2001) estimated the temporal split between
the clades to have occurred around 4.7 MYA whereas Cortés-Ortiz et al., (2003) estimated the
split to have occurred a little earlier (5.1 MYA).
Cortés-Ortiz et al., (2003) explained that the current distribution of A. caraya is not a
result of the presence any clear geographical barriers. It is parapatric with A. sara to the west,
with A. nigerrima and A. belzebul to the north, and with A. fusca to the east. It is true however
that the Goia refugia (Brown, 1982, for butterflies) in southeast Matto Grosso (Brazil) may
help to explain the distribution of A. caraya. However, Cortés-Ortiz et al., (2003) discarded
this idea because their estimated temporal split of A. caraya from the red howler clade (4
MYA) suggested a Pliocene origin for this species and not a Pleistocene refugia model of
speciation. Nevertheless, Cortés-Ortiz et al., (2003) forget that Haffer (1997, 2008) expanded
the dry-wet cycles also to the Pliocene. Additionally, Espurt et al., (2007, 2010) demonstrated
that the Nazca Ridge subduction imprint had a significant influence by means of the
Fitzcarrald Arch uplift around 4 MYA (Pliocene). This could have been decisive in the
origination of the Madeira River, a separation barrier to the ancestor of A. caraya.
Bonvicino et al., (2001) commented that the X1X2Y1Y2/X1X1X2X2 sex chromosomal
system found in A. sara and A. s. arctoidea consists of shared chromosomes that are to
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 if this were the case, the rearrangements that originated this
sex chromosome system must have occurred in the common ancestor of the red howler
species. Alternatively, these rearrangements could have occurred independently (and
involving different autosomes) in two separate ancestors, one in A. macconnelli/A.
stramineus and another in A. sara and A. s. arctoidea. Nevertheless, De Oliveira et al.,
(2002) showed that in all A. sara, A. seniculus arctoidea, A. macconnelli and A. caraya, the
autosome involved in this rearrangement corresponded to a 3c/15b association. However,
one further rearrangement involving the human chromosome 1b homologous segment was
observed in A. fusca. These results agree quite well with the fact that A. caraya is more
closely related to the red howler monkey species than A. fusca should be. This is also highly
correlated with the observation made by Cortés-Ortiz et al., (2003) but it disagrees with the
tree proposed by De Oliveira et al., (2002). As we did not include sequences of A. belzebul or
A. fusca/A. clamitans in this study, we cannot clarify which is the oldest ancestor of the
current cis-Andean howler monkey taxa. De Oliveira et al., (2002) concluded that the
ancestral sex chromosome system for the Alouatta genus should consist of X1X2/Y1Y2
chromosomes, where X1 would correspond to the homolog in humans (X, X2 to 3c/15b, Y1 to
Y/15b, and Y2 to 3c).
Alouatta shows more common fragile sites in their chromosomes than do other
Neotropical primates. Fundia et al., (2000), for instance, showed that Saimiri boliviensis
presented a low level of spontaneous breakage (six spontaneous events), whereas A. caraya
(39 spontaneous events) showed elevated frequencies with 1q23, 2q13 and 11q19 as hot-spots
for spontaneous breakage. Even, in A. caraya, one fragile site (1q31) correlated with a
breakpoint involved in a pericentric inversion (Mudry et al., 1990). Therefore, chromosomal
speciation in Alouatta could be very more frequent than in other mammals. Wienberg et al.,
(1997) showed that karyotype evolution in mammals is generally assumed to be highly
conservative. For example the number of translocations between man and cat has been
estimated to be on the order of one every 10 million years.
The most extreme karyological diversity in Alouatta is found A. fusca. De Oliveira et al.,
(1995, 1998, 2000) showed, at least, four 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). De Oliveira et al., (2000) showed
that the different karyotypes described revealed a clinal distribution for the chromosomal
variation in A. fusca clamitans. This could suggest that there are populations in different
stages of speciation, and probably reproductively isolated, due to meiotic disturbance.
Stasipatric speciation by chromosomal mutations would have been more easily fixed due to
inbreeding among small populations with low effective numbers. In Alouatta, the dominant
male copulates with the great majority of females in small groups (Wolheim, 1983),
increasing the frequency of inbreeding. De Oliveira et al., (2002) showed that A. f. fusca and
A. f. clamitans differ by two Robertsonian translocations. A hybrid between the respective
karyotypes, 2N = 49/50 and 2N = 45/46, would require the formation of two trivalents, which
could have serious problems during meiosis. Most likely, A. fusca and A. clamitans are both
full species. With the exception of one population of A. clamitans (Espiritu Santo state),
XY/XX (De Oliveira et al., 2000), and the Colombian A. palliata individuals studied by
Torres and Ramírez (2003) (56 with XY/XX), all the remaining Alouatta taxa show the Y
chromosome translocated to an autosomal chromosome. The alleged XY/XX karyotypes for
A. s. seniculus (Yunis et al., 1976) and for A. caraya (Mudry et al., 1984) were validated by
more recent studies with improved resolution (Mudry et al., 1998; Torres and Leibovici,
2001).
Therefore, the Alouatta genus should experience speciation with relative frequency 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 where chromosomal stability is higher.
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 Instituto von Humboldt (Villa de Leyva in Colombia;
Janeth Muñoz), to the Peruvian Ministry of Environment, PRODUCE (Dirección Nacional de
Extracción y Procesamiento Pesquero), Consejo Nacional del Ambiente and the Instituto
Nacional de Recursos Naturales from Peru, and to the Colección Boliviana de Fauna (Dr.
Julieta Vargas) and to CITES Bolivia for their role in facilitating the obtainment of the
collection permits in Colombia, Peru and Bolivia. The Costa Rican howler monkeys were
sampled with the collection permits conceded by the Costa Rican government to Dr. Gustavo
Gutiérrez-Espeleta. Thanks also goes to ARCAS (Guatemala) for providing hair samples of
Alouatta pigra. The first author also thanks 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,
Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri and Yucuna) by their facilities in obtaining
samples of howler monkeys.
REFERENCES
Aguiar, L., Mellek, D., Abreu, K., Boscarato, T., Bernardi, I., Miranda, J. & Passos, F.
(2007). Sympatry of Alouatta caraya and A. clamitans and the rediscovery of free-
ranging potential hybrids in Southern Brazil. Primates, 48, 245–248.
Akaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on
Automatic Control, AC, 19, 716–723.
Aquino, R. & Encarnación, F. (1994). Primates of Peru. Primate Report, 40, 1-127.
Aquino, R., Terrones, W., Navarro, R., Terrones, C. & Cornejo, F. M. (2009). Caza y estado
de conservación de primates en la cuenca del río Itaya, Loreto, Perú. Revista Peruana de
Biología, 15, 33-39.
Armada, J. L., Barroso, C. M., Lima, M. C., Muñiz, J. A. & Seuánez, H. N. (1987).
Chromosome studies in Alouatta belzebul. American Journal of Primatology, 13, 283-
296.
Ascunce, M. S., Hasson, E. & Mudry, M. D. (2003). COII: a useful tool for inferring
phylogenetic relationships among New World monkeys (Primates, Platyrrhini).
Zoologica Scripta, 32, 397-406.
Ashley, M. V. & Vaughn, T. A. (1995). Owl monkeys (Aotus) are highly divergent in
mitochondrial cytochrome c oxidase (COII) sequences. International Journal of
Primatology, 5, 793–807.
Auricchio, P. (1995). Primatas do Brasil. Terra Brasilis Editora Ltda. Sao Paulo, Brazil. pp.
1-168.
Avise, J. C., Arnold, J., Ball, R. M., Bermingham, E., Lamb, T., Neigel, J. E., Reeb, C. A. &
Saunders, N. C. (1987). Intraspecific phylogeographic: the mitochondrial DNA bridge
between population genetics and systematics. Annual Review of Ecology, Evolution, and
Systematics, 18, 489-522.
Bandelt, H-J., Forster, P. & Rohl, A. (1999). Median-joining networks for inferring
intraspecific phylogenies. Molecular Biology and Evolution, 16, 37-48.
Bender, M. A. & Chu, E. H. Y. (1963). The chromosomes of primates. Vol. 1. In Buettner-
Janusch (Ed.). Evolutionary and genetic biology of primates., Academic Press. New
York. pp. 261-310.
Bermingham, E. & Martin, A. P. (1998). Comparative mtDNA phylogeography of
neotropical freshwater fishes: testing shared history to infer the evolutionary landscape of
lower Central America. Molecular Ecology, 7, 499–517.
Bodini, R. & Pérez-Hernández, R. (1987). Distribution of the species and subspecies of
Cebids in Venezuela. Fieldiana Zoology, 39, 231-244.
Bonvicino, C. R., Fernandes, M. E. B. & Seuánez, H. N. (1995). Morphological analysis of
Alouatta seniculus species group (Primates, Cebidae). A comparison with biochemical
and karyological data. Journal of Human Evolution, 10, 169–176.
Bonvicino, C. R., Lemos, B. & Seuánez, H. N. (2001). Molecular phylogenetics of howler

