Chapter 9 Aotus

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. 287-344.
Chapter 9
CAN MITOCHONDRIAL DNA, NUCLEAR

MICROSATELLITE DNA AND CRANIAL
MORPHOMETRICS ACCURATELY DISCRIMINATE
DIFFERENT AOTUS SPECIES (CEBIDAE)? SOME
INSIGHTS ON POPULATION GENETICS PARAMETERS
AND THE PHYLOGENY OF THE NIGHT MONKEYS
Manuel Ruiz-García1,, Adriana Vallejo1, Emily Camargo1,

Diana Alvarez1, Norberto Leguizamon2 and Hugo Gálvez3
1
Laboratorio de Genética de Poblaciones-Biología Evolutiva,
Unidad de Genética. Departamento de Biología, Facultad de Ciencias,
Pontificia Universidad Javeriana, Bogotá DC., Colombia
2
Secretaría Distrital Ambiental (SDA), Bogotá DC., Colombia
3
Instituto Veterinario de Investigaciones Tropicales y de Altura (IVITA),
Estación Experimental, Iquitos, Perú
ABSTRACT
We investigated the use of diverse procedures to discriminate among Aotus taxa
(Aotus; Cebidae, Platyrrhini) and provide new insights about the systematics and
phylogenetic relationships of Aotus taxa. To carry out these tasks we measured 38
craniometric characters in 80 individuals representing eight Aotus taxa. We sequenced
190 specimens at the mitochondrial COII gene for all 13 Aotus taxa recognized to date as
well as genotyped (12 nuclear DNA microsatellites) 143 individuals belonging to seven

Correspondence: mruizgar@yahoo.es, mruiz@javeriana.edu.co.
2 Manuel Ruiz-García, Adriana Vallejo, Emily Camargo et al.
Aotus taxa. The use of skull morphometrics allowed us to differentiate the most southern
of the taxa analyzed, Aotus azarae boliviensis. Microsatellite analyses in combination
with the FST statistic, gene flow and AMOVA analyses also significantly differentiated A.
a. boliviensis. The use of morphometrics and microsatellites did not clearly differentiate
A. nancymaae (a red-necked taxon) from all of the other gray-necked Aotus taxa at the
northern side of the Amazon River. However, the other more southern Aotus taxa were
not included (A. nigriceps, A. infulatus and A. a. azarae) in these analyses.
Microsatellites did not detect any significant bottleneck in the three taxa with the highest
sample sizes (A. nancymaae, A. vociferans and A. lemurinus griseimembra). The
mitochondrial analyses revealed a higher discrimination power than the other two
procedures. Two large clusters were found with two relevant sub-clusters inside each one
of them: A. nancymaae + A. miconax and all the Northern Amazon taxa (excluding A.
trivirgatus) on one side, and A. trivirgatus and all the other Southern Amazon taxa on the
other side. In the Northern Aotus group, the morphological and molecular differentiation
is very small but the chromosome differentiation is outstanding. This indicates that the
fundamental processes for diversification are parapatric chromosomal events (more than
peripatric or stasipatric chromosomal events) following fision processes from 2n = 46,
47, 48 (A. vociferans) to 2n = 58 (A. l. lemurinus). Taking all of the data into account we
propose that within Aotus, four superspecies could be defined: 1- A. vociferans with six
karyomorphs, including vociferans, brumbacki, jorgehernandezi, griseimembra,
lemurinus and zonalis; 2- A. trivirgatus, although we are fairly ignorant about this taxon;
3- A. miconax, including A. miconax and A. nancymaae, although we don’t know the
karyotype of the original A. miconax and 4- A. azarae with diverse karyomorphs (less
differentiated than in A. vociferans), including nigriceps, azarae, boliviensis and
infulatus.
Keywords: Aotus, skull morphometrics, nuclear DNA microsatellites, mitochondrial genes,

mt COII, parapatric chromosomal speciation, superspecies, phylogenetic inferences
INTRODUCTION
The night monkeys are in the genus Aotus, a controversial Neotropical primate taxon in
reference to systematics as well as in regard to other Platyrrhini genera. Aotus was initially
considered to be a monotypic genus with a single species, Aotus trivirgatus (Hershkovitz,
1949). The genus was originally described by Humboldt in 1812. However, starting in the
1970’s, many authors identified considerable karyotypic variation (Brumback et al., 1971;
Brumback 1973, 1974, 1976; Brumback and Willenborg, 1973; Ma 1981a, b, 1983; Ma et al.,
1976a, b, 1977, 1978, 1980, 1982, 1985; Thorington and Vorek, 1976). These studies led to
the proposal of a number of new species. These pioneer works, such as those of Brumback et
al., (1971), Brumback (1973) and Ma et al., (1976a) discovered polymorphic karyotypes in
one area of Colombia (2n = 52, 53 and 54) and 2n = 54 in Peru. Indeed, Brumback (1974)
reported the finding of a karyotype 2n = 50 for an alleged individual coming from Paraguay.
Following these initial cytogenetic discoveries, Hershkovitz (1983) revised the genus and
proposed a scheme encompassing nine night monkey species and four subspecies, arranged in
two main groups, based on the coloration of the pelage on the sides of the neck. The “gray-
necked” group is distributed north of the Amazon-Solimoes River (the Northern group),
whereas the “red-necked” group occurs south of this River (the Southern group). The first
group north of the Amazon River contained four species (Aotus lemurinus [with two
Can Mitochondrial DNA, Nuclear Microsatellite DNA … 3
subspecies, A. l. lemurinus and A. l. griseimembra], A. brumbacki, A. trivirgatus and A.

vociferans). The second group contained five species (Aotus nancymaae, A. miconax, A.
nigriceps, A. infulatus and A. azarae with two subspecies, A. azarae azarae and A. a.
boliviensis) located mostly south of the Amazon River. In fact, Hershkovitz (1983) studied
the alleged Paraguayan individual of Brumback (1974), determined its origin to be in the
Meta Department in Colombia, and named it A. brumbacki.
However, Hershkovitz’s (1983) scheme was challenged by subsequent studies based on
cytogenetics, morphology and molecular data. Ford (1994), for example, using multivariate
analysis on both craniodental metrical data of 193 Aotus skulls and data on pelage
characteristics of 105 adult Aotus skins, questioned the Hershkovitz’s classification. This
author concluded that there were only seven species, two Northern species, Aotus trivirgatus
and A. vociferans (which included A. lemurinus and A. brumbacki) and five Southern species,
A. nancymaae, A. miconax (although both taxa could be a single species), A. nigriceps, A.
azarae (only A. a. azarae), and A. infulatus (including infulatus and azarae boliviensis). After
consulting with Hershkovitz, Ramírez-Cerqueira (1983) included A. hershkovitzi as an
additional fifth northern species based on an inadequate description and considering,
particularly, its diagnostic diploid number of 58 and fundamental number of 76, the highest
known for the genus. Groves (2001) listed four subspecies of Aotus lemurinus (A. lemurinus
lemurinus, A. l. griseimembra, A. l. zonalis, and A. l. brumbacki) in his influential book. He
also listed Aotus hershkovitzi, A. trivirgatus, and A. vociferans in the Northern group and A.
miconax, A. nancymaae, A. nigriceps, and A. azarae (with three subspecies: A. azarae azarae,
A. a. boliviensis and A. a. infulatus) in the Southern group. Groves (2001) accepted
brumbacki as a form of lemurinus, whereas infulatus, azarae and boliviensis formed a
complex with overlapping characteristics, close to Aotus nigriceps but specifically different.
However, earlier, Thorington and Vorek (1976) had criticized the establishment of subspecies
for Aotus on the grounds that it would complicate the recognition of discrete populations and
mosaic evolution, and that there did not seem to be species-wide phenotypes. Indeed, many
authors have emphasized that the morphological similarity of several Aotus taxa has led to
frequent misidentifications, mainly at the boundaries of their distribution (Menezes et al.,
2010).
At least, 18 distinct karyotypes are now known, with 2n varying between 46 and 58
chromosomes (Ma, 1981a, 1983; Ma et al., 1976a, 1985; Galbreath, 1983; Pieczarka et al.,
1992, 1993; Torres et al., 1998; Defler et al., 2001; Defler and Bueno, 2007; Menezes et al.,
2010). Some karyotype studies do not necessarily support the above taxonomies. For
example, Defler et al., (2001) showed that A. hershkovitzi is not differentiated from A.
lemurinus lemurinus and that the taxon described by Hershkovitz as A. l. lemurinus was in
fact A. l. zonalis. Defler and Bueno (2007) studied a specimen karyotyped by Torres et al.,
(1998), as 2n = 50, and sampled in the Quindio region of Colombia, and defined this as a new
species called A. jorgehernandezi. Ma (1981a) compared the karyotypes of A. nigriceps and
A. azarae boliviensis and showed that a fusion of the Y chromosome occurred with the short
arm of a medium-size subtelocentric autosome in both taxa. G-banding data demonstrated
that this autosome was the same in both taxa (Ma 1981a; Ma et al., 1980), although these taxa
differ by three autosomic rearrangements. Similarly, Pieczarka and Nagamachi (1988) and
Pieczarka et al., (1993) found that the karyotypes of A. infulatus and A. azarae boliviensis are
very similar, suggesting that these are the same species. On the other hand, the sufficient
chromosomal differences needed to isolate Aotus individuals should be investigated. For
instance, Giraldo et al., (1986) examined 288 Colombian A. l. griseimembra from the lower
Río San Jorge (Bolívar Department, Northern Colombia) with 2n = 52, 53 and 54 and they
found a balanced polymorphism with no problems for interbreeding among the three
karyotypes.
In consideration of these facts, we have studied three large Aotus samples from three
different perspectives (one morphometrics and two molecular ones). The first one was a
sample of 80 Aotus skulls, representing eight Aotus taxa: A. l. griseimembra, A. l. zonalis, A.
l. lemurinus, A. brumbacki, A. vociferans, A. trivirgatus, A. nancymaae and A. a. boliviensis
for a total of 38 cranial, mandibular and teeth traits. In this chapter, we use a mixed
nomenclature of Hershkovitz (1983) and Groves (2001) up until we discuss our phylogenetic
results. The second one was the largest sample size to date for studying sequences of the
mitochondrial cytochrome oxidase subunit II (mt COII). This included 190 Aotus individuals
representing, for first time, all the Aotus taxa recognized to date. The mt COII gene has been
used to infer phylogenetic relationships in primates (within the Hominoidea, Adkins and
Honeycutt, 1991; Ruvolo et al., 1991; within the Cercopithecoidea, Disotell et al., 1992;
within the Strepsirrhini, Adkins and Honeycutt, 1994, and within several Neotropical primate
genera, including Aotus (Ashley and Vaughn, 1995; Plautz et al., 2009; Menezes et al., 2010;
Ruiz-García et al., 2011, 2013), Alouatta (Figueiredo et al., 1998), Ateles (Collins and
Dubach, 2000a, b; Ruiz-García et al., 2016a, this volume), Lagothrix (Ruiz-García and
Pinedo-Castro, 2010; Ruiz-García et al., 2014a), Saimiri (Ruiz-García et al., 2015), Cebus
(Ruiz-García et al., 2010; 2012a,b; 2016b,c,d, this volume) or Saguinus and other
Callitrichinae (Sena et al., 2002; Ruiz-García et al., 2014b)). The use of the COII gene (and of
mitochondrial coding regions) for inferring primate phylogeny can be problematic because of
the rapid rate of molecular evolution at mitochondrial loci and the saturation problem
regarding a phylogenetically informative signal at the 3rd position within codons at the
intergeneric level. Nonetheless, Ascunce et al., (2003) have shown that the gene can be very
informative at the intrageneric level. However, we cannot completely exclude the possibility
that some sequences obtained in this study represent numts (mitochondrial DNA fragments
inserted into the nuclear genome) rather than true mtDNA (Chung and Steiper, 2008) as we
will show. Therefore, special care must be taken to use this marker for phylogenetic
inferences in Aotus.
The third sample consisted of 143 Aotus individuals representing seven taxa (A. l.
griseimembra, A. l. zonalis, A. brumbacki, A. vociferans, A. trivirgatus, A. nancymaae and A.
a. boliviensis). It was analyzed for 12 nuclear DNA microsatellites, which are composed of
tandem repetitive units of 2–6 base pairs in length (Weber and May, 1989). Microsatellites
are randomly distributed, highly polymorphic and frequently found inside eukaryotic
genomes. The small amount of DNA needed for these molecular analyses has helped
researchers to use noninvasive procedures when sampling wild animals which has in turn led
to a strong incorporation of molecular techniques in the study of population biology dynamics
(Bruford and Wayne, 1993). All the samples for mt COII gene sequences and microsatellites
were obtained from wild organisms. Morphological descriptions and geographical references
were recorded to avoid incorrect classifications of samples. We can see many incorrect
individuals and sequences in some articles as well as in the GenBank because they do not
correspond to their real taxa. Here we highlight some of these cases. For instance, we
detected: the sample of an alleged A. a. boliviensis from Ashley and Vaughn (1995) is really
A. a. azarae; a sample of an alleged A. trivirgatus of Collura et al., (unpublished) is really A.
l. griseimembra; the sample of an alleged A. nigriceps of Plautz et al., (2009) is really A.

