Chapter 13 Ateles

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. 435-476.
Chapter 13
HISTORICAL GENETIC DEMOGRAPHY AND SOME

INSIGHTS INTO THE SYSTEMATICS OF ATELES
(ATELIDAE, PRIMATES) BY MEANS OF DIVERSE
MITOCHONDRIAL GENES
Manuel Ruiz-García1,*, Nicolás Lichilín1,

Pablo Escobar-Armel1, Geven Rodríguez1 and
Gustavo Gutiérrez-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 sampled 283 spider monkeys (Ateles) and sequenced three of their mitochondrial
genes (Cyt-b, COI and COII). This was the largest molecular genetics sample set of
Ateles ever analyzed and included all of the morphological taxa described for this genus.
There were six main findings:
1. All the species, or main taxa studied, showed elevated mitochondrial gene diversity
levels, with the exception of A. paniscus. The hybridus taxa, which showed
considerably low gene diversity levels for nuclear DNA microsatellites (Ruiz-García
et al., 2006), but also showed relatively elevated levels of mitochondrial gene
diversity.
*
Corresponding author: mruizgar@yahoo.es, mruiz@javeriana.edu.co.
2 Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al.
2. Changes in population size: The taxa chamek and belzebuth showed strong evidence
of population expansions during the Pleistocene. Also, we detected population
expansions in fusciceps and geoffroyi. The demographic history of marginatus was
ambiguous with some of our analyses indicating population expansion while others
suggesting a declination in females. This was probably due to small sample size.
Similarly, paniscus didn’t show clear evidence of demographic changes, but
hybridus did show a declination trend in its female population.
3. Genetic heterogeneity: Taxa pairs with A. paniscus showed the highest values of
genetic differentiation—suggesting A. paniscus is the most differentiated taxon
within Ateles. Genetic heterogeneity values were only statistically significant when
all taxa were analyzed together.
4. The Kimura 2P genetic distance model showed maximum values around 4% for the
different taxa pairs. Many of these were around 2-3%, clearly lower than those of
other Neotropical primate species within genera. This indicates that the number of
Ateles species proposed by Groves (2001) is probably an overestimation (taxonomic
inflation) caused by a very typological use of the Phylogenetic species concept.
5. Our molecular phylogenetic results support the existence of two (A. paniscus and A.
belzebuth) or three (A. paniscus, A. belzebuth and A. geoffroyi) species.
6. We (both mitochondrial and microsatellites) detected a very strong phylogenetic
relationship between hybridus and some fusciceps individuals. These results don’t
agree with the view of Collins and Dubach (2000a,b) and Nieves et al., (2005) that
hybridus is a full and differentiated species.
Keywords: Ateles, mitochondrial gene sequences, demographic changes, IUCN

classifications, phylogenetic insights, two or three Ateles species
INTRODUCTION
The spider monkeys, Ateles genus, are among the largest primates in the Neotropics
along with Alouatta, Lagothrix and Brachyteles (Strier, 1992). Unfortunately, Ateles
populations are currently being threatened by many factors. They are intensively hunted for
food by indigenous people throughout Central and South America. There is also the
traditional demand for spider monkeys as attractive animals in zoos and as pets. These
demands have led to the development of intense commercial trafficking (legal and illegal) of
this genus. As large primates, spider monkeys have low reproductive rates and therefore, even
a low hunting pressure, could extirpate extensive populations throughout their distribution
range (Collins, 1999). Konstant et al., (1985) and Rosenberger and Strier (1989) revealed that
Ateles is extremely environmentally sensitive and they are the first primates to disappear after
small environmental changes disturb the rain forest. Spider monkeys live in undisturbed areas
within primary rain forests and they are highly arboreal. Furthermore, Ateles is primarily
frugivorous and feeds largely on the mature, soft parts of a wide variety of fruits that provide
more energy than leaves. Van Roosmalen (1980) estimated that 82.9 to 90% of the Ateles’s
diet is composed of fruit. This diet requires Ateles to have extensive territories to encompass
the non-uniform distributions of fruit. Thus, high sensitivity to environmental change, low
reproductive rate, and large territories help to explain why many Ateles populations are now
seriously threatened and need to be targeted for conservation. Indeed, after Brachyteles and
Historical Genetic Demography and Some Insights into the Systematics of Ateles ... 3
Leontopithecus, Ateles is considered the most endangered genus of the New World Primates
(Mittermeier et al., 1989).
In Table 1, we show the evolution of the classification of IUCN in reference to the
different Ateles taxa. Practically, all the taxa have worsened their situation throughout the last
decades. Here we provide a brief update as to the situation of each Ateles taxon—using the
terms vulnerable, endangered, and critically endangered. Vulnerable means that this taxon
declined by at least 30% over the past 45 years (three generations) due primarily to hunting
and habitat loss. Endangered means the same but with declination of at least 50%, while
critically endangered implies a declination of at least 80%.
Table 1. UICN classifications in 1991, 1996, 2000, 2003 and 2008 for the different Ateles
taxa defined in each period
Ateles taxa UICN 1991 UICN 1996 UICN 2000 UICN 2003 UICN 2008
paniscus - Low Risk Least Concern Least Concern Vulnerable
A2cd
chamek - Low Risk Least Concern Least Concern Endangered
A2cd
belzebuth Vulnerable Vulnerable Vulnerable Vulnerable Endangered
A2cd
marginatus Critical Risk Endangered Endangered Endangered Endangered
A2cd + 3cd
fusciceps Critical Risk Critical Risk Critical Risk Critical Risk Critically
fusciceps endangered
A2cd
fusciceps Endangered Vulnerable Vulnerable Vulnerable Critically
rufiventris endangered
A2cd
hybridus Endangered Endangered Endangered Critically Critically
hybridus endangered Endangered
A2cd + 3cd
hybridus - Endangered Endangered Critically Critically
brunneus endangered Endangered
A2cd + 3cd
geoffroyi - - - Least Concern Endangered
A2c
geoffroyi Endangered Low Risk - - Critically
geoffroyi Endangered
A4c
geoffroyi Critical Risk Critical Risk Critical Risk Critical Risk Critically
azuerensis endangered
A2c
geoffroyi Endangered Vulnerable Vulnerable Least Concern Vulnerable
frontatus A2c
geoffroyi - Endangered Endangered - Deficit Data
grisescens
geoffroyi Endangered Endangered - - -
panamensis
geoffroyi - Vulnerable Vulnerable Endangered Endangered
ornatus A4c
geoffroyi Vulnerable Low Risk - Critically Critically
vellerosus endangered endangered