monkeys Alouatta. Platyrrhini. A comparison with karyotypic data. Chromosoma, 110,
241-246.
Boubli, J.-P., Di Fiore, A., Rylands, A. B. & Mittermeier, R. A. (2008). Alouatta seniculus
ssp. puruensis. The IUCN Red List of Threatened Species. Version 2015.2.
<www.iucnredlist.org>. Downloaded on 26 July 2015.
Brown, K. S. (1982). Paleoecology and regional patterns of evolution in Neotropical forest
butterflies. In: Prance GT (Ed.) Biological diversification in the tropics. Columbia
University Press.
Bull, J. J. & Bulmer, M. G. (1981). Evolution of XY females in mammals. Heredity, 4, 347-
360.
Cabrera, A. (1939). Los monos de Argentina. Physis (Rev. Soc. Argentina Cienc. Natur), 3-
29.
Cabrera, A. (1957). Catálogo de los mamíferos de América del Sur. I (Metatheria,
Unguiculata, Carnívora). Revista del Museo Argentino de Ciencias Naturales “Bernardo
Rivadavia,” Zoología, 4, 1-307.
Collins, A. C. & Dubach, J. M. (2000). Phylogenetic relationships of spider monkeys (Ateles)
based on mitochondrial DNA variation. International Journal of Primatology, 21, 381–
420.
Collins, A. C. & Dubach, J. M. (2001). Nuclear DNA variation in spider monkeys (Ateles).
Molecular Phylogenetics and Evolution, 19, 67–75.
Consigliere, S., Stanyon, R., Koehler, U., Agoramoorthy, G. & Wienberg, J. (1996).
Chromosome painting defines genomic rearrangements between red howler monkeys
subspecies. Chromosome Research, 4, 264–270.
Consigliere, S., Stanyon, R., Koehler, U., Arnold, N. & Wienberg, J. (1998). In situ
hybridization (FISH) maps chromosomal homologies between Alouatta belzebul
(Platyrrhini, Cebidae) and other primates and reveals extensive interchromosomal
rearrangements between howler monkeys genomes. American Journal of Primatology,
46, 119–133.
Cortés-Ortiz, L., Bermingham, E., Rico, C., Rodriguez-Luna, E., Sampaio, I., Ruiz-García,
M. (2003). Molecular systematics and biogeography of the Neotropical monkey genus
Alouatta. Molecular Phylogenetics and Evolution, 26, 64–81.
Cortés-Ortiz, L., Duda, Jr., T. F., Canales-Espinosa. D., García-Ordun, F., Rodríguez Luna, E.
& Bermingham, E. (2007). Hybridization in large-bodied New World primates. Genetics,
176, 2421-2425.
Crockett, C. M. & Eisenberg, J. F. (1987). Howlers: variations in group size and demography.
In: Smuts, B.B., Cheney, D.L., Seyfarth, R.M., Wrangham, R.W., Struhsaker, T.T. (Eds.).
Primate Societies. (pp. 54-68). University of Chicago Press, Chicago.
Cropp, S. & Boinski, S. (2000). The Central American squirrel monkey (Saimiri oerstedii):
introduced hybrid or endemic species. Molecular Phylogenetics and Evolution, 16, 350–
365.
Cummings, M. P., Otto, S. P. & Wakeley, J. (1995). Sampling properties of DNA sequence
data in phylogenetic analysis. Molecular Biology and Evolution, 12, 814–822.
De Oliveira, E. H. C. (1996). Estudos citogenéticos e evolutivos nas espécies brasileiras e
argentinas do gênero Alouatta Lacépède, 1799 (Primates, Atelidae). MSc Dissertation.
Universidade Federal do Paraná. Curitiba. Brazil.
De Oliveira, E. H. C., Lima, M. C. & Sbalqueiro, I. J. (1995). Chromosomal variation in