trivirgatus; the sample of an alleged A. a. azarae of Plautz et al., (2009) is really A. nigriceps;
the alleged A. trivirgatus from GenBank (AY250707) is really A. l. griseimembra; two
alleged A. nancymaae from GenBank (AJ489745 and AJ489746) are really two A. l.
griseimembra and even one alleged A. a. azarae from GenBank (AF181085) is really a
Saimiri. There are even some mistakes of the Aotus 2n karyotype in the bibliography (for
example, Menezes et al., 2010, cited 2n = 49m/50h for A. nigriceps or 2n = 50 for A.
vociferans). Thus it’s extremely important to include the correct geographical origins and
morphotypes of Aotus individuals for molecular and karyotype studies.
As discussed above, the topic of night monkey systematics presents a very interesting
problem but these monkeys are also extremely interesting for another reason. The Aotus
species have been used in the search for vaccines against malaria and other tropical diseases.
However, not all of the Aotus species are useful for this task. This is due to the fact that
different Aotus taxa have different susceptibility to the malaria parasite (Schmidt, 1973). This
has generated an intense legal and illegal traffic of these monkeys to diverse laboratories of
the world. Especially important seems to be the intense illegal traffic of individuals of
different Aotus taxa (especially, A. nancymaae) from Peru with destination to Colombia
(Maldonado, 2011; Maldonado and Peck, 2014; Maldonado et al., 2009; Ruiz-García et al.,
2012a).
The main aims of the current work are therefore to determine the power of discrimination
of different characters (craniometrics, mitochondrial DNA and nuclear DNA microsatellites)
to differentiate different Aotus taxa and to bring forward new insights of the systematics of
Aotus. We especially focus on the Northern group (sensu Hershkovitz 1983) with reference to
its number of species, which ranges from only two species (Ford, 1994) to seven species
(Defler and Bueno, 2007) depending on authors.
MATERIAL AND METHODS

Morphological Samples and Procedures
We applied different multivariate techniques to analyze relationships among 80 Aotus

skulls. These 80 samples were distributed as follows: 21 skulls of A. l. griseimembra
(Colombia), 20 skulls of A. a. boliviensis (Bolivia), nine skulls of A. nancymaae (Peru and
Brazil), eight skulls of A. vociferans (Colombia), seven skulls of A. brumbacki (Colombia),
six skulls of A. l. zonalis (Colombia), four skulls of A. l. lemurinus (Colombia), four skulls of
Aotus sp (Colombia) and one skull of A. trivirgatus (Venezuela). A total of 38 cranial,
mandibular and teeth traits were measured (Table 1).
In the first analysis we did not use any type of standardization or transformation to
determine the simultaneous impact of size and shape among the individuals analyzed.
Different distance matrices (correlation, variance-covariance, Euclidean and Manhattan
distances; Sneath and Sokal, 1973; Marcus, 1990) were calculated among the individuals
analyzed. Each one of these procedures has different mathematical properties, which must be
evaluated to see the effects on the results. To establish possible relationships among the 80
Aotus skulls, a nonmetric multidimensional scaling analysis (MDS) was applied to the
distance matrices following Kruskal (1964a,b). The stress statistic was estimated to measure
the goodness of fit of the distances in the configuration space to the monotone function of the
original distances. We choose the analyses with the best values for the stress statistic
following Spence (1972) and Spence and Ogilvie (1973). A graphic matrix (“Minimum
Spanning tree”) was superimposed (Gower and Ross, 1969; Rohlf, 1970) in order to see the
probable local distortion generated by the process of dimensional reduction. These analyses
were carried out with the program NTSYS 2.02g (Rohlf, 2000).
Table 1. Cranial, mandible and dental measurements (38) analyzed in 80 skulls

from eight different Aotus taxa
1-Maximum Transversal Width

2-Zygomatic Width
3-Superior Facial Height
4-Total Facial Height
5-Nasal Width
6-Bigonian Width
7-Auricular Height
8-Greatest Skull Length
9-Nasal Height
10-Minimum Postorbital Width
11-Maximum Postorbital Width
12-Lower Face Length
13-Base Face Length
14-Basal Height
15-Palate Length
16-Palate Width
17-Foramen Magnum Length
18-Foramen Magnum Width
19-Symphisis Height
20-Maximum Length of Mandible
21-Mandibular body Height between P1 and P2
22-Mandibular body Height between M1 and M2
23-Mandibular body Height between M2 and M3
24-Mandibular Branch Width
25-Mandibular Branch Height
26-Biauricular Breadth
27-Upper Canine Length
28-Lower Canine Length
29-Upper Canine Breadth
30-Lower Canine Breadth
31-Upper Molar Length
32-Lower Molar Length
33-Upper Molar Breadth
34-Lower Molar Breadth
35-Maximum Biorbital Width
36-Orbital Height
37-Opistion-Nasal Spine-Opistion Distance (subnasal prognathisme)
38-Ectoconion-Nasion-Ectoconion Distance
In order to detect differences among the different Aotus taxa, a Canonical Population
analysis (PCA) was performed. This method separates groups along axes with high
discrimination power (canonic axes) using the Mahalanobis distance (Mahalanobis, 1936).
This analysis is based on two hypotheses: there is homogeneity between all covariance
matrices corresponding to the population groups (this was verified with a maximum-
likelihood test) and the means of the k groups must be significantly different. To contrast this
hypotheses, the Wilks’s  and the Fisher-Snedecor F associate value test by means of the Rao
(1951) approximation was used. Subsequently, a canonical transformation was made and the
eigenvalues and the significance of the first canonical axes with Barlett’s test were calculated.
Additionally, the factorial structure of the canonic variables, the canonical representation and
the confidence region radius at 90% level, were determined. The expression for the radius is
R/N1/2, where R2 = F (N – k) n / (N – k- n + 1), with P (F > F) = 1 -  for the Fisher-
Snedecor distribution with n and N – k – n – 1 degrees of freedom (N = total population
number, k = number of population groups, and n = number of variables). This analysis was
performed using the MULTICUA Software created by Cuadras (1991).
Burnaby’s size adjustment method was also applied (Burnaby, 1966). This is useful
because this procedure determines the relationships among the individuals exclusively by
shape using the program NTSYS 2.02g (Rohlf, 2000). The relationships among the
individuals were undertaken with an UPGMA tree.
These three sets of analyses were performed for all the morphometric characters studied
(38), the cranial characters (24), and the mandible and teeth characters (14).
Molecular and Sample Procedures
In the wild, we directly sampled 190 Aotus specimens for the mt COII gene as follows: 1-
A. vociferans: 43 individuals (32 from Colombia, two from Ecuador, two from Brazil and
seven from Peru); 2- A. l. zonalis: two individuals from Colombia; 3- A. l. griseimembra: 44
individuals from Colombia; 4- A. brumbacki: seven individuals from Colombia; 5- A.
jorgehernandezi: one individual from the Quindio Department (Colombia); 6- A. l. lemurinus:
five individuals from Colombia; 7- A. nancymaae: 48 individuals, all from different areas of
Peru (Tapiche, Ucayali, Huallaga, Nanay, Amazon rivers); 8- A. miconax: two individuals
from Peru (Bongaro and another individual confiscated in Chiclayo); 9- A. trivirgatus: four
individuals from Northern Brazil (Negro River and affluent); 10- A. nigriceps: 10 individuals
(six from Brazil and four from Peru); 11- A. infulatus: eight individuals from Brazil; 12- A. a.
boliviensis: five individuals (two from Rondonia, Brazil and three from the Santa Cruz
Department in Bolivia); 13- A. a. azarae: nine individuals from Argentina (Corrientes and
Formosa) and two individuals whose specific designations are dubious (one albine individual
obtained in Iquitos, Peru [A. nancymaae or A. vociferans] and one individual from the Madre
de Dios River in Southern Peru [A. nigriceps or A. a. boliviensis]). The following species
were used as outgroups: seven individuals of different subspecies of Cebus albifrons
(Colombia and Ecuador), seven individuals of C. capucinus (Colombia and Costa Rica), three
individuals of Saimiri oerstedii citrinellus (Costa Rica), five individuals of S. cassiquiarensis
albigena (Colombia), two S. sciureus (French Guiana) and four individuals of S. ustus
(Brazil).
For the microsatellite analysis, 143 Aotus specimens were analyzed: 48 individuals of A.
l. griseimembra (Colombia), 10 individuals of A. l. zonalis (Colombia), 12 individuals of A.
brumbacki (Colombia), 28 individuals of A. vociferans (Colombia and Peru), four individuals
of A. trivirgatus (Brazil), 32 individuals of A. nancymaae (Peru) and nine individuals of A. a.
boliviensis (Bolivia).
Our 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.
Mitochondrial Sequences
Samples of blood (one ml) from animals captured in Peru and Colombia as well as
samples of hair (with follicles) from individuals kept as pets by Indian communities
throughout Colombia, Peru, Ecuador, Bolivia, Brazil and Paraguay were preserved in disodic
EDTA. We obtained blood DNA using phenol-chloroform (Sambrook et al., 1989), and hair
DNA using 10% Chelex resin (Walsh et al., 1991). We used the primers L6955 (5’-
AACCATTTCATAACTTTGTCAA-3’) and H7766 (5’-CTCTTAATCTTTAACTTAAAAG-
3’) to amplify (Polymerase Chain Reaction, PCR) the mt COII gene (located in the lysine and
asparagines tRNAs) (Ashley and Vaughn, 1995; Collins and Dubach, 2000a). We performed
each PCR in a 50-l volume with reaction mixtures including 4 l of 10x Buffer, 6 l of
3mM MgCl2, 2 l of 1 mM dNTPs, 2 l (8pmol) of each primer, 2 units of Taq DNA
polymerase, 13.5 l of H20 and 2 l of DNA. PCR reactions were carried out in a Geneamp
PCR system 9600 (Perkin Elmer) and in a Bio-Rad thermocycler. We used the following
temperatures and cycles: 95°C for 5 minutes, 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 minutes at 72°C. Using the molecular weight marker
X174 DNA digested with Hind III and Hinf I, we checked all the amplifications, including
positive and negative controls, in 2% agarose gels. We purified the amplified samples with
membrane-binding spin columns (Qiagen), directly sequenced the double-stranded DNA in a
377A (ABI) automated DNA sequencer in both directions and then repeated the sequencing
of each sample to ensure accuracy.
Microsatellite Markers
We used 12 microsatellite markers (AP40, AP68, AP74, D5S111, D5S117, D6S260,