A4c
geoffroyi - Vulnerable Vulnerable Vulnerable Endangered
yucatanensis A4c
The Ateles taxa have become increasingly vulnerable and their classifications have in
many cases moved from vulnerable to endangered or even to critically endangered. For
example, A. belzebuth was classified as vulnerable in 1991 and then later reclassified as
endangered A2cd in 2008. Similarly, A. chamek was classified as low risk in 1996 and as
endangered A2cd in 2008. A. fusciceps fusciceps was classified as critically endangered in
1991 and as critically endangered A2cd in 2008. A. fusciceps rufiventris was classified as
endangered in 1991 and vulnerable in 1996 and as critically endangered A2cd in 2008. A.
hybridus was classified as endangered in 1991 and as critically endangered A2cd + 3cd in
2008. A. hybridus brunneus was classified as endangered in 1996 and as critically endangered
A2cd + 3cd in 2008. A. marginatus was classified as critically endangered in 1991 and as
endangered A2cd + 3cd in 2008. A. paniscus was classified as low risk in 1996 and as
vulnerable A2cd in 2008.
A. geoffroyi, taken as a whole, was classified as least concern in 2003 and as endangered
A2cd in 2008. Here we also provide updates on the subspecies of A. geoffroyi. A. g. geoffroyi
was classified as endangered in 1991, low risk in 1996 and critically endangered A4c in 2008.
A. g. azuerensis was listed as critically endangered in 1991 and critically endangered A2c in
2008. A. g. frontatus was listed as endangered in 1991 and vulnerable A2c in 2008. A. g.
grisescens was considered endangered in 1996 and there was insufficient data to report on it
in 2008. A. g. panamensis was listed as endangered in 1991 and there were no data reported
for this subspecies in 2008. A. g. ornatus was vulnerable in 1996 and endangered A4c in
2008. A. g. vellerosus was vulnerable in 1991 and critically endangered A4c in 2008. Finally,
A. g. yucatanensis was listed as vulnerable in 1996 and endangered A4c in 2008.
The only cases, where the classification situation did not get worse, were for A.
marginatus and for A. g. frontatus.
In fact, Mittermeier et al., (2009) enclosed A. hybridus as one of the 25 most endangered
primates in the world (2008-2010), together with two other Neotropical primates (Saguinus
oedipus and Lagothrix flavicauda). For A. hybridus, only 0.67% of the current range is
protected. However, the IUCN and other classifications did not contain genetic historical
demographic information of these species. In many cases, the current demographic situation
of the Ateles species could be a consequence of their own evolution without human
intervention. For example, see the case of the Andean bear (Ruiz-García, 2003a, 2007, 2013;
Ruiz-García et al., 2003, 2005). In contrast, if a taxon shows elevated levels of gene diversity
and population expansions are detected throughout its evolution but its current censuses sizes
are small, this would be a clear symptom of a negative human intervention. This would
probably cause a severe bottleneck. For this reason it is extremely important to reconstruct the
historical genetic demography of a taxon and to compare it with its classification by IUCN
and other institutions. The case of Ateles is interesting from this point of view.
Nevertheless, the systematics of the taxa under study could negatively affect these kinds
of comparisons. There is disagreement in the literature over the systematics of Ateles, making
it difficult to construct and present an effective conservation proposal for this genus. The
most traditionally used classification scheme recognizes four different species (Kellog and
Goldman 1944; Hill 1962): A. geoffroyi, A. belzebuth, A. fusciceps, and A. paniscus.
Each of these species has their own distinct subspecies. A. geoffroyi has nine subspecies
in Central America: A. g. ornatus (Costa Rica), A. g. panamensis (Costa Rica, Panama), A. g.
azuarensis (Panama), A. g. geoffroyi (Costa Rica, Nicaragua), A. g. frontatus (Costa Rica,
Nicaragua), A. g. yucatanensis (Mexico, Belize, Guatemala), A. g. vellerosus (Mexico,
Guatemala, Honduras, El Salvador), A. g. pan (Guatemala), and A. g. grisescens, (Colombia,
Panama).
A. belzebuth has three geographical discontinuous subspecies: A. b. belzebuth (Colombia,
Peru, Ecuador, Venezuela, Brazil), A. b. hybridus (Colombia, Venezuela), and A. b.
marginatus (Brazil).
The third species, A. fusciceps, is found in the Pacific coast of Colombia and Ecuador and
has two subspecies: A. f. fusciceps (Ecuador) and A. f. rufiventris (= robustus; Colombia,
Panama).
The fourth species, A. paniscus, has two discontinuous subspecies A. p. paniscus
(Guyana, Suriname, French Guiana, Brazil) and A. p. chamek (Peru, Bolivia, Brazil).
Tallying all subspecies across the four species there are 16 taxa. Mittermeier and
Coimbra-Filho (1977) and Konstant et al., (1985) used the same classificatory scheme,
although Konstant et al., (1985) noted the difficulty in discriminating several of the
subspecies identified by Kellog and Goldman (1944). This included the challenge of
differentiating A. g. vellerosus and A. g. yucatanensis and the validity of A. g. azuerensis. The
authors also claimed that marginatus might need to be classified within A. paniscus because
of its close relationship with A. p. chamek. Differences between species and subspecies of this
classification were based almost entirely upon pelage characteristics.
Conversely, Hershkovitz (1968, 1969, 1972), Hernández-Camacho and Cooper (1976)
and Wolfheim (1983) supported a second classification scheme and have considered all Ateles
to belong to one, wide-ranging variable polytypic species, A. paniscus. The authors based this
scheme on the varying coat color patterns within this genus. Nevertheless, Shedd and
Macedonia (1991) and Jacobs et al., (1995) have not supported the use of metachromatism to
infer phylogenetic relationships among diverse Neotropical primates because the genetic and
developmental systems that underlie the phenotypic expression of pelage traits may be
different across primate species.
In a third classificatory scheme based on cytogenetic analyses, diverse authors (García et
al., 1975; Kunkel et al., 1980) postulated that differences in the morphological chromosome
pairs 5, 6 and 7 could support the Ateles taxonomy proposed by Kellog and Goldman (1944).
This could also be interpreted as intraindividual heteromorphism and also support the view of
one unique Ateles species as suggested by Hershkovitz (1968, 1969, 1972), and Hernández-
Camacho and Cooper (1976). Pieczarka et al., (1989) and DeBoer and DeBruijn (1990)
determined that A. paniscus possessed 32 chromosomes, while all the other Ateles taxa had 34
chromosomes, suggesting the first as a separate species from the others (A. paniscus and A.
belzebuth). A fourth scheme was proposed by Medeiros et al., (1997) from the analysis of
karyotypes from four taxa: 1- A. geoffroyi (including geoffroyi and hybridus); 2- A. fusciceps
(fusciceps and rufiventris); 3- A. belzebuth (including belzebuth, chamek and marginatus) and
4- A. paniscus. The authors did not discard the notion of more than the existence of two
species (A. paniscus and A. belzebuth).
Froehlich et al., (1991) used a fifth classificatory scheme based on discriminant analysis
of 76 cranial and dental characters. These authors classified Ateles in three different species,
A. paniscus, A. belzebuth (which included A. belzebuth, A. chamek and A. marginatus), and A.
geoffroyi (which included A. geoffroyi, A. fusciceps and A. hybridus). Differently, Groves
(2001) stated that there were seven different species of Ateles. These were A. paniscus, A.
belzebuth, A. chamek, A. hybridus, A. marginatus, A. fusciceps (with two subspecies, A. f.
fusciceps and A. f. rufiventris) and A. geoffroyi (with 5 susbspecies, A. g. yucatanensis, A. g.
vellerosus, A. g. geoffroyi, A. g. ornatus, A. g. griscesens). However, Groves (1989) had
previously considered A. belzebuth and A. hybridus to be of the same species. Collins and
Dubach (2000a,b; 2001) and Collins (2008) were the first studies to use molecular markers to
try and resolve the systematics of Ateles. They used two mitochondrial genes, the
hypervariable I portion of the mitochondrial control region and the Cytochrome c Oxidase
subunit II gene, COII, and the nuclear Aldolase A Intron V gene. They concluded that four
species existed: A. paniscus, A. belzebuth, A. geoffroyi, and A. hybridus. For A. belzebuth they
only included samples of chamek and marginatus. Later, Collins (2008), included one sample
of belzebuth and thus proposed three subspecies forms of A. belzebuth. For A. geoffroyi, they
mentioned three subspecies: A. g. fusciceps, the Northern Central America population
(yucatanensis, and vellerosus) and the Southern Central American population.
We sequenced three mitochondrial genes (Cytochrome b, Cyt-b; sub-unity cytochrome
oxidase I, COI; sub-unity cytochrome oxidase II, COII) from 283 individuals to provide
insights on the demographic evolution and systematics of Ateles taxa. This is the largest
number of Ateles ever sampled (217 higher than Collins study conducted in 2008).
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 mammal populations,
including primates (Lavergne et al., 2010). 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; Plautz et al.,
2009; Ruiz-García et al., 2010, 2011, 2014).
MATERIAL AND METHODS

We sequenced three mitochondrial genes in 283 Ateles individuals. The specimens and
their geographical origins are as follows. 1-There were a total of 89 A. fusciceps rufiventris
individuals sampled in diverse departments of Colombia (28 from Antioquia, 23 from Choco,
10 from Atlantico, one from Sucre, 13 from Cordoba, five from Valle del Cauca and Cauca
and nine from Nariño). 2- Five specimens of A. fusciceps fusciceps were sampled from
diverse locations of the Pacific area of Ecuador. 3- We sampled 62 A. geoffroyi specimens—
representing all the taxa described in Meso-America (seven A. g. ornatus from Costa Rica;
nine A. g. panamensis and A. g. azuarensis from Panama and Costa Rica; 16 A. g. geoffroyi
from Costa Rica and Nicaragua; 11 A. g. yucatanensis, A. g. vellerosus and A. g. pan from
Mexico, Guatemala and Honduras; 18 A. g. frontatus from Costa Rica, and one A. g.
grisescens from Choco in Colombia). 4- There were also 24 A. hybridus. This included wild
specimens of A. h. hybridus and A. h. brunneus, four animals from Santander, three from
Magdalena, one from Norte de Santander, one from Arauca, and three from Antioquia. All of
these locations are within Colombia. There was also one individual from Maracaibo,
Venezuela. Some samples were also collected from European zoos. Three were from the
Barcelona Zoo (Catalonia), two from the Erfurt Zoo (Germany), one from the Twycross Zoo
(England) and five from Romagne Zoo (France). 5- We sampled a total of 45 A. chamek. Of
these, 24 were from diverse areas of Peru. However, two samples were obtained in the
Ecuadorian Amazon and another two were obtained in Leticia, within the Colombian
Amazon. Another 18 of the 45 were from diverse areas of Bolivia and three were from Brazil.
6- There were also a total of 35 A. belzebuth (15 from Peru, 14 from Ecuador, five from
Brazil and one from Colombia). 7- We also sampled 10 A. marginatus (all near Santarem,
Pará in Brazil). 8- Additionally, we sampled 13 A. paniscus (one from Surinam, six from
French Guiana and six from Brazil). In the early part of this chapter we use the “classical”
terminology to name the different Ateles taxa. Later, in the discussion, we revisit the names
and validate or reject them pending on our phylogenetic results.
Samples were obtained in two ways. In all the countries sampled, with the exception of
Costa Rica, we visited different indigenous communities living along major rivers. We
requested permission to collect biological materials from either carcasses or live animals that
were already present in the community. We sampled small pieces of tissue (muscle) or teeth
from hunted animals that were discarded during the cooking process, or hairs with bulbs
plucked from live pets. Communities were visited only once, all sample donations were
voluntary, and no financial or other inducement was offered for supplying specimens for
analysis. Second, in the case of Costa Rica, blood samples were obtained by darting wild
monkeys. Five Lagothrix lagotricha individuals from Colombia were used as an out-group.
These sampling procedures complied with all relevant Colombian, Peruvian, Ecuadorian,
Bolivian, Brazilian, French Guiana, Panamanian, Costa Rican, Guatemalan and Mexican
laws. These sampling procedures complied with all the protocols approved by the Ethical
Committee of the Pontificia Universidad Javeriana (No. 45677) and the laws of the Ministerio
de Ambiente, Vivienda y Desarrollo Territorial (R. 1252) from Colombia. This research also
adhered to the American Society of Primatologists’ Principles for the Ethical Treatment of
Primates.
Molecular Procedures
The DNA from blood and muscle was extracted using the phenol-chloroform procedure
(Sambrook et al., 1989), while DNA samples from hairs and teeth were extracted with 10%
Chelex resin (Walsh et al., 1991). The 283 spider 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 of 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, 2000a; Ruiz-Garcia et al., 2010, 2012a,b). The temperatures employed were as
follows: 95°C for 5 min, 35 cycles of 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) of each 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