Alouatta fusca. Neotropical Primates, 3, 181-183.
De Oliveira, E. H. C., Lima, M. C., Sbalqueiro, I. J. & Pissinatti, A. (1998). The karyotype of
Alouatta fusca clamitans from Rio de Janeiro State. Brazil: evidence for a Y autosome
translocation. Genetics and Molecular Biology, 31, 361-364.
De Oliveira, E. H. C., Suemitsu, E., da Silva, A. F. & Sbalqueiro, I. J. (2000). Geographical
variation of chromosomal number in Alouatta fusca clamitans (Primates, Atelidae).
Caryologia, 53, 163-168.
De Oliveira, E. H. C., Neusser, M., Figueiredo, W. B., Nagamachi, C., Pieczarka, JC,
Sbalqueiro, I. J., Wienberg, J. & Miller, S. (2002). The phylogeny of howler monkeys
(Alouatta, Platyrrhini): Reconstruction by multicolor cross-species chromosome painting.
Chromosome Research, 10, 669-683.
De Souza Jesus, A., Schunemann, H. E., Müller, J. & da Silva, M. A. (2010). Hybridization
between Alouatta caraya and Alouatta guariba clamitans in captivity. Primates, 51, 227–
230.
Dobzhansky, T. (1971). Evolutionary oscillations in Drosophila pseudoobscura. In: Creed, R.
(Eds.). Ecological genetics and evolution. Blackwell Scientific, Oxford, UK. pp. 109–
133.
Dos Reis, N. R., Peracchi, A. L. & Andrade, F. R. (2008). Primatas Brasileiros. Technical
Books Editora. Londrina, Brazil. Pp. 1-260.
Drummond, A. J., Ho, S. Y. W., Phillips, M. J. & Rambaut, A. (2006). Relaxed phylogenetics
and dating with confidence. PLOS Biology, 4, e88.
Drummond, A. J., Suchard, M. A., Xie, D. & Rambaut, A. (2012). Bayesian phylogenetics
with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29, 1969–1973.
Dutrillaux, B., Descaileaux, J., Viegas-Péquignot, E. & Couturier, J. (1981). Y-Autosome
translocation in Cacajao calvus rubicundus (Platyrrhini). Annales Génetique, 24, 197-
201.
Espurt, N., Baby, P., Brusset, S., Roddaz, M., Hermoza, W., Regard, V., Antoine, P. O.,
Salas-Gismondi, R. & Bolanos, R. (2007). How does the Nazca Ridge subduction
influence the modern Amazonian foreland basin? Geology, 35, 515–518.
Espurt, N., Baby, P., Brusset, S., Roddaz, M., Hermoza, W. & Barbarand, J. (2010). The
Nazca Ridge and uplift of the Fitzcarrald Arch: implications for regional geology in
northern South America. In Hoorn, C., and Wesselingh, F. (Eds.). Amazonia, Landscape
and Species Evolution: A Look into the Past. (pp. 89-102). Wiley-Blackwell, Oxford.
Estrada, A. & Coates-Estrada, R. (1984). Some observations on the present distribution and
conservation of Alouatta and Ateles in southern Mexico. American Journal of
Primatology, 7, 133-137.
Egozcue, J. & Egozcue, M. V. (1966). The chromosome complement of the howler monkey
(Alouatta caraya Humboldt, 1812). Cytogenetics, 5, 20–24.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood
approach. Journal of Molecular Evolution, 17, 368–376
Figueiredo, W. B., Carvalho-Filho, N. M., Schneider, H. & Sampaio, I. (1998). Mitochondrial
DNA sequences and the taxonomic status of Alouatta seniculus populations in
Northeastern Amazonia. Neotropical Primates, 6, 73-77.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994). DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan
invertebrates. Molecular Marine Biology and Biotechnology, 3, 294-299.
Ford, C. E., Hamerton, J. L. & Sharman, G. B. (1957). Chromosome polymorphism in the
common shrew. Nature, 180, 392-394.
Frohelich, J. W. & Frohelich, P. H. (1987). The status of Panama’s endemic howling
monkeys. Primate Conservation, 8, 58–62.
Fu, Y-X. (1997). Statistical tests of neutrality against population growth, hitchhiking and
background selection. Genetics, 147, 915-925.
Fu, Y. & Li, W. (1993). Statistical Tests of Neutrality of Mutations. Genetics, 133, 693-709.
Fundia, A., Gorostiaga, M. & Mudry, M. (2000). Expression of common fragile sites in two
Ceboidea species: Saimiri boliviensis and Alouatta caraya. (Primates: Platyrrhini).
Genetics Selection Evolution, 32, 87-97.
Garber, P. A., Estrada, A., Bicca-Marques, J. C., Heymann, E. W. & Strier, K. B. (2009).
South American Primates. Comparative perspectives in the study of behavior, ecology
and conservation. Springer Science+Business Media, New York, USA. pp. 1-564.
Gregorin, R. (2006). Taxonomia e variação geográfica das espécies do gênero Alouatta
Lacépède (Primates, Atelidae) no Brasil. Revista Brasileira de Zoologia, 23, 64-144.
Gregory, T., Carrasco Rueda, F., Deichmann, J. L., Kolowski, J. & Alonso, A. (2012).
Primates of the lower Urubamba region, Peru, with comments on other mammals.
Neotropical Primates, 19, 16-23.
Groves, C. P. (2001). Primate taxonomy. Washington, DC: Smithsonian Institution Press.