D8S165, D14S51, D17S804, PEPC3, PEPC8 and PEPC59). The AP74, AP68 and AP74
markers were designed for Alouatta palliata and PEPC3, PEPC8 and PEPC59 for Cebus
apella, while the remaining markers were designed for humans (Ellesworth and Hoelzer,
1998). These microsatellites have been successfully used in other Neotropical primates such
as Alouatta, Ateles, Lagothrix, Cebus, Saimiri, Callicebus and Saguinus (Ruiz-García, 2005;
Ruiz-García et al., 2006, 2007 and in this book). Our final PCR volume and reagent
concentrations for the DNA extraction from blood were 25 μl, with 3 μl of 3 mM MgCl2, 2.5
μl of buffer 10×, 1 μl of 0.4 mM dNTP, 1 μl of each primer (forward and reverse; 4 pmol),
13.5 μl of H2O, 2 μl of DNA, and 1 Taq polymerase unit per reaction (1 μl). For the PCR
reactions with hair, the overall volume was 50 μl, with 20 μl of DNA and twofold amounts of
MgCl2, buffer, dNTPs, primers, and Taq polymerase. We performed all PCR reactions in a
PerkinElmer Geneamp PCR System 9600 thermocycler for 5 min at 95°C, 30 1-min cycles at
95°C, 1 min at the most accurate annealing temperature (57°C for AP40, 50°C for AP68, and
52°C for the remaining markers), 1 minute at 72°C, and 5 min at 72°C. We kept amplification
products at 4°C until they were used in a denatured 6% polyacrylamide gel in a Hoefer SQ3
sequencer vertical chamber. Depending upon the size of the markers analyzed, and the
presence of 35 W as a constant, we stained the gels with AgNO3 (silver nitrate) after 2–3 h of
migration. We used the molecular markers HinfI and ϕ174 (cut with HindIII). We repeated
the PCR reactions three times for DNA extracted from hair. Thus, allelic dropout was highly
improbable, but we cannot completely exclude the existence of null alleles, which could
increase the number of false homozygous genotypes. Nevertheless, it is improbable that all
loci were affected in the same way.
Mitochondrial Phylogenetics Procedures
The Akaike information criterion (AIC; Akaike, 1974; Posada and Buckley, 2004) and
the Bayesian information criterion (BIC; Schwarz, 1978) were used to determine the best
evolutionary nucleotide model for the mt COII gene sequences. Additionally, we obtained
maximum likelihood estimates of transition/transversion bias (Tamura et al., 2013).
The Kimura (1980) 2P genetic distance matrix among all the Aotus taxa (11) pairs was
estimated with their standard errors by means of a 10,000 maximum likelihood permutation.
Phylogenetic trees were constructed by using two procedures: 1- a Maximum Likelihood
tree (ML) obtained with RAxML v.7.2.6 Software (Stamatakis, 2006) and 2- a Minimum
Evolution tree (ME) with the Kimura (1980) 2P genetic distance obtained with Mega 6.05
Software (Tamura et al., 2011). The significance of the tree nodes was measured with 100
bootstraps.
Microsatellite Statistical Analyses
The coalescence theory generated by Beaumont and Nichols (1996) was used to detect
whether the microsatellites used were effected by constrictive or diversifying natural selection
within the Aotus genus. We used the fdist program and we obtained the observed and
expected FST statistical values for each marker used throughout the samples. Both the infinite
allele (IAM) and the step-wise (SMM) mutation models were considered. A total of 5,000
iterations were completed to calculate the values that represented the relationship between the
FST statistic and the expected heterozygosity of the markers. The iterations were grouped into
batches of 200 from which the medians and the 2.5% and the 97.5% quartiles were calculated.
The observed FST and the heterozygosity values were superimposed under this distribution
and distributed by the median and the quartiles. Values that are outside of this theoretical
distribution indicate that the microsatellites in question are being affected by natural
selection.
The genetic heterogeneity among the seven Aotus taxa analyzed with microsatellites was
studied for each marker. For this, we used the gene frequencies of the 12 microsatellites
relying on exact tests with Markov chains, 10,000 dememorizations parameters, 20 batches,
and 5,000 iterations per batch. A FST population comparison pair analysis with exact tests
with Markov chains and the same parameters as above were also undertaken. A Wright F-
statistics analysis (Wright, 1951) with the procedure of Michalakis and Excoffier (1996) was
carried out. The standard deviations of the F-statistics were calculated using a jackknifing
over loci and the 99% confidence intervals were measured by means of bootstrapping over
loci. The following procedure was used to measure the significance of FST. It used 10,000
randomizations of genotypes among populations and did not assume random mating within
populations by means of the log-likelihood G test (Goudet et al., 1996). The significance of
FIS and FIT was also found by using 10,000 randomizations of alleles within samples and in
the overall sample. Additionally, the gene diversity analysis of Nei (1973) was also
undertaken. Possible theoretical gene flow estimates among all the Aotus taxa studied taken
together were measured using the private allele model (Slatkin, 1985; Barton and Slatkin,
1986) as well as by taxa pairs from FST. All these analyses were carried out by means of the
Genepop v. 4.2.1 Software (Raymond and Rousset, 1995), Arlequin 3.5.1.2 Software
(Excoffier et al., 2005) and FSTAT.2.9.1 Software (Goudet, 1995).
We used an AMOVA analysis of the Aotus taxa studied to determine the distribution of
gene diversity at different hierarchical geographical levels (Excoffier et al., 1992). Two
analyses were done, one considering the Amazon River dividing the Northern Aotus
population (A. l. zonalis, A. l. griseimembra, A. brumbacki, A. vociferans and A. trivirgatus)
and the Southern Aotus population (A. nancymaae and A. a. boliviensis) following the
Hershkovitz’s (1983) hypothesis and a second one with all the Aotus taxa in a group and A. a.
boliviensis in another group (hypothesis where this taxon is the most differentiated of all
those studied for microsatellites). The fixation indices of Wright (1951) were estimated in the
AMOVA analysis: sc (variation of populations within the groups), ct (variation among
groups) and st (variation among individuals). This analysis was carried out by means of the
Arlequin 3.5.1.2 Software (Excoffier et al., 2005).
To determine if the degree of microsatellite differentiation was extreme among the Aotus
taxa, we developed two assignment analyses by using the GENECLASS 2 Program (Piry et
al., 2004). We performed two strategies, one Bayesian method (Cornuet et al., 1999) with the
“leave one out” procedure and one genetic distance method (standard genetic distance, Nei,
1972) with the “as is” procedure. The assignation analyses were carried out without
simulations and served to estimate the probabilities of individuals belonging or being
excluded from the original populations where they were “a priori” assigned (P < 0.05).
Another assignment analysis was applied using Structure 2.3 (Falush et al., 2007). It employs
Markov Chain Monte Carlo procedures and the Gibbs sampler and uses multilocus genotypes
to infer population structure, and simultaneously assigns individuals to specific populations.
The model considers K populations, where K may be unknown, and the individuals are
assigned tentatively to one population or jointly to ≥ 2 populations (if their genotypes are
considered admixed). Two analysis groups were carried out. First, we considered the
admixture model, wherein the individuals may have mixed ancestry and the no-admixture
model, both with no prior population information to assist with clustering (USEPOPINF = 0).
In addition,  was inferred (Dirichlet parameter for degree of admixture; with an initial value
of  = 1) using uniform priors (its value was the same for all populations). The maximum
value of this parameter was 10. Allele frequencies were uncorrelated among populations,
assuming different values of FST for each population. We revealed the presence of the most
probable number of gene pools by using the increasing likelihood method. The second
analysis was undertaken with a model that incorporates informative geographic origin
individual priors to assist with the clustering of weakly structured data in order to determine
migrants or detect slightly different populations (USEPOPINF = 1) with both admixture and
no admixture models. Furthermore, in this case, in order to apply the same conditions to that
of the previous case, we introduced LocPrior = 1, Gensback = 2, and Migprior = 0.05. The
program was run with 1,000,000 iterations after a burn period of 100,000 iterations for each
analysis. Each analysis was performed twice with convergent results.
The last analyses were focused on the detection of recent bottleneck events using the
theory generated by Cornuet and Luikart (1996) and Luikart et al., (1998). The population,
which experienced a recent bottleneck, simultaneously decreases the allele number and the
expected levels of heterozygosity. Nevertheless, the allele number (ko) is reduced faster than
the expected heterozygosity. Therefore, the value of the expected heterozygosity calculated
throughout the allele number (Heq) is lower than the obtained expected heterozygosity (He).
For neutral markers, in a population in gene mutation drift equilibrium, there is an equal
probability that a given locus has a slight excess or deficit of heterozygosity in regard to the
heterozygosity calculated from the number of alleles. In contrast, in a bottlenecked
population, a large fraction of the loci analyzed will exhibit a significant excess of the
expected heterozygosity. To measure this probability, four diverse procedures were used as
follows: sign test, standardized difference test, Wilcoxon´s signed rank test and graphical
descriptor of the shape of the allele frequency distribution. A population, which did not suffer
a recent bottleneck event, will yield an L-shape distribution (such as expected in a stable
population in mutation-gene drift equilibrium), whereas a recently bottlenecked population
will show a mode-shift distribution. The Wilcoxon´s signed rank test probably has its greatest
power when the number of loci analyzed is low, such as in the current case. The
BOTTLENECK Software (Piry et al., 1999) was used for this task. Another procedure used to
detect any possible bottleneck was that created by Garza and Williamson (2001). This
procedure is based on the ratio M = k/r, where k is the total number of alleles detected in a
locus given and r is the spatial diversity; that is, the distance between alleles in number of
repeats and the overall range in allele size. When a population is reduced in size, this ratio
will be smaller than in equilibrium populations. To calculate this M value, the program will
simulate an equilibrium distribution of M and give assumed values for three parameters of the
two phase mutation model ( = 4Ne, ps = mean percentage of mutations that add or delete