We used the following statistics to determine the genetic diversity within the Ateles taxa:
number of polymorphic sites (S), haplotypic diversity (Hd), nucleotide diversity (), average
number of nucleotide differences (k) and  statistic by sequence. These gene diversity
statistics were undertaken in the program DNAsp 5.10 (Librado and Rozas, 2009).
To determine possible historical population changes we relied on three procedures we
describe here. 1- The mismatch distribution (pairwise sequence differences) was obtained
following the method of Rogers and Harpending (1992) and Rogers et al., (1996). We
compared the curves obtained assuming constant and non-constant sizes to the empirically
observed distribution. We used the raggedness rg statistic (Harpending et al., 1993;
Harpending 1994) to determine the similarity between the observed and the theoretical
curves. 2- 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), the Strobeck’s S statistic (Strobeck, 1987) and the
R2 statistic (Ramos-Onsins and Rozas, 2002), to determine possible population size changes
in the fusciceps, geoffroyi, hybridus, chamek, belzebuth, marginatus and paniscus taxa
(Simonsen et al., 1995; Ramos-Onsins and Rozas, 2002). Confidence interval at the 95% and
probabilities were obtained with 1,000 coalescence permutations. 3- A Bayesian skyline plot
(BSP) was obtained for the concatenated mitochondrial sequences by means of the BEAST v.
1.6.2 and Tracer v1.5 software. The Coalescent-Bayesian skyline option in the tree priors was
selected with four steps and a piecewise-constant skyline model with 40,000,000 generations
(the first 4,000,000 discarded as burn-in). In the Tracer v1.5, the marginal densities of
temporal splits were analyzed and the Bayesian Skyline reconstruction option was selected
for the trees log file. A stepwise (constant) Bayesian skyline variant was selected with the
maximum time as the upper 95% high posterior density (HPD) and the trace of the root height
as the treeModel.rootHeigh. To determine the time ranges for possible demographic changes
of each Ateles taxa, we employed the time splits estimated by Collins and Dubach (2000b).
We estimated possible demographic changes for fusciceps (in last 1.5 MY), geoffroyi (2 MY),
hybridus (1.5 MY), chamek (2.5 MY), belzebuth (2.5 MY), marginatus (1.5 MY) and
paniscus (3 MYA).
Phylogenetic Analyses
The sequence alignments were carried out manually and with the DNA alignment
program (Fluxus Technology Ltd.). Modeltest Software (Posada and Crandall, 1998) and
Mega 6.05 Software (Tamura et al., 2013) were applied to determine the best evolutionary
mutation model for the sequences analyzed for the three concatenated gene sequences.
Akaike information criterion (AIC; Akaike, 1974) and the Bayesian information criterion
(BIC; Schwarz, 1978) were used to determine the best nucleotide evolutionary model. Upon
selection of the best model we obtained maximum likelihood estimates of
transition/transversion bias as well as maximum likelihood estimates of the gamma parameter
for site rates (Tamura et al., 2013).
Two kinds of procedures were carried out to estimate genetic heterogeneity, and
theoretical gene flow estimates, among the diverse Ateles taxa. They were applied to
haplotypic frequencies (GST statistic) and to nucleotide sequences (ST, NST and FST statistics,
Hudson et al., 1992; Weir and Hill, 2002).
Only one phylogenetic tree was obtained by means of a neighbor-joining tree (NJ; Saitou
and Nei, 1987) with the Kimura 2P genetic distance (Kimura, 1980) and is presented here. A
future publication will provide additional phylogenetic results of Ateles (Ruiz-García et al.,
2016a). Bootstrap analyses (1,000) were performed to determine the more consistent clades.
Table 2. Gene diversity statistics for all the Ateles taxa defined and for the
mitochondrial genes studied. 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
Ateles taxa NH Hd  K 
paniscus 5 0.628 + 0.143 0.0014 + 0.0001 0.974 + 0.706 1.289 + 0.768
chamek 24 0.957 + 0.014 0.0095 + 0.0012 6.636 + 3.192 18.295 + 5.48
belzebuth 28 0.970 + 0.020 0.0142 + 0.0009 9.911 + 4.644 28.653 + 8.82
marginatus 8 0.933 + 0.077 0.0101 + 0.0003 7.044 + 3.615 9.544 + 4.186
fusciceps 38 0.901 + 0.023 0.0132 + 0.0005 9.231 + 4.281 19.159 + 5.01

hybridus 10 0.783 + 0.070 0.0147 + 0.0008 10.297 + 1.41 23.833 + 8.02
geoffroyi 17 0.883 + 0.023 0.0075 + 0.0002 5.239 + 2.567 12.137 + 3.54
RESULTS
Gene Diversity and Historical Demographic Evolution
in Ateles Taxa
Paniscus yielded the lowest levels of gene diversity (Hd = 0.628,  = 0.0014; k = 0.974)
of all the Ateles taxa. A large fraction of remaining taxa showed high levels of gene diversity
(Table 2). One taxon (geoffroyi) had intermediate gene diversity values (Hd = 0.883,  =
0.0076; k = 5.239). The hybridus taxon, one of the 25 most endangered primates of the world,
did not show especially low levels of mitochondrial gene diversity.
Historical demographic analyses by taxon revealed seven trends (Table 3 and Figures 1
and 2):
1. Analyses of paniscus indicated demographic change but it was inconsistent across

tests. The Tajima’s D, Fu and Li’s D*, Fu and Li’s F* and the Strobeck’s S statistics
did not reveal any appreciable demographic change in this taxon. The BSP analysis
showed a constant, but moderate increase in female population size within the last
2.25 MY. However, the Fu’s FS, R2 statistics together with the mismatch pairwise
distribution and its associated rg statistic showed a significant population expansion
in paniscus.
2. chamek showed multiple indications of population expansion. Five statistics
(Tajima’s D, Fu and Li’s D*, Fu and Li’s F*, Strobeck’s S and Fu’s FS) showed
significant evidence of population expansion as well as the mismatch pairwise
distribution and its associated rg statistic did. Also, the BSP analysis yielded a
population expansion in the last 0.225 MY. Only the R2 statistic did not reveal any
demographic change trend in chamek.
3. belzebuth practically showed the same demographic dynamics as did chamek. Five
statistics (Tajima’s D, Fu and Li’s D*, Fu and Li’s F*, Strobeck’s S and Fu’s FS)
showed significant evidence of population expansion, as did the mismatch pairwise
distribution and its associated rg statistic. Also the BSP analysis yielded a population
expansion, although in a more complex pattern than chamek did. A first population
expansion episode was detected around 2.2 MYA with a moderate increase in the
number of females up until about 1.5 MYA. Then, a striking and exponential growth
began and lasted until about 1 MYA, when a very slight increase was detected that
continued until the present. Only the R2 statistic did not reveal any change of
demographic trend in belzebuth.
4. For marginatus the mismatch pairwise distribution and its associated rg statistic as
well as the R2 statistic indicated population expansion. The other five statistics also
supported population expansion but they were not statistically significant. A.
marginatus had the lowest sample size of all the Ateles taxa we sampled and it may
have been insufficient to reach statistical significance. However, its demographical
evolution could be similar to that observed in chamek and belzebuth. Nevertheless,
the BSP analysis for marginatus was different to those of both previously mentioned
Amazon Ateles taxa. Its female size was constant until around 50,000 YA where the
female population decreased until present day.
Table 3. Tests for possible historical demographic expansions or contractions in the
different Ateles taxa studied by means of three concatenated mitochondrial genes
(A)
Fu & Li Fu & Li raggedness
Tajima D Fu’s Fs R2
D* F* rg
paniscus P[D < - P[D* < - P[F* < - P[Fs < - P[rg < P[R2 <
0.829] = 0.509] = 0.672] = 1.82 ] = 0.0281] = 0.108] =
0.231 0.269 0.265 0.041* 0.0032** 0.0043**
chamek P[D < - P[D* < - P[F* < - P[Fs < - P[rg < P[R2 <
2.313] = 3.812] = 3.897] = 7.414] = 0.038] = 0.189] =
0.001** 0.002** 0.003** 0.019* 0.330 0.530
belzebuth P[D < - P[D* < - P[F* < - P[Fs < - P[rg < P[R2 <
2.526] = 4.189] = 4.291] = 12.675] = 0.046] = 0.174] =
0.001** 0.001** 0.001** 0.001** 0.522 0.458
marginatus P[D < - P[D* < - P[F* < - P[Fs < - P[rg < P[R2 <
1.251] = 1.406] = 1.543] = 1.113] = 0.018] = 0.082] =
0.113 0.115 0.095 0.232 0.026* 0.028*
fusciceps P[D < - P[D* < - P[F* < - P[Fs < - P[rg < P[R2 <
1.714] = 3.646] = 3.406] = 9.318] = 0.031] = 0.121] =
0.019* 0.005** 0.005** 0.019* 0.627 0.719
hybridus P[D < - P[D* < - P[F* < - P[Fs < P[rg < P[R2 <
2.267] = 3.983] = 4.042] = 2.537] = 0.043] = 0.123] =
0.004** 0.001** 0.001** 0.863 0.395 0.335
geoffroyi P[D < - P[D* < - P[F* < - P[Fs < - P[rg < P[R2 <
1.949] = 2.895] = 3.032] = 1.520] = 0.057] = 0.117] =
0.006** 0.009** 0.007** 0.335 0.257 0.638
* P < 0.05 and ** P < 0.01, significant population expansions. For the statistics of Tajima D, Fu and Li
D*, Fu and Li F*, Fu’s Fs, rg and R2 (A); for the statistic of Strobeck S (B)
(B)
Ateles taxa Strobeck S
paniscus P = 0.101
chamek P = 0.0001**
belzebuth P = 0.0001**
marginatus P = 0.174
fusciceps P = 0.0001**
hybridus P = 0.085
geoffroyi P = 0.073
5. A. hybridus only showed three statistics that indicated a possible population

expansion (Tajima’s D, Fu and Li’s D* and Fu and Li’s F*). However, the other
statistics (Strobeck’s S, Fu’s FS and R2) did not detect any relevant population
change. Neither the mismatch pairwise distribution nor its associated rg statistic
revealed any significant population expansion. The BSP analysis detected a

continuous but slight increase in the female population size from 1 MY to 0.25 MY.
Since that time the female population has either been constant or has slightly
decreased as it has in the last 15,000-20,000 years.
6. Five different statistical tests supported fusciceps as having population expansions
(Tajima’s D, Fu and Li’s D*, Fu and Li’s F*, Strobeck’s S and Fu’s FS). In contrast,
the mismatch pairwise distribution and its associated rg statistic and the R2 statistic
did not reveal any significant population expansion. The BSP analysis revealed a
constant, or very slight decrease, in the last 1.5 MY up until about 30,000 YA when a
very striking increase in the size of the female population began and continued until
today.
7. geoffroyi showed a similar situation with that observed in fusciceps. Four statistics
revealed population expansion (Tajima’s D, Fu and Li’s D*, Fu and Li’s F* and
Strobeck’s S), but the mismatch pairwise distribution and its associated rg statistic,
the Fu’s F and the R2 statistics did not. The BSP analysis was practically identical to
that of fusciceps with more or less a constant female population up until 30,000-
40,000 YA. After this point the female population size hardly increased.
Therefore, we can distinguish four different demographic trajectories in these Ateles taxa
(1- paniscus, 2- chamek, belzebuth and marginatus, 3- hybridus and 4- fusciceps and
geoffroyi).
(A)
(B)
(C)
(D)
(E)
Figure 1. (Continued)
(F)
(G)
Figure 1. Mismatch distributions (pairwise sequence differences) at the three concatenated

mitochondrial DNA genes (COI, COII and Cyt-b) for the different Ateles taxa considered: paniscus (A),
chamek (B), belzebuth (C), marginatus (D), hybridus (E), fusciceps (F) and geoffroyi (G).
Molecular Phylogenetic Inferences
The BIC and the Akaike information criterion highlighted the best nucleotide substitution
model for the three concatenated genes we sequenced. For BIC, the best model was HKY + G
(16,404.049), whilst for the Akaike information criterion it was GTR + G (10,479,003). The
maximum likelihood estimate of transition/transversion bias was 3.01 (maximum log
likelihood = 4,731.25).
(A)
(B)
(C)
(D)
(E)
(F)
(G)
Figure 2. Bayesian skyline plot analysis (BSP) to determine possible demographic changes across the
natural history of the different Ateles taxa considered: paniscus (A), chamek (B), belzebuth (C),
marginatus (D), hybridus (E), fusciceps (F) and geoffroyi (G). On the x-axis, time in millions of years;
on the y-axis, effective population size of females.
Table 4. Genetic heterogeneity and gene flow (Nm) statistics among

all the Ateles taxa considered
Genetic differentiation estimated P Gene flow

 = 1629.633 df = 756 NS
HST = 0.0860 0.0000* γST = 0.5938 Nm = 0.34
KST = 0.5858 0.0000* NST = 0.6747 Nm = 0.24
KST* = 0.3944 0.0000* FST = 0.6729 Nm = 0.24
ZS = 8,130.8037 0.0000*
ZS* = 8.3433 0.0000*