Haffer, J. (1969). Speciation in Amazonian forest birds. Science, 165, 131-137.
Haffer, J. (1982). General aspects of the refuge theory. In Prance, G. T., (Ed.). Biological
diversification in the tropics. (pp. 6-24). New York: Columbia University.
Haffer, J. (1997). Alternative models of vertebrate speciation in Amazonia: an overview.
Biodiversity Conservation, 6, 451-476.
Haffer, J. (2008). Hypotheses to explain the origin of species in Amazonia. Brazilian Journal
of Biology, 68, 917-947.
Harpending, H. C. (1994). Signature and ancient population growth in a low-resolution
mitochondrial DNA mismatch distribution. Human Biology, 66, 591-600.
Harpending, H. C., Sherry, S. T., Rogers, A. R. & Stoneking, M. (1993). Genetic structure of
ancient human populations. Current Anthropology, 34, 483-496.
Hasegawa, M., Kishino, H. & Yano, T. (1985). Dating of human-ape splitting by a molecular
clock of mitochondrial DNA. Journal of Molecular Evolution, 22, 160-174.
Hebert, P. D. N., Ratnasingham, S. & de Waard, J. R. (2003). Barcoding animal life:
cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings
of the Royal Society of London B, 270 (Suppl.), S96-9.
Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T. & Francis, C. M. (2004). Identification of birds
through DNA barcodes. PLoS Biology, 2, 1657-63.
Hershkovitz, P. (1949). Mammals of northern Colombia. Preliminary report no. 4: monkeys
(primates), with taxonomic revisions of some forms. Proceedings of the United States
National Museum, 98, 323–427.
Hill, W. C. O. (1962). Primates Comparative Anatomy and Taxonomy. V. Cebidae. Part. B.
Edinburgh University Press, Edinburgh.
Hudson, R. R., Boss, D. D. & Kaplan, N. L. (1992). A statistical test for detecting population
subdivision. Molecular Biology and Evolution, 9, 138-151.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions
through comparative studies of nucleotide sequences. Journal of Molecular Evolution,
16, 111-120.
King, M. (1993). Species Evolution. Cambridge University Press, Cambridge.
Koiffmann, C. P. (1982). Variabilidadi cromossomica em Cebidae. In P. H (Ed.). Genética
comparada de primates Brasileiros. (pp. 113-132).
Koiffmann, C. P. & Saldhana, P. H. (1974). Cytogenetics of Brazilian monkeys. Journal of
Human Evolution, 3, 275–282.
Lavergne, A., Ruiz-García, M., Catzeflis, F., Lacote, S., Contamin, H., Mercereau-Puijalon,
O., Lacaste, A. & Thoisy B. (2010). Taxonomy and phylogeny of squirrel monkey (genus
Saimiri) using cytochrome b genetic analysis. American Journal of Primatology, 72, 242-
253.
Lewis, H. (1966). Speciation in flowering plants. Science, 152, 167-172.
Librado, P. & Rozas, J. (2009). DnaSP v5: A software for comprehensive analysis of DNA
polymorphism data. Bioinformatics, 25, 1451-1452 | doi: 10.1093/bioinformatics/btp187.
Lima, M. C. & Seuánez, H. N. (1989). Cytogenetic characterization of Alouatta belzebul with
atypical pelage coloration. Folia Primatologica, 52, 97 -101.
Lima, M. C., Sampaio, M. I., Schneider, M. P., Scheffrahn, W., Schneider, H. & Salzano, F.
M. (1990). Chromosome and protein variation in red howler monkeys. Revista Brasileira
de Genética, 13, 789 - 802.
Lima, M. M. C. & Seuánez, H. N. (1991). Chromosome studies in the red howler monkey,
Alouatta seniculus stramineus (Platyrrhini, Primates): description of an
X1X2Y1Y2/X1X1X2X2 sex-chromosome system and karyological comparisons with
other subspecies. Cytogenetics and Cell Genetics, 57, 151–156.
Lundberg, J. G., Marshall, L., Guerrero, J., Horton, B., Malabarba, C. & Wesselingh, F.
(1998). The stage for neotropical fish diversification: history of a tropical South
American river. In Malabarba, L.R., Reis, R., Vari, R., Lucena, Z., and Lucena, C.A.,
(Eds.). Phylogeny and classification of neotropical fishes. (pp. 13-48). Porto Alegre,
Brazil: Pontificia Universidade Católica Do Rio Grande Do Sul.
Ma, N. S., Jones, T. C., Thorington, R. W., Miller, A. & Morgan, L. (1975). Y-autosome
translocation in the howler monkey, Alouatta palliata. Journal of Medical Primatology,
4, 299-307.
Ma, N. S., Jones, T. C., Miller, A. C., Morgan, L. M. & Adams, E. A. (1976). Chromosome
polymorphism and banding patterns in the owl monkey (Aotus). Laboratory Animal
Science, 26, 1022-1036.
Ma, N. S. F., Renquist, D. M., Hall, R., Sehgal, P. K., Simeone, T., Jones, T. C. (1980).
XX/XO sex determination systems in a population of Peruvian owl monkeys, Aotus.
Journal of Heredity, 71, 336–342.
Meireles, C. M., Czelusniak, J., Schneider, M. P., Muniz, J. A. P. C., Brigido, M. C., Ferreira,
H. S. & Goodman, M. (1999). Molecular phylogeny of Ateline New World Monkeys