only one repeat unit, and g = mean size of larger mutations). Once M is obtained, it is ranked
relative to the equilibrium distribution. Using conventional criteria, there is evidence of a
significant reduction in population size if less than 5% of the replicates are below the
observed value. The average values used in this analysis were obtained from the MISAT
program by Nielsen (1997). This analysis was carried out with the M-P-Val and Critical-M
programs from Garza and Williamson (2001).
Figure 1. Multidimensional Scaling Analysis (MDS) by using correlations with all 38 cranial, mandible
and teeth characters measured in different Aotus taxa. A Minimum Spanning Tree (MST) was
superimposed to connect the different specimens studied. All = Aotus l. lemurinus; Alz = A. l. zonalis;
Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A.
trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.
RESULTS
Morphological Results
Different morphological analyses are shown in Figures 1 (MDS analysis with correlations
with all the characters measured), 2 (MDS analysis with the Euclidean distance only for
cranial characters), 3 (MDS analysis with the Euclidean distance only for mandible and teeth
characters), 4 (PCA with cranial characters), 5 (PCA with mandible characters), 6 (UPGMA
with all the characters measured with the Euclidean distance with the Burnaby’s procedure), 7
(UPGMA with the cranial characters with correlations and the Burnaby’s procedure) and 8
(UPGMA with the mandible and teeth characters with correlations and the Burnaby’s
procedure). All the analyses, independent of the effects of size + shape or only shape in the
cranial or mandible characters (or both together), yielded the same results. All the individuals
of the different Aotus taxa were intermixed. Only a large fraction of A. a. boliviensis
individuals were differentiated from the other Aotus taxa. This was especially detected in the
PCA analyses as well as in the UPGMA trees with the Burnaby’s procedure, where a nested
cluster with a large fraction of the individuals of A. a. boliviensis was observed. Therefore,
with the exception of A. a. boliviensis, morphometrics of skulls and mandibles did not
differentiate Aotus taxa.
Figure 2. Multidimensional Scaling Analysis (MDS) by using the Euclidean distance with only the 24
cranial characters measured in different Aotus taxa. A Minimum Spanning Tree (MST) was
Figure 3. Multidimensional Scaling Analysis (MDS) by using the Euclidean distance with only the 14
mandible and teeth characters measured in different Aotus taxa. A Minimum Spanning Tree (MST) was
Figure 4. Population Canonic Analysis (PCA) with only the 24 cranial characters measured in different
Aotus taxa. Ab = A. brumbacki; Alg = A. l. griseimembra; Alz = A. l. zonalis; Av = A. vociferans; An =
A. nancymaae; Aa = A. a. boliviensis.
Figure 5. Population Canonic Analysis (PCA) with only the 14 mandible and teeth characters measured
in different Aotus taxa. Ab = A. brumbacki; Alg = A. l. griseimembra; Alz = A. l. zonalis; Av = A.
vociferans; An = A. nancymaae; Aa = A. a. boliviensis.
Mitochondrial Analyses
With BIC, the best substitution model for the mt COII sequences was Tamura 92 + G
(24,792.431), whereas for the AIC it was General Time Reversible + G (20,396.182). The
estimated transition/transversion bias was 4.19 (maximum Log likelihood = -9,883.810).
Table 2. Kimura (1980) 2P genetic distances at the mitochondrial COII gene among the
different Aotus taxa considered. Below, genetic distance values in percentages (%).
Above, standard errors in percentages.
1 = A. l. griseimembra-A. l. zonalis; 2 = A. l. lemurinus;
3 = A. jorgehernandezi; 4 = A. brumbacki; 5 = A. vociferans;
6 = A. nancymaae; 7 = A. miconax; 8 = A. trivirgatus; 9 = A. nigriceps;
10 = A. azarae; 11 = A. infulatus-A. a. boliviensis
Taxa 1 2 3 4 5 6 7 8 9 10 11
1 - 0.3 0.4 0.3 0.5 0.7 0.6 0.7 0.7 0.7 0.7
2 1.8 - 0.5 0.4 0.6 0.7 0.7 0.7 0.7 0.7 0.6
3 3 2.9 - 0.3 0.4 0.6 0.7 0.8 0.7 0.8 0.7
4 2.7 2.5 2.2 - 0.4 0.6 0.6 0.8 0.7 0.8 0.7
5 4.3 4.2 3.4 3.5 - 0.5 0.5 0.8 0.7 0.8 0.7
6 5.8 5.1 5.8 5.6 5.7 - 0.3 0.8 0.8 0.9 0.7
7 4.4 3.7 4.4 4.3 4.4 2.0 - 0.8 0.8 0.9 0.7
8 5.6 4.7 6.6 5.8 6.1 6.6 5.2 - 0.7 0.9 0.7
9 4.7 3.9 4.7 4.5 5.2 6.2 4.7 4.3 - 0.6 0.4
10 5.7 5.1 5.8 5.4 6.1 7.3 5.8 6.2 3.8 - 0.5
11 4.8 4.1 5.0 4.7 5.6 6.6 5.1 4.6 1.7 3.0 -
Figure 6. UPGMA phenogram with all 38 cranial, mandible and teeth characters measured in different
Aotus taxa by using the Euclidean distance with the Burnaby’s procedure to delete the effect of the size.
All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A.
vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A.
sp.
Figure 7. UPGMA phenogram with only the 24 cranial characters measured in different Aotus taxa
using correlations with the Burnaby’s procedure to delete size effect. All = Aotus l. lemurinus; Alz = A.
l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At =
A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.
Figure 8. UPGMA phenogram with only the 14 mandible and teeth characters measured in different
Aotus taxa using correlations with the Burnaby’s procedure to delete size effect. All = Aotus l.
lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An =
A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.
Table 2 shows the Kimura 2P genetic distance among Aotus taxa pairs. The lowest
genetic distance pairs were for A. nigriceps-A. infulatus (1.7%), A. l. griseimembra-A. l.
lemurinus (1.8%), A. nancymaae-A. miconax (2.0%), A. brumbacki-A. jorgehernandezi
(2.2%), A. brumbacki-A. l. lemurinus (2.5%), A. l. griseimembra-A. brumbacki (2.7%), A.
jorgehernandezi-A. l. lemurinus (2.9%), A. l. griseimembra-A. jorgehernandezi (3.0%), A. a.
azarae-A. infulatus and bolivianus (3.0%), A. vociferans-A. jorgehernandezi (3.4%) and A.
vociferans-A. brumbacki (3.5%). The highest values of genetic distance pairs were for A.
vociferans-A. a. azarae (6.1%), A. vociferans-A. trivirgatus (6.1%), A. nancymaae-A.
nigriceps (6.2%), A. a. azarae-A. trivirgatus (6.2%), A. jorgehernandezi-A. infulatus and
bolivianus (6.6%), A. jorgehernandezi-A. trivirgatus (6.6%) and A. jorgehernandezi-A. a.
azarae (7.3%).
The ML tree (Figure 9a) and the ME tree with the Kimura 2P genetic distance (Figure 9b)
basically showed the same trends. The most differentiated cluster was composed of 22
sequences where mixed Aotus taxa were represented (A. vociferans, A. l. griseimembra, A.
nigriceps, A. a. boliviensis, A. brumbacki, A. l. lemurinus and A. nancymaae). The most
striking fact is that this cluster is outside of the relationship between the designed outgroup
(Cebus and Saimiri) and the remaining Aotus. The fact, that this cluster was constituted by
animals of different Aotus taxa and that they were more differentiated from the other Aotus
individuals than the Cebus and Saimiri were, suggests that the amplifications of these 22
Aotus individuals should correspond to numts (mitochondrial DNA fragments inserted into
the nuclear genome). This is extremely interesting because we don’t detect numts for the mt
COII gene in many other genera of Neotropical primates that we have studied (Alouatta,
Ateles, Callicebus, Cebus, Lagothrix, Pithecia, Saguinus and Saimiri). If so, the sequencing
of the mt COII gene in Aotus should be done very carefully to not enclose numts sequences
which could affect phylogenetic inferences. Within the Aotus cluster, the first big cluster [99
(99)%, these numbers are the bootstraps for the two commented trees] was comprised of four
well defined subclusters: trivirgatus (99 [99]%), azarae (79 [73]%), nigriceps (77 [71]%) and
infulatus with boliviensis (97 [93]%). The second big cluster was integrated by all the A.
nancymaae’s individuals sampled plus the two A. miconax’s individuals sampled [99 (99)%].
The third large cluster contained mixed haplotypes of A. vociferans, A. l. griseimembra, A. l.
lemurinus, A. l. zonalis, A. brumbacki and A. jorgehernandezi [75 (73)%]. The outgroups,
Cebus (integrated by C. capucinus and different traditional subspecies of C. albifrons: pleei,
versicolor, cesarae, albifrons, aequatoralis and malitosus) and Saimiri (integrated by the
traditional oerstedii citrinellus, albigena, sciureus and ustus) showed elevated percentages of
bootstraps (99 [99] and 99 [99]%, respectively). Therefore, the mitochondrial gene sequences
differentiated more clearly many taxa of Aotus with reference to the discriminatory power of
the cranial biometric characters, showing three or four large groups of Aotus.
Microsatellite Analyses
The application of the fdist program showed that, independently of the mutational model
(IAM and SMM), the markers D5S111, D5S117, PEPC59 and D6S269 could be influenced
by constrictive natural selection. The remaining microsatellites didn’t deviate from a neutral
model (Kimura, 1983) (Figure 10a, b).
The genic differentiation of the seven Aotus taxa (with microsatellites and 12 markers) is
displayed in Table 3. All the markers, with the exception of AP40, significantly discriminated
the seven taxa when they were taken as a whole (Bonferroni’s correction). Table 4 shows the
gene differentiation by Aotus taxa pairs by using the FST statistic and exact probabilities. The
significant pairs were A. brumbacki-A. vociferans, A. l. zonalis-A.a. boliviensis, A.
brumbacki-A. a. boliviensis, A. vociferans-A. a. boliviensis and A. nancymaae-A. a.
boliviensis. Clearly, A. a. boliviensis was the most differentiated Aotus taxon of all those
analyzed with microsatellites, similar to the morphometric analysis.
Figure 9. (Continued).
Figure 9. Maximum likelihood (ML) tree for 190 Aotus individuals representing the 13 Aotus taxa
recognized to date by using mitochondrial COII gene sequences. Number in the nodes are bootstrap
percentages (A). Minimum Evolution (ME) tree with the Kimura (1980) 2P genetic distance for 190
Aotus individuals representing the 13 Aotus taxa recognized to date by using mitochondrial COII gene
sequences. Number in the nodes are bootstrap percentages (B).
Figure 10. Fdist analysis to detect possible natural selection on the 12 nuclear DNA microsatellites
applied to seven Aotus taxa. For an infinite allele mutation model (IAM) (A). For a step-wise mutation
model (SMM) (B).
Globally, the gene flow estimate with the private allele method for all seven Aotus taxa
taken simultaneously was Nm = 0.305. This indicates very low gene flow values for the
existence of a unique Aotus taxon. Table 5 shows gene flow estimation pairs for all the Aotus
taxa analyzed. All the gene flow estimation pairs were higher than Nm = 1, with the
exception of the pairs, A. l. zonalis-A.a. boliviensis (Nm = 0.302), A. brumbacki-A. a.
boliviensis (Nm = 0.446) and A. vociferans-A. a. boliviensis (Nm = 0.413). This validates A.
a. boliviensis as the most genetically disconnected of the Aotus taxa analyzed with
microsatellites.
Table 3. Genic differentiation at 12 nuclear DNA microsatellites taken simultaneously