Snn = 0.9198 0.0000*
*Significant Probability (P < 0.05); NS = No significant
All of the genetic heterogeneity tests that simultaneously compared the “a priori” Ateles
taxa were significant with the exception of the contingency 2 table (Table 4). The
heterogeneity statistics NST (= 0.675) and FST (= 0.673) showed considerable sequence
differentiation among the Ateles taxa. Indirect historical gene flow estimates were lower than
1 (Nm = 0.24 for both statistics), and thus low enough to consider these taxa as different
species. However, the haplotype genetic heterogeneity statistic, GST = 0.089, was
considerably lower and the associated gene flow estimate (Nm = 5.10) showed an appreciable
genetic connectivity among these Ateles taxa. Indeed, if we look for genetic heterogeneity by
Ateles taxa pairs, two considerations can be made. First, all comparisons implicating paniscus
were those which showed the highest levels of genetic heterogeneity (FST values higher than
0.75), which means that paniscus is the most differentiated Ateles taxon. Second, there were
some comparisons with FST values lower than 0.50 (medium value of genetic differentiation).
These were the cases, geoffroyi-fusciceps (both Northern Ateles taxa) and chamek-belzebuth,
chamek-marginatus and belzebuth-marginatus (all Amazonian Ateles taxa).
The Kimura 2P genetic distances among Ateles taxa are shown in Table 5. The largest
genetic distances were around 4% and were between the following pairs: fusciceps fusciceps-
belzebuth (4.3%), fusciceps fusciceps-hybridus (4.0%), geoffroyi-hybridus (4.0 %) and
belzebuth-hybridus (3.9%). The smallest values (around 2%) were between belzebuth-
marginatus (2.4%), geoffroyi-fusciceps rufiventris (2.1%), chamek-belzebuth (1.9%),
fusciceps rufiventris-fusciceps fusciceps (1.9 %) and chamek-marginatus (1.7 %).
We generated an NJ tree with the Kimura 2P genetic distances (Figure 3) of the 283
Ateles individuals analyzed. Clearly, the 13 individuals of paniscus (Surinam, French Guiana
and two different areas from northern Brazilian Amazon) formed a monophyletic clade
(100% bootstrap) well differentiated from the remaining Ateles clades. Two other large clades
were found (58%).
The first differentiated clade (56%) mainly contained three Amazon Ateles taxa: chamek,
belzebuth and marginatus. The first two morphological taxa were intermixed across all the
clades. There were eight sub-clades with bootstrap percentages higher than 70%. 1- One
chamek group contained eight individuals from Bolivia and Peru (70%). 2- A chamek-
belzebuth group had five individuals from Peru (86%). 3- One little chamek cluster contained
two individuals from the Nanay River (Peru) (70%). 4- Another chamek group included three
individuals from Peru and Bolivia (88%). 5- A small belzebuth cluster contained two
individuals from Ecuador (88%). 6- A belzebuth clade consisted of seven individuals from
Ecuador, Peru and Brazil (86%). 7- A small belzebuth cluster contained two individuals from
Peru (76%). 8- Finally, one chamek group had five individuals from Bolivia and Brazil
(74%). Within this large clade, there was a monophyletic clade consisting of 10 marginatus
individuals although the bootstrap was only 38% (within this clade, there was a cluster of
three marginatus individuals from the area of Santarem, Brazil, with 72%). Another
interesting fact is that this large Amazonian clade contained three individuals, which
belonged to the northern Caribbean Colombia and Venezuela and trans-Andean Ateles
populations. One geoffroyi grisescens individual was sampled in the Colombian Choco and
appeared within the chamek haplotypes. Also, there were two fusciceps rufiventris individuals
(one from Monte Libano, Cordoba, Colombia and other from Antioquia, Colombia) which
presented haplotypes related to belzebuth haplotypes.
Table 5. Kimura (1980) 2P genetic distances among the different Ateles taxa considered.
Below, genetic distance values in percentages (%); above, standard errors in
percentages (%)
Ateles taxa 1 2 3 4 5 6 7 8 9
1 - 0.7 0.7 0.6 0.6 0.6 0.7 0.6 1.4
2 3.0 - 0.3 0.4 0.6 0.7 0.7 0.6 1.5
3 3.5 1.9 - 0.5 0.7 0.7 0.7 0.7 1.6
4 2.9 1.7 2.4 - 0.6 0.7 0.7 0.7 1.6
5 3.1 3.4 3.8 3.4 - 0.3 0.6 0.4 1.5
6 3.7 3.8 4.3 2.4 1.9 - 0.6 0.4 1.6
7 3.7 3.7 3.9 3.6 3.7 4.0 - 0.7 1.5
8 3.0 3.2 3.7 3.4 2.1 2.6 4.0 - 1.5
9 11.5 11.8 12.8 12.7 12.4 13 12.9 12.1 -
1 = A. paniscus; 2 = A. chamek; 3 = A. belzebuth; 4 = A. marginatus; 5 = A. fusciceps
rufiventris; 6 = A. fusciceps fusciceps; 7 = A. hybridus; 8 = A. geoffroyi; 9 = Lagothrix lagotricha
66 Ateles fusciceps rufiventris 99 Uraba COL

56
Ateles fusciceps rufiventris 75 Antioquia COL
64 Ateles fusciceps rufiventris 15 Risaralda COL
36 Ateles fusciceps rufiventris 47 Antioquia COL
6 Ateles fusciceps rufiventris 35 Valle Cauca COL
Ateles fusciceps rufiventris 59 Nariño COL
22
Ateles fusciceps fusciceps 122 Manabí Ecuador
40 Ateles fusciceps rufiventris 38 ValleCauca COL
10 Ateles fusciceps rufiventris 23 Choco COL
76 Ateles fusciceps rufiventris 39 ValleCauca COL

14
40
Ateles fusciceps rufiventris 21 Cordoba COL
6 Ateles fusciceps rufiventris 27 Cordoba COL

56
Ateles fusciceps fusciceps 124 Santa Elena Ecuador
Ateles fusciceps rufiventris 14 Choco COL
Ateles fusciceps rufiventris 22 Chocó COL
Ateles fusciceps rufiventris 36 ValleCauca COL
Ateles fusciceps rufiventris 29 Darien COL
40 Ateles fusciceps rufiventris 94 PNLosKatios COL
66 Ateles chamek 116 Yarinacocha PER
Ateles fusciceps rufiventris 91 Atlantico COL
40 Ateles fusciceps rufiventris 26 Chocó COL
Ateles fusciceps rufiventris 27 Atlántico COL

74
Ateles fusciceps rufiventris 62 SahagunCordoba COL
Ateles fusciceps rufiventris 66 LosCordobas Cordoba COL
Ateles fusciceps rufiventris 68 Montelibano Cordoba COL

54Ateles fusciceps rufiventris 10 Choco COL
Ateles
34 fusciceps rufiventris 45 Antioquia COL
54 Ateles fusciceps rufiventris 12 Choco COL
22
Ateles fusciceps rufiventris 37 Cauca COL
Ateles fusciceps rufiventris 61 Montilibano Cordoba COL
92 Ateles fusciceps rufiventris 86 Turbo Antioquia COL
Ateles geoffroyi ornatus 51 CostaRica
82 Ateles geoffroyi ornatus 52 CostaRica
36
Ateles geoffroyi panamensis/azuarensis 56 CostaRica
Ateles geoffroyi panamensis/azuarensis PAN
16 Ateles geoffroyi panamensis/azuarensis 53 CostaRica
24 Ateles geoffroyi panamensis/azuarensis 43 CostaRica
44
100 Ateles geoffroyi panamensis/azuarensis 11 Boquete PAN
46
Ateles geoffroyi panamensis/azuarensis 11B Boquete PAN
Ateles geoffroyi panamensis/azuarensis 13 Boquete PAN
Ateles geoffroyi geoffroyi 3 CostaRica
72 Ateles geoffroyi geoffroyi 18 CostaRica
26 Ateles geoffroyi geoffroyi 38 CostaRica
Ateles geoffroyi geoffroyi 49 Nicaragua
60 Ateles geoffroyi vellerosus 14 HONDURAS
Ateles geoffroyi yucatensis Yucatan MEX
36
Ateles geoffroyi vellerosus 18 Peten GUAT
Ateles geoffroyi pan 74 Peten GUAT
46
Ateles geoffroyi yucatensis 67 Yucatan MEX
Ateles geoffroyi yucatensis 66 Yucatan MEX
Ateles geoffroyi frontatus 12 CostaRica

56
52 Ateles geoffroyi frontatus 33 CostaRica
Ateles fusciceps rufiventris 89 Sucre COL

90 Ateles fusciceps rufiventris 28 Atlantico COL
Ateles belzebuth 14 Mocogua PER
Ateles hybridus 7 BarcelonaZOO SPA
68 Ateles hybridus 10 Santander COL
Ateles hybridus 13 ErfurtZOO GER
58 Ateles hybridus 12 TwycrossZOO UK
Ateles hybridus 11 RomagneZOO FRA
60
66
60 Ateles hybridus 6 RomagneZOO FRA
Ateles hybridus 2 Magdalena COL
Ateles hybridus 25 Arauca COL
Ateles hybridus 17 Maracaibo VEN
18 Ateles hybridus 4 RomagneZOO FRA
68 36 Ateles hybridus 24 PTOWilches Santander COL
44 Ateles hybridus 9 PTOWilches Santader COL
Ateles fusciceps fusciceps 59 ECU
94 Ateles hybridus 11 Norte Santader COL
Ateles hybridus 68 Norte Santader COL
26
20
Ateles hybridus 2 Antioquia COL
6 Ateles hybridus 14 ErfurtZOO GER
Ateles chamek 60 PER