(Platyrrhini, Atelinae) based on -globin gene sequences: evidence that Brachyteles is the
sister group of Lagothrix. Molecular Phylogenetics and Evolution, 12, 10–30.
Mercado, N. I. & Wallace, R. B. (2010). Distribución de primates en Bolivia y áreas
prioritarias para su conservación. Tropical Conservation Science, 3, 200-217.
MINAM. (2011). Informe Final del Estudio de Especies CITES de Primates Peruanos.
Ministerio del Medio Ambiente, Lima, Perú. pp. 1-219.
Minezawa, M., Harada, M., Jordan, O. C. & Borda, C. J. V. (1985). Cytogenetics of Bolivian
endemic red howler monkeys (Alouatta seniculus sara): accessory chromosomes and Y-
autosome translocation related numerical variations. Kyoto Univ. Overs. Res. Rep. N. W.
Monkeys, 5, 7–16.
Mittermeier, R. A. & Coimbra-Filho, A. F. (1981). Systematics: species and subspecies. In
Coimbra-Filho, A. F., and Mittermeier, R. A. (Eds.). Ecology and behavior of
Neotropical Primates., Vol 1. (pp. 29-109).
Mittermeier, R. A., Rylands, A. B., Coimbra-Filho, A. & Fonseca, G. A. B. (1988). Ecology
and behavior of Neotropical Primates., Volume 2. World Wildlife Fund, Washington, D.
C., USA. pp. 1-610.
Montgelard, C., Catzeflis, F. M. & Douzery E. (1997). Phylogenetic relationships of
artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S
rRNA mitochondrial sequences. Molecular Biology and Evolution, 14, 550–559.
Moore, W. (1995). Inferring phylogenies from mtDNA variation: mitochondrial-gene trees
versus nuclear-gene trees. Evolution, 49, 718–726.
Morral, N., Bertrantpetit, J. & Estivill, X., et al. (1994). The origin of the major cystic fibrosis
mutation (delta F508) in European populations. Nature Genetics, 7, 169-175.
Mudry, M. D., Laval, M. L., Colillas, O. J. & Brieux, S. (1984). Banding patterns of Alouatta
caraya. Revista Brasileira de Genética, 2, 373 - 379.
Mudry, M. D., Slavutsky, I. & Vinuesa, M. L. (1990). Chromosome comparison among five
species of Platyrrhini (Alouatta caraya, Aotus azarae, Callithrix jacchus, Cebus apella,
and Saimiri sciureus). Primates, 31, 415–420.
Mudry, M., Ponsa, M., Borell, A., Egozcue, J. & Garcia, M. (1994). Prometaphase
chromosomes of the howlermonkey (Alouatta caraya): G, C, NOR and restriction
enzyme (Res) banding. Am. J. Primatol., 33, 121-134.
Mudry, M. D., Rahn, M., Gorostiaga, M., Hick, A., Merani, M. S. & Solari, A. J. (1998).
Revised karyotype of Alouatta caraya (Primates: Platyrrhini) based on synaptonemal
complex and banding analyses. Hereditas, 128, 9-16.
Opazo, J. C., Wildman, D. E., Prychitko, T., Johnson, R. M. & Goodman, M. (2006).
Phylogenetic relationships and divergence times among New World monkeys
(Platyrrhini, Primates). Molecular Phylogenetetics and Evolution, 40, 274-280.
Pacheco, V., Cadenillas, R., Salas, E., Tello, C. & Zeballos, H. (2009). Diversidad y
endemismo de los mamíferos del Perú. Revista Peruana de Biología, 16, 5-32.
Perelman, P., Johnson, W. E., Roos, C., Seuanez, H. N., Horvath, J. E., Moreira, M. A. M.,
Kessing, B., Pontius, J., Roelke, M., Rumpler, Y., Schneider, M. P. C., Silva, A.,
O’Brien, S. J. & Pecon-Slattery, J. (2011). A molecular phylogeny of living primates.
PLoS Genetics, 7, e1001342.
Pieczarka, J. C. & Nagamachi, C. Y. (1988). Cytogenetic studies of Aotus from eastern
Amazonia Y autosome rearrangement. American Journal of Primatology, 14, 255–264.
Pope, T. R. (1992). The influence of dispersal patterns and mating system on genetic
differentiation within and between populations of the red howler monkeys (Alouatta
seniculus). Evolution, 46, 1112–1128.
Posada D, Crandall KA. (1998). MODELTEST: testing the model of DNA substitution.
Bioinformatics, 14, 817-818.
Posada, D. & Crandall, K. A. (2001). Intraspecific gene genealogies: trees grafting into
networks. Trends Ecology and Evolution, 16, 37–45.
Rambaut, A. (2012). FigTree v1.4. http://tree.bio.ed.ac.uk/software/figtree/.
Rambaut, A. & Drummond, A. J. (2013a). LogCombiner v1.8.0. http://beast.bio.ed.ac.uk/.
Rambaut, A. & Drummond, A. J. (2013b). TreeAnnotator v1.8.0. http://beast.bio.ed.ac.uk/.
Rambaut, A., Suchard, M. A., Xie, W. & Drummond, A. J. (2013). Tracer v1.6.
http://tree.bio.ed.ac.uk/software/tracer/.
Ramos-Onsins, S. E. & Rozas, J. (2002). Statistical Properties of New Neutrality Tests
Against Population Growth. Molecular Biology ad Evolution, 19, 2092–2100.
Reig, O. (1980). Modelos de especiación cromosómica en las casiraguas (Género
Proechimys) de Venezuela. In Reig, O (Ed.). Ecología y Genética de la especiación
animal. pp. 149-190). Equinoccio, Editorial de la Universidad Simón Bolívar, Caracas,
Venezuela.
Rogers, A. R. & Harpending, H. C. (1992). Population growth makes waves in the