seven Aotus taxa by means of exact tests. * Significant probability with Bonferroni
correction
Locus Probability Standard Error

PEPC3 0.000001* 0.00000
PEPC8 0.000001* 0.00000
PEPC59 0.00316* 0.00046
AP68 0.000001* 0.00000
AP40 0.01890 0.00000
D5S111 0.000001* 0.00000
D5S117 0.000001* 0.00000
D6S269 0.000001* 0.00000
D8S165 0.000001* 0.00000
D14S51 0.000001* 0.00000
D17S804 0.000001* 0.00000
2 = Infinite
Degree of freedom = 24
Overall probability with the Fisher method= Highly significant
Table 4. Genetic differentiation by means of the FST statistic (with correction by sample
size) by Aotus taxa pairs (seven taxa) throughout the results of 12 nuclear DNA
microsatellites. 1 = A. l. griseimembra; 2 = A. l. zonalis; 3 = A. brumbacki;
4 = A. trivirgatus; 5 = A. vociferans; 6 = A. nancymaae; 7 = A. azarae boliviensis.
* P < 0.0001, significant heterogeneity
Aotus taxa 1 2 3 4 5 6 7
1 -
2 0 -
3 0 0 -
4 0.151 0 0.007 -
5 0 0 0.101* 0.173 -
6 0.026 0 0.016 0.097 0.023
7 0.467* 0.453* 0.359* 0.133 0.377* 0.159* -
Table 5. Gene flow estimates (Nm) by means of the FST statistic (with correction by
sample size) by Aotus taxa pairs (seven taxa) throughout the results of 12 nuclear DNA
microsatellites. 1 = A. l. griseimembra; 2 = A. l. zonalis; 3 = A. brumbacki; 4 = A.
trivirgatus; 5 = A. vociferans; 6 = A. nancymaae; 7 = A. azarae boliviensis. Inf = Infinite
Aotus taxa 1 2 3 4 5 6 7
1 -
2 Inf -
3 Inf Inf -
4 2.815 Inf 62.823 -
5 Inf Inf 4.414 2.390 -
6 18.871 Inf 31.572 4.640 21.012 -
7 0.552 0.603 0.891 3.246 0.826 2.644 -
Table 6. Genetic diversity analysis for night monkeys (Aotus) by means of the Nei’s
statistics (Ho = observed gene diversity in the total Aotus population; HS = average gene
diversity within the Aotus taxa; HT = expected gene diversity in the total Aotus
population; DST = absolute gene differentiation among Aotus taxa; GST = relative genetic
differentiation among Aotus taxa with regard to the total gene diversity; HT,’ DST’ and
GST’= the same as the previous statistics but corrected by sample size
Locus Ho HS HT DST DST’ H T’ GST GST’

PEPC3 0.296 0.586 0.809 0.223 0.278 0.864 0.276 0.322
PEPC8 0.239 0.574 0.884 0.310 0.387 0.961 0.351 0.403
PEPC59 0.639 0.741 0.871 0.130 0.173 0.914 0.149 0.189
AP68 0.237 0.371 0.820 0.449 0.539 0.909 0.548 0.593
AP40 0 0.081 0.083 0.002 0.003 0.083 0.028 0.034
AP74 0.308 0.688 0.946 0.257 0.3 0.989 0.272 0.304
D5S111 0.403 0.769 0.910 0.141 0.169 0.938 0.155 0.180
D5S117 0.283 0.641 0.880 0.239 0.318 0.960 0.271 0.332
D6S269 0.326 0.611 0.913 0.302 0.362 0.913 0.330 0.372
D8S165 0.423 0.814 0.898 0.084 0.098 0.912 0.093 0.107
D14S51 0.431 0.715 0.915 0.2 0.240 0.955 0.219 0.251
D17S804 0.376 0.774 0.859 0.086 0.1 0.874 0.1 0.115
All the loci taken 0.330 0.614 0.816 0.202 0.252 0.866 0.247 0.291
together
The Nei’s gene diversity analysis (Table 6) and the Wright F statistics analysis (Table 7)
showed an overall significant genetic heterogeneity: GST = 0.247-0.291, FST = 0.238 + 0.037
with jackknifing over loci, FST = 0.159-0.339 (99% confidence interval). However, some
individual microsatellites did not show a significant genetic differentiation with 10,000
randomizations and the log-likelihood G test and the Bonferroni’s correction. This was true
for PEPC59, AP40, D5S111, D5S117 and D6S269. The FIT and FIS statistics generally
showed significant homozygote excess, with the exception of PEPC59 and AP40. These
results agree well with the existence of different species of Aotus and with some genetic
fragmentation within. This is true at least, in some of these species but with the differences
among these species not being very high. If A. a. boliviensis is extracted from these analysis,
the degree of genetic differentiation considerably decreases.
The AMOVA with two groups (north and south of the Amazon River) for all the 12
microsatellites analyzed showed that 94.78% of the genetic variance was among the
considered taxa (FST = 0.0522; P = 0.123 + 0.008). Only 8.59% of the genetic variance was
due to being north and south from the Amazon River (FCT = 0.085; P = 0.141 + 0.009). The
genetic variance among taxa within the groups was practically null (FSC = -0.03; P = 0.596 +
0.017). If we only take the three most polymorphic loci (AP74, D6S269 and D17S804), the
levels with significant genetic variance were within the considered taxa (75.72% of genetic
variance; FST = 0.248; P = 0.000 + 0.000) and among taxa within the groups (27.53% of
genetic variance; FSC = 0.266; P = 0.000 + 0.000). However, the genetic variance between
those north and south of the Amazon River was practically nonexistent (FCT = -0.032; P =
0.569 + 0.012).
Table 7. Wright F statistics, estimated by jackknifing over loci, for the seven Aotus taxa
studied at 12 nuclear DNA microsatellites. * P < 0.05 for FIT; + P < 0.05 for FIS; # P <
0.05 for FST and no assuming random mating within populations by means of the log-
likelihood G test (Goudet et al., 1996)
PEPC3
FIT FST FIS
0.528* 0.215# 0.410+ Average
0.122 0.138 0.156 Standard Error
PEPC8
FIT FST FIS
0.619* 0.224# 0.503+ Average
PEPC59
FIT FST FIS
0.104* -0.027 0.120 Average
AP68
FIT FST FIS
0.732* 0.503# 0.480+ Average
AP74
FIT FST FIS
0.683* 0.228# 0.592+ Average
D5S111
FIT FST FIS
0.488* 0.057# 0.460+ Average
D5S117
FIT FST FIS
0.702* 0.295 0.573+ Average
D6S269
FIT FST FIS
0.477* 0.144# 0.361+ Average
D8S165
FIT FST FIS
0.534* 0.099# 0.481+ Average
D14S51
FIT FST FIS
0.661* 0.303# 0.515+ Average
D17S804
FIT FST FIS
0.665* 0.185# 0.595+ Average
All the loci
FIT FST FIS
0.586* 0.238# 0.456+ Average

Figure 11. Assignment of diverse Aotus individuals belonging to seven night monkey taxa by means of
the Geneclass 2.0 Software by using the Bayesian method of Cornuet et al., (1999) and the “leave one
out” procedure. Assignment of A. l. griseimembra (A); Assignment of A. l. zonalis (B); Assignment of
A. brumbacki (C); Assignment of A. trivirgatus (D); Assignment of A. vociferans (E); Assignment of A.
nancymaae (F); Assignment of A. a. boliviensis (G). Alg = A. l. griseimembra; Alz = A. l. zonalis; Ab =
A. brumbacki; At = A. trivirgatus; Av = A. vociferans; An = A. nancymaae; Aa = A. a. boliviensis.

Figure 12. Assignment of diverse Aotus individuals belonging to seven night monkey taxa by means of
the Geneclass 2.0 Software by using the Nei’s (1972) standardized genetic distance and with the “as is”
procedure. Assignment of A. l. griseimembra (A); Assignment of A. l. zonalis (B); Assignment of A.
brumbacki (C); Assignment of A. trivirgatus (D); Assignment of A. vociferans (E); Assignment of A.
nancymaae (F); Assignment of A. a. boliviensis (G). Alg = A. l. griseimembra; Alz = A. l. zonalis; Ab =
A. brumbacki; At = A. trivirgatus; Av = A. vociferans; An = A. nancymaae; Aa = A. a. boliviensis.
The AMOVA with all the Aotus taxa in one group and A. a. boliviensis in another group
with the 12 microsatellites analyzed showed that 76.71% of the genetic variance was among
the considered taxa (FST = 0.233; P = 0.142 + 0.009). Whereas the genetic variance between
the groups (all Aotus taxa versus A. a. boliviensis) increased with reference to the previous
AMOVA in 26.37% of the genetic variance (FCT = 0.264; P = 0.153 + 0.012). The genetic
variance among taxa within the groups was practically null (FSC = -0.041; P = 0.746 + 0.011).
If we only take the three most polymorphic loci (AP74, D6S269 and D17S804), the levels
with significant genetic variance were among the considered taxa (66.28% of genetic
variance; FST = 0.337; P = 0.000 + 0.000), among taxa within the groups (17.86% of genetic
variance; FSC = 0.212; P = 0.000 + 0.000) and between all the Aotus taxa and A. a. boliviensis
(15.86% of genetic variance FCT = 0.158; P = 0.005 + 0.001). Thus, there is more genetic
differentiation between A. a. boliviensis and the other Aotus taxa considered than the genetic
differentiation caused by the Amazon River between the Northern and Southern Aotus
populations. This disagrees with the Hershkovitz’s view of the Amazon River separating the
two main Aotus groups.
Figure 13. Structure 2.3 Software analyses with specimens of seven Aotus taxa. Without origin and
without admixture (A); without origin and with admixture (B); with origins and with admixture (C);
with origins and without admixture (D).
Although, microsatellite data showed genetic heterogeneity among many pairs of Aotus
taxa, this differentiation is not absolute. The Geneclass 2.0 Software showed this fact. The
Bayesian method with the technique of Cornuet et al., (1999) and the “leave one out”
procedure (58.86% of correct assignment, 83/141) showed that 18 individuals of other taxa
were enclosed in A. l. griseimembra, nine individuals of other taxa in A. l. zonalis, four