Ateles marginatus 5 Santarem Pará BRA
38
60
20 Ateles marginatus 2 Santarem Pará BRA
Ateles marginatus 2B Santarem Pará BRA
38 42
58 Ateles marginatus 1B Santarem Pará BRA
44
72
56 Ateles chamek 3 STACruz BOL

52
Ateles chamek 4 STACruz BOL
Ateles chamek 26 Javari River BRA
Ateles chamek 3 BOL
52
74
Ateles chamek 24 Branco River Acre BRA
70 Ateles chamek 6 STACruz BOL
Ateles belzebuth 69 Pastaza ECU
Ateles belzebuth 9 Napo ECU
48
Ateles belzebuth 54 Morona Santiago ECU
2
Ateles belzebuth 11 Nanay River PER
16
48 Ateles belzebuth 5 Negro River BRA
54 Ateles belzebuth 15 Huila COL
Ateles belzebuth 46 Amazon River PER

Ateles belzebuth 62 Orellana ECU
18 10 Ateles belzebuth 39 SanMartin PER
100 18 Ateles belzebuth 45 Amazon River PER
42 Ateles belzebuth 38 SanMartin PER
Ateles belzebuth 42 Negro River BRA

34 42 Ateles belzebuth 65 Pastaza ECU
10
26 Ateles belzebuth 51 Pastaza ECU
Ateles belzebuth 48 Amazon River PER
76 Ateles belzebuth 47 Amazon River PER
0 76
Ateles belzebuth 64 Napo ECU
Ateles belzebuth 61 PER
86
8
Ateles belzebuth 8 Orellana ECU
Ateles belzebuth 40 SanMartin PER
24 8 Ateles belzebuth 57 Napo ECU
88 Ateles belzebuth 56 Morona Santiago ECU
Ateles fusciceps 67 Montelibano Cordoba COL
Ateles belzebuth 13 Huallaga River PER
26 58
Ateles belzebuth 41 Huallaga River PER
Ateles chamek 41 Mocagua PER
Ateles chamek 32 Chazuta PER
88
68
Ateles chamek 79 BOL
20 Ateles chamek 42
Ateles chamek 51 Ucayali River PER
36
48 Ateles chamek 53 Ucayali River PER
42
Ateles chamek 45 Madre de dios PER
Ateles chamek 64 Santa Rosa BOL

60
Ateles chamek 11 Beni River BOL
54
Ateles chamek 55 Nanay River PER
Ateles chamek 58 Ballivian BOL
Ateles geoffroyi grisescens 68 Choco COL
56
Ateles chamek 30 In. Leticia PER
Ateles chamek 43 Loreto PER
Ateles chamek 59 STA Ana Yacuma BOL
Ateles chamek 81 STA Ana Yacuma BOL
Ateles chamek 5 STA Cruz BOL
Ateles chamek 63 Madidi BOL
Ateles chamek 25 Maderia River BRA
70
10 Ateles belzebuth 44 Nanay River PER
86
Ateles chamek 26 In. Leticia PER
32
Ateles chamek 7 STA Cruz BOL
8 Ateles chamek 80 BOL
Ateles paniscus 24 Surinam
46 Ateles paniscus 25 Trombetas River BRA
Ateles paniscus 1 French Guiana
26 Ateles paniscus 2 French Guiana
24 Ateles paniscus 6 Uatama River BRA
Ateles paniscus 8 Uatama River BRA
Ateles paniscus 9 Uatama River BRA
Lagothrix lagotricha 2
88 Lagothrix lagotricha 1
100
72 100 Lagothrix lagotricha 3
Figure 3. Neighbor-Joining tree with the Kimura (1980) 2P genetic distance with the 283 spider
monkeys (Ateles) studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The
number in the nodes are bootstrap percentages.
The second large differentiated clade mainly contained Ateles individuals from the
Northern Caribbean in Colombia and Venezuela. It also had trans-Andean Ateles individuals
(hybridus, fusciceps rufiventris, fusciceps fusciceps and different geoffroyi taxa). Many
individuals of these taxa were intermixed within this large clade.
There were six clusters within this large clade. 1- One cluster contained fusciceps
fusciceps-fusciceps rufiventris-hybridus and had 16 individuals from Ecuador and Colombia
and two hybridus individuals from one French zoo and Germany zoo respectively (94%); 2-
There was a small cluster of fusciceps rufiventris composed of two individuals from the
Atlantic Department (Colombia) (90%); 3- One large cluster contained 55 geoffroyi
individuals. They belonged to seven described morphological taxa of the Central American
Ateles (yucatanensis, vellerosus, pan, frontatus, geoffroyi, panamensis, and azuarensis).
There was also one fusciceps rufiventris from the Sucre Department (Colombia) (72%); 4-
One geoffroyi cluster only had individuals from Costa Rica (ornatus) (96%); 5- Another little
cluster of fusciceps rufiventris had two individuals from Turbo (Antioquia) and from the
Choco Department, both in Colombia (92%). 6- Finally, there was another large cluster
containing fusciceps rufiventris from all the Colombian Departments where this species lives
as well as all the fusciceps fusciceps individuals from Ecuador (except one). Similar to the
large Amazonian clade, there were also some individuals of Amazon origins in the big
Northern Caribbean Colombia and Venezuela/trans-Andean clade. One individual was
classified as belzebuth from Caballococha (Amazon River, Peru), and related to haplotypes of
hybridus, fusciceps fusciceps and fusciceps rufiventris. Also, one individual was classified as
chamek from Yarinacocha, Ucayali River (Peru). It was within the main fusciceps rufiventris-
fusciceps fusciceps haplotype clade.
DISCUSSION
Current IUCN Classifications and the Evolutionary Demographics of Ateles
IUCN classified paniscus as being in the least dangerous situation relative to all other
Ateles taxa. It was listed at low risk in 1996, of least concern in 2000 and 2003, and in
Nucleus II and vulnerable in 2008. However, our mitochondrial data showed this taxon to
have the lowest gene diversity levels and no clear evidence of population expansions
throughout its evolutionary history. Therefore, its evolutionary potential could be one of the
lowest of the Ateles taxa, and this is not recorded in the IUCN classifications.
A. chamek was classified by IUCN as in low risk (1996), least concern (2000, 2003), in
Nucleus II and endangered (2008). However, the mitochondrial data showed that this taxon
had a relatively high gene diversity levels and clear evidence of significant population
expansions across its evolutionary history. Additionally, Ruiz-García et al., (2006) showed
this taxon to have the highest gene diversity levels (expected heterozygosity and average
number of alleles) for a set of nuclear DNA microsatellites. The estimated size of the wild
population for this species was greater than 10,000 animals and was considered to be in a safe
status according to Stevenson et al., (1992). Ruiz-García et al., (2006) used nuclear
microsatellites to estimate the historically effective population size to be 8,000-61,000 and
6,000-47,600 based on coalescence and maximum likelihood procedures, respectively. If
IUCN has changed the status of this taxon in the last two decades it is probably due to intense
hunting pressure and habitat deforestation, even though this taxon inhabits the Western
Amazon (Bolivia, Peru and western Brazilian Amazon), where the Amazon is still fairly
preserved. However, the current genetic findings do not consider these circumstances and
IUCN’s classification does not take into consideration that this is an Ateles taxon with great
evolutionary potential.
A similar situation is found for belzebuth. IUCN had classified this taxon as vulnerable
but now it is considered endangered. However, both mitochondrial and nuclear DNA
microsatellite markers showed elevated gene diversities. They also showed strong population
expansions. The effective population size ranged from 4,000 to 36,000 and from 8,300 to
66,400 for the two procedures (Ruiz-García et al., 2006). Additionally, chamek and belzebuth,
although they have very different coat colors, belonged to the same species. This complicates
IUCN’s classifications. Thus, the evolutionary potential of belzebuth could be as important as
that of chamek.
IUCN has always classified marginatus as endangered. It is not affected by current
human pressure but perhaps by climatological changes during the last 50,000 YA. About
80,000-70,000 YA, it was extremely cold during the early Pleni-glacial, which was the first
extreme cold period in the fourth glaciation. Absy et al., (1991), Van der Hammen (1992) and
Van der Hammen and Absy (1994) analyzed the vegetation in Carajas (Eastern Amazon)
covering a time period starting approximately 65,000-51,000 YA. They determined that this
current area of the Amazon forest was a savannah that probably formed during an extension
of the early Pleni-glacial period. Liu and Colinvaux (1985) and Colinvaux and Liu (1987)
analyzed Ecuadorian Andean valleys and the Amazon and concluded that the elevation limit
of vegetation descended 27,000-34,000 YA. The temperature also descended 4.5°C during the
middle and the upper Pleni-glacial periods. For example, one of the dryer and colder periods
of the middle Pleni-glacial period occurred 30,000 YA. Bogota lagoon was dried at that
period (Van der Hammem, 1992). Afterwards, another period of extreme cold occurred
around 23,000 to 20,000 YA (Dryas I) (MacNeish, 1979). The Last Glacial Maximum (LGM)
occurred around 19,000-16,500 YA. It was the moment where the extension of the snow
reached the maximum in the Central Andes. Finally, around 14,500 to 12,000 YA (Dryas II),
the cold was extreme at times. The glaciers reached their maximum surface area in the
Manachaque Valley (Cordillera Blanca), in the Upismayo-Jalacocha at the Vilcanota
Cordillera in Peru, in the Nevado of ChoqueYapu in Bolivia and in the Chimborazo in
Ecuador. Rodbell and Seltzer (2000) showed that the glaciers in the Cordillera Blanca (San
Martin Department in Peru) reached their maximum elevation (3,170-3,827 masl)
approximately 12,000 YA. Today its elevations is 4,600 masl. Maslin and Burns (2000)
showed that the Amazon River reached its maximum dry peak around 16,000-15,000 YA.
This dry period continued until about 12,000 YA. The analysis with O18 isotopes revealed
that the mouth of the Amazon River had only 40% of the water that it has today. In the
tropical forest, the temperature decreased by 2°C to 6°C and in some areas as the Fuquene
Lagoon near Bogota, the precipitation was only half of what it is today (Van der Hammen,
1992). The temperature of the Atlantic Ocean near the Brazilian coasts descended 6°C (Clark,
2002). These climatological changes could be more responsible for the limited geographic
distribution of marginatus and its population decrease compared to current factors.
The IUCN classification is probably more useful to hybridus because of its critically
endangered status in 2008. Interestingly, the mitochondrial data showed that hybridus is not
particularly impoverished in its gene diversity. It also has elevated amounts of mitochondrial
gene diversity. However, Ruiz-García et al., (2006) found that A. hybridus showed the lowest
levels of expected heterozygosity and average number of alleles for a set of microsatellites
compared to other taxa of Ateles. Stevenson et al., (1992) claimed that only 100 to 1,000
individuals of this taxon still exist in the wild. Ruiz-García et al., (2006) estimated the
effective historical population size (based on the two procedures) to be 3,000-26,000 and
2,900-23,300 respectively. Ruiz-García (2003b), using other theoretical models, determined
possible effective numbers for hybridus. These estimates from the average heterozygosity
with the infinite allele and the step-wise mutation models showed values from 997 to 5,334.
The fact that the mitochondrial gene diversity is not especially low for this taxon but the
nuclear DNA microsatellite diversity is very low should agree quite well with a very recent
genetic bottleneck in hybridus caused by human activity. Note, mitochondrial diversity can
show ancestral diversity in a taxon and microsatellite diversity can show more recent
diversity. However, the beginning of this decrease in genetic variability along with low
effective numbers, could have begun around 15,000-20,000 YA as the BSP analysis
registered. As we previously explained Dryas I, LGM and Dryas II could have affected
hybridus because many forest of current Northern Colombia and Venezuela were highly
affected. Correlated with this, Ruiz-García et al., (2015) detected a strong population decrease
14,000 YA for the lowland tapir population of Northern Colombia and Northwestern
Venezuela (Tapirus terrestris). This is same area where hybridus inhabits, agreeing quite well
with the situation of this population, which was considered critically threatened in 2004 by
the IUCN (Constantino et al., 2006).
This parallel population decrease of hybridus and T. terrestris in the same geographical
area is related to the massive extinction of mammals across the Earth. This corresponds with
the Younger Dryas (Dryas III), typical of Northern Europe and Scandinavia (Clapperton,
1993). The Younger Dryas was more drastic in the northern part than the southern part of
South America. Thus, the situation of hybridus as one of the 25 most endangered primates in
the world (2008-2010) (Mittermeier et al., 2009) seems justified.
The case of fusciceps is more complex. IUCN has always classified fusciceps fusciceps as
critically endangered and its populations has dropped by 90% in the last 45 years. Ateles
fusciceps rufiventris was classified as vulnerable (1996, 2000, 2003), and then was re-
categorized as critically endangered in 2008. The geographic distribution of fusciceps
fusciceps is restricted to some very small areas of the Pacific coast of Ecuador and this could
correlate with its classification as critically endangered. However, the current study is the first
to analyze this taxon at the molecular scale and we demonstrate that fusciceps fusciceps is
undifferentiated from fusciceps rufiventris. A. fusciceps showed elevated mitochondrial gene
diversity as well as elevated nuclear microsatellite diversity. Stevenson et al., (1992)
estimated the wild population of this taxon to be about 1,000-3,000 individuals, which is
extremely low compared to the effective number obtained by Ruiz-García et al., (2006)
(12,000-95,000 and 12,300-99,000 respectively). As we will discuss in brief, both
mitochondrial and microsatellites results detected two different groups of fusciceps rufiventris
individuals (all had black coats), one more related to geoffroyi and other the more related to
hybridus. The second group yielded the highest levels of microsatellite diversity. This was
probably due to gene admixture involving hybridus which in turn overestimate the real
effective numbers of fusciceps rufiventris. Nevertheless, the IUCN classification did not
reflect the internal diversity within fusciceps or the high evolutionary potential of this Ateles
taxon.
Finally, geoffroyi (taken as a whole) was classified by the IUCN as a taxon of least
concern in 2003, but as endangered in 2008. Both the mitochondrial gene and nuclear
microsatellite diversities of goeffroyi were low. It was the second lowest after paniscus in the
mitochondrial case and after hybridus in the microsatellite case. Also, after hybridus, it
yielded the second lowest effective size numbers: 3,000-28,000 and 2,800-22,600. Its
classification as endangered could reflect its census situation as well as its evolutionary
potential.
We can appreciate that the IUCN’s classification of a taxon helps to determine its
potential endangered status. Sometimes the classification agrees with gene diversity levels
and predictions of evolutionary potential (for instance, hybridus and geoffroyi). Other times it
does not (for instance, chamek, belzebuth, fusciceps, paniscus).
New Insights on the Systematics of Ateles
Our molecular results, the most complete to date for mitochondrial analyses, suggest the
existence of only two (more conservative perspective) or three Ateles species, both valid for
the biological and phylogenetic species concepts. If we take into consideration only the
existence of monophyletic clades in a tree for defining species, then, only two Ateles species
are recognized: A. paniscus (Linnaeus, 1758) and A. belzebuth (Geoffroy, 1806). A. paniscus
(Linnaeus, 1758) was the first derived taxon from the hypothetical ancestor of the current
spider monkeys. All the other haplotypes of different geographical origins and morphotypes
were intermixed to some degree and the bootstrap percentages of the main clades within this
large cluster were relatively low. As we showed, one of the main sub-clades contained mostly
Amazonian individuals and one geoffroyi grisescens (Colombian Choco) and two fusciceps
rufiventris individuals (Colombia). A second main sub-clade consisted of Northern South
American and trans-Andean individuals. Additionally, there was one belzebuth from the
Amazon River (Peru) and one chamek from Ucayali River (Peru). These are cases of genetic
introgression more than recent hybridization (Ruiz-García et al., 2014) because there are large
geographical distances among the specimens. This means that the split among these taxa is
very recent. The elapsed time is probably short and no post and pre-zygote isolation
mechanisms have emerged, constituting a unique species (Barton and Bengtsson, 1986;
Coyne et al., 1994; Antonovics, 2006). This view agrees absolutely with the result of
Pieczarka et al., (1989) with A. paniscus having 32 chromosomes, while all the other Ateles
taxa had 34 chromosomes. Following this perspective within A. belzebuth some subspecies
could be proposed. Within the Amazonian area four subspecies could be distinguished and are
described here.
1. A. b. belzebuth (Geoffroy, 1806), includes the traditional belzebuth and chamek