distribution of pairwise genetic differences. Molecular Biology and Evolution, 9, 552-
569.
Rogers, A. R., Fraley, A. E., Bamshad, M. J., Watkins, W. S. & Jorde, L. B. (1996).
Mitochondrial mismatch analysis is insensitive to the mutational process. Molecular
Biology and Evolution, 13, 895-902.
Rosenbaum, G., Giles, D., Saxon, M., Betts, P. G., Weinberg, R. F. & Duboz, C. (2005).
Subduction of the Nazca Ridge and the Inca Plateau: Insights into the formation of ore
deposits in Peru. Earth Planetary Science Letters 239, 18–32.
Ruiz-García, M. & Pinedo-Castro, M. (2010). Molecular systematics and phylogeography of
the genus Lagothrix (Atelidae, Primates) by means of mitochondrial COII gene. Folia
Primatologica, 81, 109–128.
Ruiz-García, M., Escobar-Armel, P., Alvarez, D., Mudry, M., Ascunce, M., Gutierrez-
Espeleta, G. & Shostell, J. M. (2007). Genetic variability in four Alouatta species
measured by means of nine DNA microsatellite markers: Genetic structure and recent
bottlenecks. Folia Primatologica, 78, 73–87.
Ruiz-García, M., Castillo, M. I., Vásquez, C., Rodriguez, K. & Pinedo-Castro, M. et al.
(2010). Molecular phylogenetics and phylogeography of the white-fronted capuchin
(Cebus albifrons; Cebidae, Primates) by means of mtCOII gene sequences. Molecular
Phylogenetics and Evolution, 57, 1049-1061.
Ruiz-García, M., Castillo, M. I., Lichilin, N. & Pinedo-Castro, M. (2012a). Molecular
relationships and classification of several tufted capuchin lineages (Cebus apella, C.
xanthosternos and C. nigritus, Cebidae), by means of mitochondrial COII gene
sequences. Folia Primatologica, 83, 100-125.
Ruiz-Garcia, M., Castillo, M. I., Ledezma, A., Leguizamon, N. & Sánchez, R. et al. (2012b).
Molecular systematics and phylogeography of Cebus capucinus (Cebidae, Primates) in
Colombia and Costa Rica by means of the mitochondrial COII gene. American Journal of
Ruiz-García, M., Pinedo-Castro, M. & Shostell, J. M. (2014). How many genera and species
of woolly monkeys (Atelidae, Platyrrhine, Primates) are there? The first molecular
analysis of Lagothrix flavicauda, an endemic Peruvian primate species. Molecular
Phylogenetics and Evolution, 79, 179–198.
Ruiz-García, M., Vásquez, C., Sandoval, S., Kaston, F., Luengas-Villamil, K. & Shostell, J.
M. (2015a). Phylogeography and spatial structure of the lowland tapir (Tapirus terrestris,
Perissodactyla: Tapiridae) in South America. Mitochondrial DNA, 26, 1-9, on line DOI:
10.3109/19401736.2015.1022766.
Ruiz-García, M., Luengas, K., Leguizamón, N., Thoisy, B. & Gálvez, H. (2015b). Molecular
Phylogenetics and Phylogeography of all the Saimiri species (Cebidae, Primates) inferred
from mt COI and COII gene sequences. Primates, 56, 145-161.
Ruiz-García, M., Escobar-Armel, P., Thoisy, B., Martínez-Agüero, M., Pinedo-Castro, M. &
Shostell, J. M. (2016). Biodiversity in the Amazon: origin hypotheses, intrinsic capacity
of species colonization, and comparative phylogeography of river otters (Lontra
longicaudis and Pteronura brasiliensis, Mustelidae, Carnivora) and pink river dolphin
(Inia sp, Iniidae, Cetacea). Biological Journal of the Linnean Society (in press).
Ruvolo, M., Disotell, T. R., Allard, M. W., Brown, W. M. & Honeycutt, R. L. (1991).
Resolution of the African hominoid trichotomy by use of a mitochondrial gene sequence.
Proceedings of the National Academy of Sciences of the USA, 88, 1571–1574.
Rylands, A. B., Mittermeier, R. A. & Rodríguez-Luna, E. (1995). A species list for the New
World primates (Platyrrhini): distribution by country, endemism, and conservation status
according to the Mace-Land system. Neotropical Primates, 3, 113–160.
Rylands, A. B., Rodríguez-Luna, E. & Cortés-Ortiz, L. (1996/1997). Neotropical primate
conservation-The species and the IUCN/SSC primate specialist group network. Primate
Conservation, 17, 46-69.
Rylands, A. B. & Brandon-Jones, D. (1998). Scientific nomenclature of the red howlers from
the northeastern Amazon in Brazil, Venezuela and the Guianas. International Journal of
Rylands, A. B., Schneider, H., Langguth, A., Mittermeier, R. A., Groves, C. P. & Rodríguez-
Luna, E. (2000). An assessment of the diversity of New World primates. Neotropical
Primate, 8, 61–93.
Rylands, A. B. & Mittermeier, R. A. (2009). The diversity of the New World primates
(Platyrrhini): An annotated taxonomy. In South American Primates. Comparative
perspectives in the study of behavior, ecology and conservation. Garber, P. A., Estrada,
A., Bicca-Marques, J. C., Heymann, E. W., Strier, K. B. (Eds.). Springer
Science+Business Media, New York, USA. Pp. 23-54.