individuals of other taxa in A. brumbacki, one individual of other taxon in A. trivirgatus, 11
individuals of other taxa in A. vociferans, 14 individuals of other taxa in A. nancymaae and
one individual of other taxon in A. a. boliviensis (Figure 11 a, b, c, d, e, f, g). The
standardized genetic distance of Nei (1972) with the “as is” procedure (86.52% of correct
assignment, 122/141) showed seven individuals of other taxa in A. l. griseimembra, three
individuals of other taxon in A. l. zonalis, three individuals of other taxa in A. vociferans and
five individuals of other taxa in A. nancymaae. In the cases of A. brumbacki, A. trivirgatus
and A. a. boliviensis, none were mis-classified (Figure 12 a, b, c, d, e, f, g).
Table 8. Number of possible different gene pools for seven Aotus taxa (A. l.
griseimembra; A. l. zonalis; A. brumbacki; A. trivirgatus; A. vociferans; A. nancymaae; A.
azarae boliviensis) analyzed using the Structure Program with 12 microsatellite loci. * =
Most probable number of populations. K = number of populations. Without “a priori”
origins and with admixture (A); Without “a priori” origins and without admixture (B);
With “a priori” origins and with admixture (C); With “a priori” origins and without
admixture (D)
(A)
Populations Ln Likelihood
K=1 -1,774.9
K=2 -1,604.6
K=3 -1.544,4
K = 4* -1,536.1
K=5 -1,539.2
K=6 -1,558.3
K=7 -1,576.6
K=8 -1,594.1
K=9 -1,612.3
K = 10 -1,629.5
K = 11 -1,646.3
K = 12 -1.663.3
(B)
K=1 -1,774.8
K=2 -1,591.9
K=3 -1.525.4
K=4 -1,505.4
K = 5* -1,490.2
K=6 -1,499.7
K=7 -1,505.5
K=8 -1,513.1
K=9 -1,519.9
K = 10 -1,524.5
K = 11 -1,530.3
K = 12 -1.535.8
(C)
K=1 -1,774.8
K=2 -1,609.0
K=3 -1.577.3
K=4 -1,525.4
K = 5* -1,508.6
K=6 -1,515.9
K=7 -1,516.7
K=8 -1,523.5
K=9 -1,548.0
K = 10 -1,534.7
K = 11 -1,537.6
K = 12 -1.542.7
(D)
K=1 -1,774.8
K=2 -1,611.5
K=3 -1.545.7
K=4 -1,531.1
K = 5* -1,506.4
K=6 -1,512.2
K=7 -1,512.4
K=8 -1,528.1
K=9 -1,517.6
K = 10 -1,521.3
K = 11 -1,518.2
K = 12 -1.517.7
For the Structure Software (Table 8 and Figure 13 a, b, c, d), four (without origin and
with admixture) or five (without origin and without admixture; with origins with and without
admixture) populations were detected as the more probable results. Without origins, it’s
clearly difficult to classify one animal in a determined taxon. When we provide the origins of
the individuals to the software, then it is easier to classify the exemplars to the corresponding
taxa, especially when admixture is allowed. This last analysis distinguished (1) A. l.
griseimembra and A. l. zonalis, (2) A. brumbacki, (3) A. vociferans, (4) A. nancymaae and (5)
A. trivirgatus and A. a. boliviensis. Therefore, although we started this microsatellite analysis
with seven “a priori” Aotus taxa, the Structure analyses detected only four or five different
taxa.
We applied Bottleneck Software to detect possible demographic changes for the three
Aotus taxa with the greatest sample sizes (A. l. griseimembra, A. vociferans and A.
nancymaae) (Table 9). For A. l. griseimembra, only the Wilcoxon test with the IAM (p =
0.027) supports a bottleneck. The remaining tests did not detect any evidence in favor of a
bottleneck (Sign test: IAM [p = 0.282] and SMM [p = 0.209]; Standardized difference test:
IAM [T2= 0.171, p = 0.189] and SMM [T2 = -0.883, p = 0.019]; Wilcoxon test: SMM [p =
0.902]). The significant negative value of the Standardized difference test is more related to a
population expansion or to population fragmentation within this taxon. For A. vociferans, no
tests detected any support in favor of a bottleneck (Sign test: IAM [p = 0.332] and SMM [p =
0.273]; Standardized difference test: IAM [T2= 0.055, p = 0.478] and SMM [T2 = -2.301, p =
0.011]; Wilcoxon test: IAM [p = 0.289] and SMM [p = 0.927]). As in the previous case, the
significant negative value of the Standardized difference test is more related with a population
expansion or to population fragmentation within this taxon. For A. nancymaae, no tests
detected any proof in favor of a bottleneck [Sign test: IAM (p = 0.087) and SMM (p = 0.151);
Standardized difference test: IAM (T2= -1.154, p = 0.124) and SMM (T2 = -0.021, p = 0.221);
Wilcoxon test: IAM (p = 0.527) and SMM (p = 0.215)]. The graphic descriptors for the three
species (Figure 14 a, b, c) and an analysis by Garza and Williamson (2001) did not detect any
sign of recent bottlenecks in these Aotus taxa (Table 10). Therefore, no evidence of
bottlenecks were detected in the Aotus taxa analyzed for the microsatellites.
Table 9. Application of the Bottleneck Software to detect possible recent bottleneck

events in three Aotus taxa sampled. This Software employed the Cornuet and Luickart
(1996)’s procedure. IAM = Infinite allele model; SMM = Stepwise mutation model
Aotus lemurinus griseimembra

Locus Expected heterozygosity in the Expected heterozygosity under Expected heterozygosity under
sample the IAM the SMM
PEPC3 0.742 0.759 0.789
PEPC8 0.561 0.607 0.695
AP40 Monomorphic - -
AP74 0.718 0.683 0.785
D5S111 0.828 0.760 0.838
D5S117 0.563 0.551 0.626
D8S165 0.900 0.864 0.885
D145S51 0.812 0.716 0.777
D17S804 0.879 0.847 0.868
Aotus vociferans
PEPC3 0.385 0.430 0.490
PEPC8 0.733 0.774 0.812
PEPC59 0.642 0.791 0.832
AP68 0.680 0.549 0.624
AP40 Monomorphic - -
AP74 0.826 0.766 0.819
D5S111 0.867 0.834 0.841
D5S117 0.844 0.862 0.875
D6S269 0.908 0.877 0.898
D8S165 0.742 0.759 0.790
D14S51 0.233 0.293 0.321
D17S804 0.736 0.645 0.720
Aotus nancymaae
PEPC3 0.735 0.630 0.710
PEPC8 0.871 0.829 0.869
PEPC59 0.541 0.775 0.824
AP68 0.440 0.775 0.824
AP40 Monomorphic
AP74 0.714 0.642 0.685
D5S111 0.848 0.778 0.826
D6S269 0.933 0.915 0.918
D8S165 0.864 0.824 0.845
D15S51 0.821 0.753 0.769
D17S804 0.803 0.828 0.847
Figure 14. Graphic descriptors to detect possible bottlenecks in three Aotus taxa. For A. l. griseimembra
(A); for A. vociferans (B); for A. nancymaae (C).
Table 10. Average M value in simulations with the Garza and Williamson (2001)
procedure, Critical Mc value, average M value of analyzed data, and microsatellites
showing an M value under Mc for six Aotus taxa
DISCUSSION
Morphometrics, Molecular Population Genetics and Discrimination of Aotus
Taxa
It is important to take into account that the craniometric and the microsatellite analyses
did not contain all the Aotus taxa recognized from a morphological or karyotype perspective.
Also, the mt COII analyses contained all the described Aotus taxa to date. This is the first
molecular analysis to enclose all of these taxa.
Clearly, all the craniometrics analyses revealed that the skulls of A. l. zonalis, A. l.
griseimembra, A. l. lemurinus, A. brumbacki, A. vociferans and A. nancymaae were
intermixed. This was independent of the combined influence of size and shape or just shape.
A. a. boliviensis was the unique taxon which showed some craniometric differentiation in
MDS (the most external individuals), PCA (a uniquely different and significant group) and in
UPGMA trees (they formed a nested group within all the other intermixed Aotus taxa skulls,
although some A. a. boliviensis skulls were also intermixed with the other skulls). Only one
skull of A. trivirgatus was analyzed and it was intermixed with the remaining skulls. Thus,
craniometrics has a comparatively lower power to differentiate the Northern Aotus taxa
(among and between taxa and A. nancymaae). Only the most Southern taxon (A. a.
boliviensis) was positively discriminated.
The microsatellite analyses with the same Aotus taxa showed a higher degree of
differentiation among the Aotus taxa than did craniometrics. However, basically, the most
differentiated taxon was again A. a. boliviensis, which was specially observed in the FST taxa
pair comparisons, gene flow estimates, GeneClass 2.0 results and in the AMOVA analyses.
The mitochondrial analyses revealed the highest discrimination power of all the analyses
carried out. This procedure analyzed the most individuals including all the described Aotus
taxa. However, if, in this last procedure we remove A. a. azarae, A. nigriceps and A. infulatus
from the analyses, then A. a. boliviensis individuals appear as the most differentiated ones.
Another commonality among the three procedures is that the differentiation among all the
Northern Aotus taxa, excluding A. trivirgatus, is extremely limited. However, they do have
different chromosome numbers (from 2n = 46, 47, 48 of A. vociferans to 2n = 58 of A. l.