morphotypes (they are color variations of the same taxa; belzebuth [Geoffroy, 1806],
has priority over chamek, [Humboldt, 1812]). From a coat pelage perspective, Elliot
(1913) identified specimens with pelage characteristics of belzebuth in the territory
of chamek (Chamicuros in the Huallaga River, Peru). We support their observations
of hybrids between A. chamek and belzebuth at the Loreto Region in the Peruvian
Amazon. Indeed, one of these specimens was enclosed in this study (phenotype of
belzebuth with mitochondrial DNA of chamek). Belzebuth and chamek did not show
monophyletic clades within the Amazonian clade and they were intermixed. Chamek
presented the highest levels of gene sequence diversity at the aldolase A intron gene
of all the taxa (Collins and Dubach, 2001). This aligns with the fact that chamek was
the taxon with the highest microsatellite gene diversity (Ruiz-García et al., 2006),
thus suggesting chamek as the ancestral spider monkey clade. Medeiros et al., (1997)
also concluded that chamek could represent the ancestral karyotype for Ateles. The
finding of the 6b chromosome in A. chamek provided an important element to the
identification of this taxon as the ancestor within this genus. Dutrillaux et al., (1986)
found that 6b corresponded to 2 chromosomes within Lagothrix and Brachyteles.
Therefore, since the 6b form only occurs in chamek, Lagothrix and Brachyteles it
was interpreted as the ancestral form of Ateles. However, our mitochondrial gene
diversity level for chamek is similar to other Ateles taxa. But, chamek individuals are
the first to diverge within the Amazon clade. Thus, chamek (or better, the black
morphotype of A. b. belzebuth) could be more related to the origins of the Amazon
Ateles clade, but it is clear that the ancestor of A. paniscus was the first of the current
Ateles taxa. In addition, Ruiz-García et al., (2006) revealed a strong connection
between chamek and belzebuth by means of nuclear microsatellites. Therefore,
chamek could also be the origin of belzebuth studied in Peru, Ecuador and Colombia.
In time, the ancestor of belzebuth originated hybridus and probably crossed the
Eastern Andes Cordillera. It originated the second fusciceps rufiventris population
(Antioquia, Sucre, Bolivar, Córdoba, Atlantico). We have evidence that belzebuth
crossed the Eastern Andes Cordillera. Brother Apolimar Maria (1913) recorded a
specimen from the Tolima Department in the upper Magdalena Valley which was
practically identical to A. belzebuth individuals that inhabited the eastern piedmont of
the Eastern Andes. We have even located Ateles bones from the Huila Department in
various Colombian museums on the other side of the Eastern Andes Cordillera.
Indeed, we sampled and enclosed in this analysis one belzebuth individual from the
Huila Department. This exemplar was molecularly undifferentiated from northern
Brazilian and Ecuadorian belzebuth individuals. Hernández-Camacho and Cooper
(1976) mentioned the existence of a zoological passage east of the Eastern Andes
Cordillera into the upper Magdalena Valley. Several species of Primates such as
Cebus apella, Lagothrix lagotricha lugens and Saimiri cassiquiarensis albigena, as
well as other vertebrate species, have crossed this passage. It’s also possible that
belzebuth originated fusciceps, and later, as our mitochondrial results support, the
ancestor of Atlantic fusciceps originating hybridus.
2. A. b. marginatus (Geoffroy, 1809), which formed a monophyletic clade within the
Amazonian group.
3. A. b. hybridus (Geoffroy, 1829) is the first derived taxon from the original ancestor
of Northern South American and trans-Andean spider monkeys. One belzebuth
haplotype was related to hybridus, which could mean that A. b. belzebuth originated
to A. b. hybridus, although it could also be generated from fusciceps. This taxon
showed an intense recent hybridization with the black spider monkeys (traditionally
named fusciceps) that live on the other side of the Magdalena River. Probably, the
black coat of fusciceps is dominant to the clear coat of hybridus and these hybrids are
black (and thus morphologically classified as fusciceps) but have the mitochondrial
DNA of A. b. hybridus. These frequent cases of recent hybridization (gene flow)
were between fusciceps males x hybridus females. The reverse crossing of hybridus
males with fusciceps females, cannot be detected by only studying mitochondrial
DNA because the individuals have a black coat and contain mitochondrial fusciceps
DNA. The gene flow between both morphotypes is still probably higher than we
detected. Ruiz-García et al., (2006), by means of nuclear microsatellites, also
detected a strong hybridization between hybridus and fusciceps. They showed that
the AP74 microsatellite yielded a wide range of allele sizes. Hybridus basically only
presented small sized alleles (130-132 base pairs), whereas belzebuth, chamek and
the Atlantic fusciceps rufiventris population yielded these same alleles in addition to
some larger sized alleles. However, other Ateles taxa only showed large sized alleles
(fusciceps fusciceps, paniscus, geoffroyi and the Choco Pacific fusciceps rufiventris
population). This supports that hybridus intensively hybridizes with the nearest
Atlantic fusciceps populations.
By using the nuclear microsatellite results, Ruiz-García et al., (2006) speculated on
the origins of hybridus. Two hypotheses were described. It could be derived from
belzebuth or from the Atlantic fusciceps rufiventris population by founder effect,
although founder effect affects microsatellites less than other markers such as
isoenzymes or plasma proteins. The possibility that hybridus arose because of
founder effect and genetic drift could provide an explanation of why Collins and
Dubach (2000a) found different relationships among the hybridus and the other
Ateles clades. The parsimony analysis of the control region did not relate hybridus to
any other clade, whereas the distance-based analysis clustered hybridus with A.
geoffroyi/A. f. rufiventris with a bootstrap support of 65%. In contrast, the combined
mitochondrial gene neighbor-joining analysis placed hybridus next to the clades of A.
b. chamek/A. b. marginatus and A. geoffroyi/A. f. rufiventris. Now, we know that the
differential results of Collins and Dubach (2000a) were influenced by a reduced
number of hybridus specimens. Therefore, they did not detect the “gradient” effect
which closely relates hybridus with fusciceps.
Now, the microsatellite results, together with the new mitochondrial data, show that
Amazonian haplotypes (like we found in belzebuth) could have originated hybridus.
It could also be derived from a fusciceps population, which in turn was generated by
belzebuth. This agrees with some authors, who concluded that hybridus could have
originated directly from belzebuth. Or, it could be from the Magdalena River Valley
and Atlantic fusciceps rufiventris population. From a chromosomal view, both
origins are possible because hybridus has 6a, 6c, 7b and 14b chromosomes, while 7b
is found in fusciceps rufiventris and 6c is found in belzebuth. On the other hand,
these nuclear and mitochondrial results do not support the link between paniscus and
hybridus as it was proposed by Medeiros et al., (1997) and based on chromosomal
analysis (both taxa shared the 7b chromosome). The similarities of chromosome pair
7 between paniscus and other trans-Andean Ateles forms are not compatible with the
molecular results. However, this chromosome could be polymorphic in A. belzebuth
and in other Ateles individuals not yet studied and the apparent relationship between
A. paniscus and the trans-Andean Ateles could be spurious. In fact, Collins and
Dubach (2000a) showed that the haplotypes of hybridus did not exhibit the 25-base
pair control region deletion that occurs in all the haplotypes of A. paniscus. On the
other hand, Kunkel et al., (1980) detected the same 6c and 7b chromosomes in
geoffroyi and hybridus in the same way as Froehlich et al., (1991) had revealed with
morphometrics. This last study supported that hybridus and fusciceps were
conspecifics just as our mitochondrial results supported.
Thus, our results showed that the western cordillera of the Andes and the Cauca
River were not barriers to gene flow between fusciceps rufiventris and hybridus, such
as was claimed by Collins and Dubach (2000a). Kunkel et al., (1980) and Medeiros
et al., (1997) claimed that hybridus and fusciceps could be two different taxa such as
karyotype analyses have revealed. These authors speculated that fusciceps rufiventris
could be reproductively isolated from geoffroyi and hybridus because of the
differences in chromosome pairs (5 and 6) but we have clearly showed high gene
flow between fusciceps and hybridus. Also, Rossan and Baerg (1977) determined
hybridization between fusciceps rufiventris and geoffroyi panamensis and they noted
the location of a hybridization zone. Also, recall that no river has formed a barrier
among Ateles species with the exception, may be, of some black-water rivers
draining the Guianan highlands for A. paniscus (Collins and Dubach, 2000b). Indeed,
Collins (2008) suggested, following Brown (1987) and Colinvaux (1993, 1996), that
at the beginning of the Pliocene the gene flow across the Amazon Basin among
Ateles populations should have been high. The river draining of this basin was not as
massive as it is today and later the black-water drainage and associated unsuitable
habitat of the Guianan shield (Froehlich et al., 1991; Ayres and Clutton-Brock, 1992;
Norconk et al., 1996) split the ancestor of A. paniscus from the ancestor of the
remaining Ateles (3.3-3.6 MYA). Probably, the split of A. paniscus should be more
related to the existence of a relatively recent Amazon Lake in the Late Pliocene until
the middle Pleistocene (by eustatic marine changes). This event connected the
Orinoco and Amazon basins after its separation during the Miocene (Hoorn et al.,
2010) and it could isolate the Guianan ancestor of the current A. paniscus. This
agrees quite well with the views of Campbell (1990), Frailey et al., (1998), Klammer
(1984), Marroig and Cerqueira (1997), Nores (1999, 2004) and Rossetti et al.,
(2005). A point supporting this large Amazon lake is the existence of a Pliocene (5
MYA) sea rise of 100 m for a duration of 0.8 MY (Haq et al., 1987).
Thus, marine transgressions could have had a great influence on biotic diversification
within the Amazon and it could also have affected the diversification in Ateles in the
beginning. However, Medeiros et al., (1997) did not discount the possibility that
animals with different karyotypes produce fertile offspring. This integrative evidence
suggests that hybridus, fusciceps and geoffroyi constituted a unique species if we
agree with our three Ateles hypothesis. In Table 6, we show the results of karyotypes
found in different Ateles taxa. In some cases, the possible karyotype evolution
correlated well with the molecular results. Nevertheless, in other cases, there was no
agreement between karyotypes and molecules. Contrarily, for the Alouatta genus
both karyotype and molecular results were very similar and this allows us to
reconstruct the phylogeny and systematics of the howler monkeys with a very high
precision (see Ruiz-García et al., 2016b in this book). This was not the case for
Ateles, probably because the polymorphisms of several chromosomes in this genus is
higher than the currently detected due to the small sample studied. Defler (2003)
distinguished two possible subspecies of hybridus, A. h. hybridus, from the major
part of the geographical range of this species, and A. h. brunneus from along the
lower Cauca and Magdalena Rivers in the departments of Bolivar, Antioquia and
Caldas. Animals of the second subspecies have different coat colors from those of the
first subspecies. However, in the current study we did not find evidence of molecular
differences between individuals of A. hybridus from both geographical areas for