Saillard, J., Forster, P., Lynnerup, N., Bandelt, H-J. & Norby, S. (2000). mtDNA variation
among Greenland Eskimos: the edge of the Beringian expansion. American Journal of
Human Genetics, 67, 718-726.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing
phylogenetic trees. Molecular Biology and Evolution, 4, 405–425.
Sambrock, J., Fritsch, E. & Maniatis, T. (1989). Molecular Cloning: A Laboratory manual.
2nd edition. V1. Cold Spring Harbor Laboratory Press. New York.
Sampaio, I., Schneider, M. P. & Schneider, H. (1996). Taxonomy of the Alouatta seniculus
group: biochemical and chromosome data. Primates, 37, 65–73.
Schwarz, G. E. (1978). Estimating the dimension of a model. Annals of Statistics, 6, 461-464.
Simonsen, K., Churchill, G. & Aquadro, C. (1995). Properties of Statistical Tests of
Neutrality for DNA Polymorphism Data. Genetics, 141, 413-429.
Smith, J. D. (1970). The systematic status of the black howler monkey, Alouatta pigra
Lawrance. Journal of Mammalogy, 51, 358–369.
Soini, P., Aquino, R., Encarnación, F., Moya, L. & Tapia, J. (1989). Situación de los primates
en la Amazonía Peruana. In Saavedra, C. J., Mittermeier, R. A., and Santos, I. B. (Eds.).
La Primatología en Latinoamérica. Symposio de Primatología. Arequipa, Perú, 1983.
(pp. 13-21). World Wildlife Fund, Littera Maciel Ltda. Minas Gerais, Brazil.
Springer, M. S., Meredith, R. W., Gatesy, J., Emerling, C., Park, J., Rabosky, D. L., Stadler,
T., Steiner, C., Ryder, O., Janecka, J. E., Fisher, C. & Murphy, W. J. (2012).
Macroevolutionary dynamics and historical biogeography of primate diversification
inferred from a species supermatrix. PLoS One, 7, e49521.
Stanyon, R., Tofanelli, S., Morescalchi, M. A., Agoramoorthy, G., Ryder, O. A. & Wienberg,
J. (1995). Cytogenetic analysis shows extensive genomic rearrangements between red
howler (Alouatta seniculus, Linnaeus) subspecies. American Journal of Primatology, 35,
171–183.
Swofford, D. L. (2002). PAUP*. Phylogenetic analysis using parsimony and other methods.
http://paup.csit.fsu.edu. pp. 1-142.
Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA
polymorphism. Genetics, 123, 585-595.
Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. (2013). MEGA6: Molecular
Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 2725-
2729.
Torres, O. M. & Leibovici, M. (2001). Caracterización del cariotipo del mono aullador
colorado Alouatta seniculus que habita en Colombia. Caldasia, 23, 537-548.
Torres, O. M. & Ramírez, C. (2003). Estudio citogenético de Alouatta palliata (Cebidae).
Caldasia, 25, 193–198.
Vassart, M., Guédant, A., Vié, J. C., Kéravec, J., Séguéla, A. & Volobouev, V. T. (1996).
Chromosomes of Alouatta seniculus (Platyrrhini, Primates) from French Guiana. The
Journal of Heredity, 87, 331-334.
Villalobos, F., Valerio, A. A. & Retana, A. P. (2004). A phylogeny of howler monkeys
(Cebidae: Alouatta) based on mitochondrial, chromosomal and morphological data.
Revista de Biología Tropical, 52, 665-677.
Von Dornum, M., Ruvolo, M. (1999). Phylogenetic relationships of the New World monkeys
(Primates, Platyrrhini) based on nuclear G6PD DNA sequences. Molecular Phylogenetics
and Evolution, 11, 459–476.
Voss, R. S. & Fleck, D. W. (2011). Mammalian Diversity and Matses Ethnomammalogy in
Amazonian Peru. Part 1: Primates. Bulletin of the American Museum of Natural History,
351, 1-81.
Wallace, R., B. & Rumiz, D. I. (2010). Atelidae. In Wallace, R. B., Gómez, H., Porcel, Z. R.,
and Rumiz, D. I. (Eds.). Distribución, Ecología y Conservación de los Mamíferos
medianos y grandes de Bolivia. (pp. 1-884). Centro de Ecología Difusión. Fundación
Simón I. Patiño.
Walsh, P. S., Metzger, D. A. & Higuchi, R. (1991). Chelex 100 as a medium for simple
extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10, 506-
513.
Weir, B. S. & Hill, W. G. (2002) Estimating F-statistics. Annual Revue of Genetics, 36, 721–
750.
Wienberg, J., Stanyon, R., Nash, W. G., O’Brien, P. C. M., Yang, F., O’Brien, S. J. &
Ferguson-Smith, M. A. (1997). Conservation of human vs. feline genome organization
revealed by reciprocal chromosome painting. Cytogenetics and Cell Genetics, 77, 211–
217.
Wolfheim, J. H. (1983). Primates of the New World: Distribution, Abundance and
Conservation. Seattle, University of Washington Press.
Yunis, E. J., Torres, O. M., Ramírez, C. & Ramírez, E. (1976). Chromosomal variations in
the primate Alouatta seniculus seniculus. Folia Primatologica, 25, 215 - 224.
Zuñiga Leal, S. A. & Defler, T. R. (2013). Sympatric distribution of two species of Alouatta
(A. seniculus and A. palliata: Primates) in Chocó, Colombia. Neotropical Primates, 20, 1-
11.