lemurinus) (Table 11). One fundamental difference between mitochondrial DNA and
microsatellites is that the former could clearly differentiate A. vociferans from A. nancymaae,
whereas the second had more limited power for this task. For instance, FST microsatellite
comparision pairs and microsatellite gene flow estimates did not clearly differentiate both
taxa. Similarly, the genetic assignment analysis with the worst assignation percentage
(Bayesian with the Cornuet et al., 1999’s procedure) showed the nancymaae group including
five A. vociferans individuals and the A. vociferans group including one specimen of A.
nancymaae. The best percentage assignment analysis (with the Nei’s genetic distance)
showed the A. nancymaae group including three individuals of A. vociferans. The Structure
analysis could differentiate the multi-genotypes of A. vociferans and A. nancymaae when the
different geographical origins were given to the software, but they were not differentiated
when geographical origins were not specified. The differentiation of A. nancymaae and A.
vociferans specimens is critical because of the intense illegal traffic of A. nancymaae from the
Peruvian Amazon to Colombia. The illegal traffic is mainly due to malaria vaccine
experiments in Colombia (note the Patarroyo case with its legal considerations; see
Maldonado, 2011; Maldonado and Peck, 2014; Maldonado et al., 2009; Ruiz-García et al.,
2012a). Thus, mitochondrial analyses seem to be more effective in differentiating individuals
from both taxa. Also, mitochondrial analyses are completed more rapidly and are less
expensive than those with microsatellites. Nevertheless, we revealed 22 Aotus sequences out
of the 190 analyzed, which amplified numts in many taxa of the genus. Aotus is the first
Neotropical primate genus where we detected numts. These sequences were characterized by
missing and ambiguous data. The amino acid translations of the sequences did not have initial
start and terminal stop codons but did have premature stop codons, typical of the numts
(Chung and Steiper, 2008). Thus, caution must be taken with the use of mt COII gene
sequences with Aotus for individual classifications and phylogeny. For instance, Menezes et
al., (2010) analyzed the Aotus phylogeny, and concluded that Aotus diverged some 4.62
millions of years ago (MYA; with 95% HPD intervals of 3.07-6.43 MYA). Ashley and
Vaughn (1995) and Plautz et al., (2009) professed similar dates (3.3-3.6 MYA and 4.7 MYA,
respectively) for the initial divergence of Aotus. However, Ruiz-García et al., (2011) and
Babb et al., (2011) estimated divergence times considerably older for the original
diversification of Aotus (8.47 MYA, if the average temporal split between A. vociferans-A.
nancymaae and A. a. boliviensis is estimated, and 8.95 MYA, respectively). These higher
temporal splits could be an artifact caused by the presence of numts among the sequences
studied both of these works. Nevertheless, paleontological evidence of A. didensis fossils
(11.8–13.5 MYA) from La Venta, Colombia (Setoguchi and Rosenberger, 1987; Rosenberger
et al., 2009; Takai et al., 2009) seems to be correlated with these last estimations. The first
cited estimations are also not congruent with the temporal split of 22 MYA for an Aotus-
platyrrhine divergence based on nuclear DNA data (Opazo et al., 2006), or 15 MYA for the
origins of Aotus based on mitochondrial genomes (Hodgson et al., 2009). If the first estimates
(around 4 MYA) for the diversification of Aotus were accurate, this could be correlated with
more than 10 MYA of molecular stasis and nearly 14 MYA of morphological stasis (14 MYA
to present). This could explain the very limited morphological and morphometric differences
we found in Aotus. Maybe the fact that Aotus is nocturnal, and the consequence of little
selection for phenotypic differences, could explain the small degree of morphological changes
in the last 14 MYA (morphological stasis). The question of the temporal origin and the
molecular diversification of Aotus needs to be deeply studied.
Microsatellites did not detect any evidence of bottleneck affecting the three Aotus taxa
with the highest sample sizes (A. l. griseimembra, A. vociferans and A. nancymaae).
However, some tests detected possible population fragmentation within A. l. griseimembra
and within A. vociferans. This agrees quite well with the Structure analysis without given
origins. In other Aotus taxa, such as A. a. azarae, Babb et al., (2011) detected a high level of
haplotypic diversity and a recent expansion of Azara’s owl monkeys into the Argentinean
Chaco.
Table 11. Aotus taxa, number of diploid chromosomes (2n), fundamental numbers (FN)
estimated by Defler and Bueno (2007)/Ford (1994), Karyomorph nomenclature
of Ma (1981a) and Ma et al., (1976a) (K1), of De Boer and Reumer (1978) and Reumer
and De Boer (1980) (K2), and of Torres et al., (1998) (K3) and phenotypes defined
by Ma et al., (1976a) (Ph). Nomenclature for Aotus taxa followed Hershkovitz (1983)
and Groves (2001)
Aotus taxa 2n FN K1 K2 K3 Ph
lemurinus lemurinus 58 (BS) 76/- - - 8 B
lemurinus zonalis 55,56 (BS) 72/62 VIII, IX 1 1 B
lemurinus griseimembra 52, 53, 54 (BS) 72/62 II, III, IV 2 2 B
jorgehernandezi 50 (BS) - - - 9 -
brumbacki 50 (BS) 70/58 - 6 6 B
vociferans 46, 47, 48 (BS) 70/60 V, X, XI 7 7 B
trivirgatus 50? 54? 51-52m/52f? - - - - -
nancymaae 54 (BS) -/72 I 3 3 A
miconax - - - - - -
nigriceps 51m/52f -/66 VII 4 - C
infulatus 49m/50f -/60-61 - - 10 D
azarae boliviensis 49m/50f -/60-61 VI 5 5 D
azarae azarae 49m/50f -/60-61 - - - D
Phylogenetics and Systematics of Aotus
There has been much disagreement among primatologists about the systematics of Aotus.
However, when we consider the previous morphological, karyotypic and molecular studies
along with the current results it is relatively easy to understand why disagreements exist.
Primatologists often believe that the evolution and the speciation processes within different
Neotropical primate genera can be similar and in parallel. However, this is not certain. Here
are three examples that contest this. 1- The species of Alouatta (howler monkeys) are very
homogeneous morphologically speaking (although they have a variety of remarkably different
colors), but there are considerable karyotypic differences which are correlated with
considerable molecular differences. Thus, there is a certain morphological stasis with a long
evolution history reflected in parallel both in karyotypes and molecules (both revealed the
same evolutionary history); 2- The taxa of Ateles (spider monkey), with the exception of A.
paniscus (which is morphologically, karyotypically and molecularly different), are

morphologically very similar (but with very different color pelages), with small molecular
differences (which in turn reveals a short evolution) and some minor karyotype differences,
which don’t correlate with the molecular evolution. Therefore, the Ateles evolution is
relatively recent which is revealed by morphology (although color differences have been
extremely emphasized by traditional primatologists) and molecular biology. The minor
karyotypic differences within this genus have been of little value for reconstructing its
phylogeny (a similar situation is found for Saimiri, the squirrel monkeys and in Lagothrix, the
Humboldt woolly monkeys); 3- The taxa of Cebus (including Sapajus), capuchin monkeys,
are characterized by considerable morphological differences, but very similar karyotypes and
moderate molecular differences. Indeed, in the robust forms, the morphological differences
are very outstanding but the molecular and karyotypic differences are relatively small. The
temporal evolution of capuchin monkeys is between that observed in Alouatta and that
observed in Ateles, but characterized by a rapid morphological evolution. Nevertheless, the
Aotus case has a different history. The taxa of Aotus are extremely homogeneous from a
morphological perspective (stasis), but with extreme karyotype differences. There are also
very small molecular differences, at least, within the Northern and Southern groups. However
the chromosomal differences within these groups are outstanding. This means that different
speciation mechanisms are affecting these Neotropical primate genera. Thus we cannot apply
the same rules to define species in these different Neotropical primate genera.
The molecular genetic distances are very small especially in the Northern Aotus group,
except for A. trivirgatus. For the mt COII gene in primates, Ascunce et al., (2003) and Collins
and Dubach (2000b) reported an average genetic distance around 5.82% + 1.64% among
species within a genus, around 2-4% for subspecies within species and around 15.68% +
1.73% among genera. The genetic distances for all the Northern Aotus taxa (A. vociferans, A.
brumbacki, A. jorhehernandezi, A. l. griseimembra, A. l. lemurinus and A. l. zonalis) ranged
from 1.8% to 4.3%, and are in the range of subspecies within a species, although each one if
these named taxa have different 2n chromosomes (exception of A. brumbacki and A.
jorgehernandezi). Conversely, the genetic distance percentages between A. trivirgatus and all
the other Northern Aotus taxa ranged from 4.7% to 6.6%, typical of different species. The
genetic distance percentages between the Northern group and A. trivirgatus in respect to A.
nancymaae were 5.1-5.8% and 6.6%, respectively, which are in the range of different species.
However, A. nancymaae and A. miconax only showed 2% of genetic differences, typical of
subspecies. The genetic distance percentages among four taxa of the Southern Aotus group
(A. a. azarae, A. a. boliviensis, A. nigriceps and A. infulatus) ranged from 1.7% to 3.8%,
typical of subspecies. The genetic distance percentages between the Northern group, A.
trivirgatus, A. nancymaae-A. miconax with reference to this Southern group were 3.9-6.1%,
4.3-6.2% and 4.7-7.3% , respectively, being in the range of different species. Our results
agree extremely well with the results of Plautz et al., (2009). These authors found that A. l.
griseimembra, A. vociferans, and A. brumbacki formed a very coherent group, with relatively,
very low nucleotidic divergence. Their maximum parsimony and likelihood trees showed
unresolved polytomy among these three taxa, while the NJ tree pointed to A. vociferans as the
sister clade of the A. brumbacki–A. l. griseimembra cluster. The lack of resolution in this case
does not necessarily reflect any specific shortcomings of the mt COII sequences, but may
simply reflect relatively rapid dispersal and divergence in the Northern Aotus group.
The small genetic distances (neutral markers) and mitochondrial haplotypes shared in the
Northern group, although they have high karyotypic differences, as well as that found in the
Southern group (although in this group the karyotypic differences are not so extreme) provide
arguments in favor of recent chromosomal speciation in closely related Aotus populations.
The extreme importance of chromosomal evolution in Aotus has been demonstrated by
reciprocal chromosome painting between humans and A. nancymaae as well as between A.
nancymaae and woolly monkey whole chromosome probes. Stanyon et al., (2004) showed
that for the A. nancymaae karyotype, only three human syntenic groups were conserved. They
coexisted with 17 derived human homologous associations. A minimum of 14 fissions and 13
fusions were required to derive the A. nancymaae karyotype from that of the ancestral
karyotype of Neotropical primates. A. nancymaae is considered the ancestral karyotype by
some authors because it is the least derived karyotype of all the current Aotus taxa.
Meanwhile for Ateles, Lagothrix, Saimiri and Cebus we can invocate typical allopatric
speciation following vicariant or peripatric events or even non-allopatric speciation with
parapatric events, in the case of Aotus, the main process is chromosomal speciation following
stasipatric, peripatric or parapatric models (Lewis, 1966; White, 1968, 1978; Reig, 1980;
King, 1993). The Northern group case is very similar to the cases of some rodents such as
Spalax ehrenbergii from the Middle East with 2n = 48, 52, 54, 56 and 58 chromosomes
(Nevo, 1991) or the Venezuelan rodent Proechimys guairae with six karyomorphms forming
a “rassenkreis” (2n = 42, 44, 46, 48, 50 and 62) (Reig, 1980). Of the three chromosomal
speciation models, the stasipatric model seems to be globally the most improbable because
within the geographical range of the Northern Aotus taxa, there isn’t a wide distributed
karyotype with other karyotypes differentiated within the territory of the main distributed
karyotype. Within the geographical range of A. vociferans, there are not specimens with
karyotypes of A. brumbacki or A. l. griseimembra, for instance. However, the existence of
several chromosome polymorphisms within several of these Aotus taxa should be cases of
stasipatric events. For instance, consider A. vociferans with 2n = 46, 47, 48 and A. l.
griseimembra with 2n = 52, 53, 54, and A. l. zonalis with 2n = 55, 56. Indeed, both
microsatellites and mitochondrial DNA did not discriminate these two last forms, which
means that they are, molecularly speaking, the same taxon. Nevertheless, the karyotypic
differentiation for the Northern group taken as a whole is more related to parapatric or
peripatric chromosomal differentiation. We suggest that there is a greater (major) influence of
the chromosomal parapatric differences compared to peripatric ones, although both could
have occurred during the evolution of these Aotus forms. Our reasoning for this suggestion is
because each chromosome form is in different biomes or ecotones: A. vociferans in the
Amazon rain forest, A. brumbacki in the Eastern Llanos, A. jorgehernandezi in the Andean
areas of the Colombian Central Andean cordillera, A. l. griseimembra in the Magdalena River
Basin, A. l. lemurinus in the high Andean areas of the Colombian Eastern Cordillera and A. l.
zonalis in the Choco and Darien jungles of Western Colombia and Panama. The selective
pressures of these different ecotones could have selected the different karyotypes of the Aotus
of these areas. Additionally, in the three taxa of the Northern group where we analyzed
relatively large samples (A. vociferans, A. brumbacki and A. l. griseimembra), the gene
diversity for both microsatellite and mitochondrial genes was elevated and there was no
evidence of bottlenecks. Both of these results support parapatric chromosomal differentiation.
Ford’s (1994) study indicating morphometric variables, coat colors and karyotypes with clinal
patterns provides more evidence in favor of parapatric chromosomal differentiation.
However, if large samples of A. jorgehernandezi, A. l. lemurinus and A. l. zonalis will be