either microsatellites or mitochondrial DNA. Therefore, at a molecular level we
could not differentiate between these two possible hybridus taxa. It’s likely that
brunneus shows some pelage differences from the other hybridus population because
its degree of mixture with fusciceps rufiventris is also higher.
4. A. b. geoffroyi (Kuhl, 1820) consisted of all Meso-American spider monkey
individuals plus the fusciceps individuals where no hybridization with hybridus was
detected. Therefore, two “fusciceps” groups were detected, one strongly mixed with
hybridus and other more related to the Meso-American spider monkeys (the
traditional geoffroyi). No mitochondrial differentiation was observed between the
Colombian-Panamanian fusciceps rufiventris (= robustus) and the endemic
Ecuadorian fusciceps fusciceps. The black or brown head pelage and the dorsum
glossy black and the dorsum brownish black of both morphotypes probably do not
have any phylogenetic signal. Under this molecular perspective and due to the
limitations of the Linnaeus typological systematic nomenclature, the continuous
transition of the fusciceps taxon between A. b. hybridus and A. b. geoffroyi is lost
because hybridus (Geoffroy, 1829) and geoffroyi (Kuhl, 1820) precede fusciceps
(Gray, 1866). Moreover, this classification questions all the subspecies based on
pelage by Kellog and Goldman (1944). Indeed, this questioning was made by Silva-
Lopez et al., (1996) who showed that the typical white forehead patch of A. belzebuth
is also present in many individuals of A. g. vellerosus. Furthermore, in most cases it
is impossible to differentiate the pelage of A. g. vellerosus from that of A. g.
yucatanensis.
Maybe under a less strict point of view three Ateles species could be sustained agreeing
absolutely with the morphological classificatory scheme of Froehlich et al., (1991):
1. A. paniscus. Our results ratify the molecular results of Collins and Dubach (2000a,b)
and the isozyme analysis of Sampaio et al., (1993), who determined a genetic
distance of 0.149 between paniscus and chamek, which is incompatible with the fact
that both forms were subspecies of A. paniscus. Thus, A. paniscus is a monotypic
species.
2. A. belzebuth (which should include the previous A. b. belzebuth, which in turn
includes chamek, and A. b. marginatus). It’s interesting to remark that all the
Amazonian Ateles forms could be a unique species, despite their different coat
colors. Collins and Dubach (2000b) estimated that the initial diversification within A.
belzebuth began around 2.4 MYA (beginning in the Pleistocene). Maybe the different
color patterns could be related with some Pleistocene refugia (Napo Refugia, or
island, for the belzebuth coat pattern, Inambari Refugia, or island, for the chamek
coat pattern and the Pará Refugia, or island, for the marginatus coat pattern) if we
follow the Pleistocene Refugia hypothesis (Haffer, 1969, 1982, 1987, 1997, 2008) or
even if we follow the Recent Amazon Lake hypothesis (Nores, 1999, 2004).
However, the Ateles populations, which survived in these refugia, should be of large
size because the spider monkeys need specific habitat requirements (for instance,
primary, evergreen and never flooded forests) and large territories (frugivore
specialist). Thus, these populations could contain elevated levels of gene diversity.
This could prevent some modes of speciation (Mayr, 1954, 1963) and when refugia
were again reconnected the different morphotypes colonized other refugia and it
helping to mix the different Ateles populations and thus speciation did not occur. The
chromosomal analysis of Nieves et al., (2005) showed a trichotomy among
belzebuth, chamek and marginatus, which is also interpreted as a unique species.
Collins and Dubach (2000b) and Collins (2008) claimed that the Pleistocene refugia
hypothesis provides little support for the speciation events in Ateles, because some
important splits in the spider monkeys were during the Pliocene. Still, Haffer (1997,
2008) showed that the dry-wet cycles in the Amazon (and other areas) were also
present during Miocene and Pliocene. Thus, the importance of these dry-wet refugia
could not be discarded for the beginning of the speciation processes in Ateles.
3. A. geoffroyi. In this case, this species contained three sub-species, A. g. hybridus, A.
g. fusciceps and A. g. geoffroyi, with intense gene flow occurring among the three
subspecies. This fact was also revealed through morphological observations. Indeed,
Kellog and Goldman (1944) detected variable pelage characteristics within
populations and intergraded where population distributions overlapped. This
intergradation occurs among geoffroyi panamensis, fusciceps rufiventris and
geoffroyi grisescens at the interface of Colombia and Panama. Rossan and Baerg
(1977) located sympatric populations of geoffroyi and fusciceps in Eastern Panama,
where both populations had hybridized to some extent along a contact zone. In
addition, an Ateles specimen collected inside the territory of fusciceps robustus,
(Catival, San Jorge River in Colombia) had a strong admixture of light-colored hairs
on the back, similar to the hybridus phenotype (Hernández-Camacho and Cooper,
1976). Collins and Dubach (2000a) placed the two former species A. geoffroyi
(Central America) and A. fusciceps, (Pacific Colombia and Ecuador and Northern
Colombia) in the same group. The aldolase A gene also clustered two A. geoffroyi
yucatanensis haplotypes with two A. f. robustus haplotypes but with a very low
bootstrap support of 48%. Therefore, the aldolase A gene provided limited support
compared to the mitochondrial genes for the inclusion of both the Central America A.
geoffroyi and the South American A. fusciceps as one species (Collins and Dubach,
2001). Additionally, Froehlich et al., (1991) concluded in their morphological
variation study that A. geoffroyi and A. fusciceps belonged to the same species. The
coat colors of the two taxa are distinct overall, with the general trend of the
production of darker pelages at the extremes of the Central American Isthmus
(Konstant et al., 1985).However, although microsatellite results supported statistical
significant differences among fusciceps rufiventris, geoffroyi and hybridus (Ruiz-
García et al., 2006), our mitochondrial sequence analysis revealed a strong
connection among these three taxa—considering them as a possible unique species.
The Choco Pacific fusciceps rufiventris population diverged clearly from the Atlantic
fusciceps rufiventris population for both microsatellites and mitochondrial DNA.
However, the first population was practically identical to fusciceps fusciceps
individuals studied from Ecuador for microsatellites. Our mitochondrial DNA that
we previously discussed, also supported this. On the other hand, either of the two
Colombian fusciceps rufiventris populations could have originated geoffroyi.
In whichever case, our molecular evolutionary and population point of view (Darwin,
1859; Dobzhansky, 1937; Mayr, 1942, 1963; Simpson, 1944) applied to the systematics of
Ateles is not compatible with the use of hybridus brunneus, fusciceps fusciceps, fusciceps
rufiventris, and all the traditional subspecies within A. geoffroyi. This problem stems from the
fact that the Linnaeus classificatory scheme is Aristotelian, scholastic and the scheme was
created to classify static organisms that people thought would never evolve because they were
created by God. Thus, in many cases there would be conflict between this typological
philosophy and the evolutionary process which is continuous. As Wendt (1958) explained,
there was intense criticism from French philosophers regarding reductive typological
philosophy of Linnaeus (Montaigne, Gassendi, La Mettrie, but especially Buffon, 1749-1788:
“Nature knows no systems”). However, the Linnaeus´s view was rapidly adopted by the
scientists of his time because it was easy to understand and follow. Interestingly, some years
later Linnaeus was aware that species evolved (case of Peloria monstruosa and the hybrid
research of Réaumur). Later, he deleted comments about the invariability of species from
Systema Naturae (1759). For instance, if we molecularly study three or four persons, with
very marked physical characteristics from different continents (Africans, Europeans, Asians,
Australians and Amerindians), we will probably generate a tree where each monophyletic
clade is integrated by the individuals of each continent. In this case, we will probably claim
that each continent contains a discrete race, or if we are working with other organisms,
subspecies. This is the situation we find in many molecular systematic works. But what
happens if we sample thousands and thousands of individuals, and not in just a few very
discrete populations, but in many locations that span the greater geographical part of each
continent? The perfect monophyletic clades for each core area will disappear. It’s probably
that some populations share some characteristics (maybe morphological or biochemical, and,
probably, for some characters but not for others) but the differences among distant
populations should be more continuous or clinal than discrete ones. This is especially true if
these distant populations correspond to individuals of the same species and the gene flow is
not interrupted. This does not mean that some geographical races do not exist. Only some
morphological characters (affected by positive natural selection or by gene drift) showed
differences but not discretely, but rather as a gradient and the degree of pre-speciation was
very limited. For this last reason, it may be better to use the term, geographic races, which
doesn’t imply pre-speciation processes, as does the subspecies term. This is the situation of
the human species (see Lewontin, 1972), and probably also of Ateles. It is very difficult to
explain gradient differences with a typological discrete classification scheme such as the
Linnaeus system. In this work (283 Ateles individuals), as well as that by Ruiz-García et al.,
(2016c this volume) (452 capuchin individuals), the number of individuals and the number of
localities (distribution ranges) were greatly increased. With these additions we can begin to
detect the “gradient” effect versus the “discrete” effect.
In the case of Ateles, this is especially evident for hybridus. Collins and Dubach,
(2000a,b, 2001), Collins, (2008) (molecular results) and Nieves et al., (2005) (chromosomal
results) concluded that hybridus is a full species because this taxon conformed a
monophyletic clade differentiated from fusciceps and geoffroyi. But they only analyzed three
hybridus and four fusciceps specimens, in the molecular analysis. When we sequenced 94
fusciceps and 24 hybridus, we easily detected numerous hybrids.
Table 6. Karyomorphs of Ateles taxa