Chapter 12 Alouatta Sur Peru

Încărcat de

Informații document

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Chapter 12 Alouatta Sur Peru

Încărcat de

Drepturi de autor:

Formate disponibile

Book Title: Phylogeny, Molecular Population Genetics, Evolutionary Biology

and Conservation of the Neotropical Primates. Edited by M. Ruiz-García and J.

WHICH HOWLER MONKEY (ALOUATTA, ATELIDAE,

Manuel Ruiz-García1,*, Angela Cerón1,

species A. juara and A. puruensis seem to be molecularly un-differentiable from A. s.

since he considered A. s. macconnelli and A. s. amazonica to be synonymous to A. s.

MATERIAL AND METHODS

Molecular Population Analyses

phylogenetic analyses. We applied procedures to haplotypic frequencies (GST statistic) and to

We analyzed for possible historical demographic changes in A. seniculus, A. sara and A.

Figure 1. Mismatch distributions (pairwise sequence differences) at the three concatenated

Molecular Phylogenetic Insights into the Systematics of Alouatta

in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana

Bonvicino, C. R., Lemos, B. & Seuánez, H. N. (2001). Molecular phylogenetics of howler

De Oliveira, E. H. C., Lima, M. C. & Sbalqueiro, I. J. (1995). Chromosomal variation in

S-ar putea să vă placă și