Chromosome Chromosome Chromosome Chromosome Chromosome

Ateles taxa
5 6 7 13 14
A. paniscus Form c Forms a, e Form b Form b Form b
A. chamek Form a Forms b, e Form a Form a Form a
A. belzebuth Form a Forms c, d Form a Form a Form a
A. marginatus Form a Form d Form a Form a Form a
A. fusciceps Form b Form d Form b Form a Form a
rufiventris
A. hybridus Form a Form a, c Form b Form a Form b
A. geoffroyi Forms a, b Form c Form b Form a Form a
The most relevant conclusion of this work is that the real number of Ateles species (two
or three) is considerably lower that the seven species currently accepted by the vast majority
of the systematics and primatologists (Groves, 2001). What is a reasonable explanation of this
difference? We suggest that earlier authors may have adopted a reductive view of the
phylogenetic species concept without first securing enough samples and localities studied
from a neutral molecular perspective. Indeed, the highest genetic distances among Ateles taxa
is around 4% and many taxa pair comparisons are around 2-3%. For the three genes studied,
other authors determined genetic distance values for different species within a genus of
around 11% (for Cyt-b and COI; Bradley and Baker, 2001; Kartavtsev, 2011) and 6% (for
COII; Ascunce et al., 2003). Clearly this shows the strong genetic relationships among the
Ateles taxa.
Maybe, the most important systematic contribution of this work is to show that hybridus
is not a full and independent species as suggested by Collins and Dubach (2000a,b) and
Nieves et al., (2005) because multiple individuals of hybridus and fusciceps shared similar or
identical mitochondrial haplotypes.
ACKNOWLEDGMENTS
Thanks to Dr. Diana Alvarez, Armando Castellanos, Andrés Laguna, Fernando Nassar,
Luz Mercedes Botero, Marcela Ramírez, Luis Carrillo, Dr. B de Thoisy and Andrés Eloy
Bracho for their respective help in obtaining spider monkey samples during the last 20 years.
Thanks to Instituto von Humboldt (Villa de Leyva in Colombia; Janeth Muñoz), to the
Ministerio del Ambiente (permission HJK-9788) in Coca (Ecuador), 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 spider monkeys were sampled with the collection permits approved by the
Costa Rican government to Dr. Gustavo Gutiérrez-Espeleta. Likely, All animal sampling in
French Guiana was carried out in accordance with French animal care regulations and laws.
Thanks also go to ARCAS (Guatemala) for providing hair samples of Ateles. Thanks also to
the 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), Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya,
Yuri and Yucuna) and Ecuador (Kichwa, Huaorani, Shuar and Achuar) who helped us to
obtain samples of spider monkeys.
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Book Title: Phylogeny, Molecular Population Genetics, Evolutionary Biology

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

HISTORICAL GENETIC DEMOGRAPHY AND SOME

Manuel Ruiz-García1,*, Nicolás Lichilín1,

Keywords: Ateles, mitochondrial gene sequences, demographic changes, IUCN

vellerosus endangered endangered

MATERIAL AND METHODS

TAAACTTCAGGGTGACCAAAAAATCA-3’) (657 base pairs, bp) (Folmer et al., 1994)

Molecular Population Analyses

fusciceps 38 0.901 + 0.023 0.0132 + 0.0005 9.231 + 4.281 19.159 + 5.01

1. Analyses of paniscus indicated demographic change but it was inconsistent across

5. A. hybridus only showed three statistics that indicated a possible population

revealed any significant population expansion. The BSP analysis detected a

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

Molecular Phylogenetic Inferences

Table 4. Genetic heterogeneity and gene flow (Nm) statistics among

Genetic differentiation estimated P Gene flow

ZS* = 8.3433 0.0000*

66 Ateles fusciceps rufiventris 99 Uraba COL

Ateles fusciceps rufiventris 42 Antioquia COL

Ateles fusciceps fusciceps 119 Manabí Ecuador

Ateles fusciceps rufiventris 27 Atlántico COL

Ateles fusciceps rufiventris 44 Antioquia COL

Ateles geoffroyi frontatus 12 CostaRica

Ateles geoffroyi frontatus 47 CostaRica

Ateles chamek 60 PER

56 Ateles chamek 3 STACruz BOL

Ateles belzebuth 46 Amazon River PER

Ateles belzebuth 42 Negro River BRA

Ateles chamek 64 Santa Rosa BOL

New Insights on the Systematics of Ateles

1. A. b. belzebuth (Geoffroy, 1806), includes the traditional belzebuth and chamek

differences between individuals of A. hybridus from both geographical areas for

Table 6. Karyomorphs of Ateles taxa

Chromosome Chromosome Chromosome Chromosome Chromosome

Colinvaux, P. (1996). Quaternary environmental history and forest diversity in the

Jacobs, S. C., Larson, A. & Cheverud, J. M. (1995). Phylogenetics relationships and

Rosenberger, A. L. & Strier, W. G. (1989). Adaptative radiation of the atelinae primates.

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