studied for molecular markers and they reveal low gene diversity levels or bottlenecks, as we
suspected, these cases could then be related to peripatric chromosomal differentiation.
Therefore, although the parapatric chromosomal differentiation seems to be the most
important process in the Northern Aotus group, both stasipatric and peripatric chromosomal
differentiation could have simultaneously occurred. However, the molecular differences are
extremely small for all these Aotus forms because their temporal splits are very recent. This
could indicate a very rapid and successful colonization of many different biomes in the
current Colombian territory by the night monkeys. The small molecular differences could also
be due to the absence of total reproductive barriers although these forms have different
chromosomal numbers. In fact, total reproductive barriers could be more important to the
fundamental number (FN) than to the 2n (Martin, 1990). Defler and Bueno (2007) suggested
that there would probably be no successful interbreeding among the putative subspecies of A.
lemurinus, nor between any of them and A. brumbacki, nor between A. vociferans and
populations of A. brumbacki or A. lemurinus. However, Ma et al., (1976a) and Pieczarka and
Nagamachi (1988) claimed that these different chromosomal numbers in the Northern Aotus
group do not constitute a reproductive barrier. In fact, there is no selection against
heterokaryomorphms in A. l. griseimembra with diploid numbers of 52, 53 and 54 (Giraldo et
al., 1986). A similar situation is true for A. vociferans with 2n = 46, 47 and 48 (Descailleaux
et al., 1990) and perhaps for A. zonalis with 2n = 55 and 56 (in this taxon 2n = 54 which is
theoretically possible but has not yet to be observed). If this last one is certain, there should
be chromosomal number continuity in A. l. griseimembra and A. l. zonalis of 2n = 52, 53, 54,
55 and 56. Recall that from a molecular point of view, both mitochondrial DNA and
microsatellites are extremely similar between both Aotus taxa. Defler and Bueno (2007)
affirmed that A. l. zonalis is more closely related to A. l. lemurinus than it is to A. l.
griseimembra, because A. l. zonalis and A. l. griseimembra differ in two distinct
translocations of one chromosome (Ma et al., 1978). This put forward the extreme
relationships among the most Northern Aotus taxa. It is clear that these three taxa (A. l.
griseimembra, A. l zonalis and A. vociferans) appear to maintain their karyological identity
with multiple chromosome differences. Only the study of possible hybridization or tension
zones between some of these Aotus populations could help to distinguish between both
hypotheses (Hewitt, 1989, 1993).
Our chromosomal parapatric diversification model agrees well with the proposal by Ma
(1981a). It suggests that geographic isolation in different geographic niches led to karyotypic
diversity in the night monkeys. Related with this, Plautz et al., (2009) hypothesized that
climatic and geological changes in the last 5 MYA (eustatic sea level changes, for instance)
allowed the development of Aotus taxa in three refuge groups, one comprising A. vociferans,
A. lemurinus and A. griseimembra in the Andean foothills. A second group contains A.
trivirgatus in the northwestern Guyanan shield. The third refuge group consists of A.
nigriceps, A. azarae, A. infulatus and A. nancymaae in the Brazilian shield refuge. Menezes et
al., (2010) suggested that A. nancymaae should be included in the Andean foothill refuge
rather than in the Brazilian shield refuge. We provide evidence more compelling than
Menezes et al., (2010) that A. nancymaae was highly related with the Andean foothill refuge.
Menezes et al., (2010) also claimed that A. nigriceps, A. azarae and A. infulatus must have
diverged after the rise of sea level (5 MYA) while grey neck species and A. nancymaae could
have diverged before this event. Babb et al., (2011) also detected older diversification
processes in the Northern group, around 7.34 MYA, whereas that of the Southern group was
6.22 MYA. Similarly, the diversification process for A. a. azarae was 1.78 MYA (95% HPD:
0.24–3.99 MYA), whereas for A. nancymaae, it was 4.68 MYA (95% HPD: 1.93–8.10
MYA). The more recent diversification of the Southern Aotus group could be related with the
more recent establishment of southern rivers and the draining of the South American Chaco
(Rosenberger et al., 2009). Hershkovitz (1977, 1983), using the theory of metachromism,
claimed that the red-necked Aotus species had to have derived from the gray-necked species
in the North. Following this theory, these pigment changes are one-way and always proceed
from the loss of eumelanin (although the molecular results contradicts this because the
ancestor of A. nancymaae, a red-necked taxon, is in the origin of all the gray-necked Aotus
taxa, except A. trivirgatus). However, our results don’t clearly show which of the two clades,
A. nancymaae-miconax + Northern group or A. trivirgatus + Southern group, was the first to
diverge.
Two possible schemes could explain the order of appearance of the different Northern
Aotus forms for the parapatric chromosomal differentiation:
1. The first hypothesis: A. nancymaae (2n = 54)  A. vociferans (2n = 46, 47, 48) 
A. brumbacki (2n = 50) and/or A. jorgehernandezi (2n = 50) (and A. brumbacki  A.
jorgehernandezi or A. jorgehernandezi  A. brumbacki)  A. l. griseimembra (2n =
52, 53, 54)  A. l. zonalis (2n = 55, 56) and A. l. lemurinus (2n = 58). This scheme
shows that the parapatric chromosome differentiation is basically by fission events
(with the exception of the first step) and not by fussion events as was suggested by
Defler and Bueno (2007). These authors concluded that A. l. lemurinus with 2n = 58
was the original karyotype of these Aotus forms. However, this hypothesis is
untenable because this form has a peripheric distribution and lives in a very
specialized habitat (high Andes). It has the highest 2n chromosome number of all the
Aotus and is more related to a derived form than to an original form. A higher
number of chromosomes facilitates a higher degree of recombination which could be
very important when adapting to new environmental conditions. Defler and Bueno
(2007) and Defler et at., (2001) claimed that A. l. lemurinus has an acrocentric
chromosome that is involved in two different rearrangements, in A. brumbacki and A.
l. griseimembra. However, molecular and other karyotype characters show the
reverse, A. l. lemurinus is the most derived of all the Northern Aotus group.
2. The second hypothesis should agree with the fact that some authors (Ma, 1981a and
Galbreath, 1983) have claimed that the original Aotus had 54 chromosomes.
Therefore: A. l. griseimembra (2n = 52, 53, 54)  A. l. zonalis (2n = 55, 56) and A. l.
lemurinus (2n = 58) is on one side, and, A. l. griseimembra (2n = 52, 53, 54)  A.
brumbacki (2n = 50) and/or A. jorgehernandezi (2n = 50) (and A. brumbacki  A.
jorgehernandezi or A. jorgehernandezi  A. brumbacki)  A. vociferans (2n = 46,
47, 48) is on the other side. Ma et al., (1985) suggested that A. vociferans was
derived from A. brumbacki by a single fusion event, and that the latter represents an
intermediate form between A. vociferans and A. l. griseimembra. However, this
second hypothesis is less credible than the first one because it means that in the area
north of A. l. griseimembra’s distribution there were only fission events and in the
area south of A. l. griseimembra’s distribution there were only fussion events, which
is less probable. It’s the same fact detected for the Venezuelan rodent, Proechimys
guairae (Reig, 1980).
It’s remarkable that within the Northern Aotus taxa with a unique 2n, there is also
considerable chromosomal heterogeneity. This is the case of A. brumbacki. None of the three
previously published descriptions of chromosomal morphology for A. brumbacki (Brumback,
1974; Yunis et al., 1977; Torres et al., 1998) agreed completely in the chromosomal
characteristics (all with 2n = 50) and showed considerable variation in the identification of
numbers of metacentric, submetacentric and acrocentric chromosomes.
Our mitochondrial analysis is the first including two individuals of A. miconax. This
taxon was inside the large A. nancymaae cluster. Since the A. miconax’s karyotype is
unknown, we cannot know which relationship exists with the A. nancymaae’s karyotype. But,
the fact that both forms are distributed in contiguous but very different biomes (A. nancymaae
in the lowland Amazon forest and A. miconax in the transition forests from high Andes to
lowland Amazon), could be related with peripatric or parapatric chromosome differentiation.
Thus, we believe that A. miconax should have a different karyotype from A. nancymaae.
However, it must be investigated.
The mitochondrial analysis revealed that the remaining four Southern taxa formed a
monophyletic group with low genetic distances and relatively minor karotypic differences
with regard to the Northern group (all the Southern taxa possess 2n = 49(male)/50(female)
chromosomes, with the exception of A. nigriceps with 2n =51m/52f). A. nigriceps, A. a.
boliviensis, and A. a. azarae show a difference in chromosome numbers between males and
females as a consequence of a fusion of the Y chromosome with an autosome. Ma et al.,
(1980) and Ma (1981a) compared the karyotypes of A. nigriceps and A. a. boliviensis. They
found that the fusion of the Y chromosome occurs with the short arm of a medium-size
subtelocentric autosome. Ma et al., (1980) and Ma (1981a, 1984), by using G-banding and
gene mapping, showed evidence that this autosome is the same on both karyotypes. In
addition, in A. a. boliviensis the fusion of the Y with the short arm of the autosome is
followed by a pericentric inversion, whereby the short arm bearing the Y chromosome
becomes a portion of the long arm of the resulting submetacentric. In A. a. azarae, the Y
fusion is to a small acrocentric autosome, but all other chromosomes are identical on the G-
and C-banding level to A. a. boliviensis chromosomes (Mudry et al., 1984). Galbreath (1983)
assumed on the basis of geographic distribution and similarities in fur pattern that the
karyotype of A. infulatus should not be very different from the karyotypes of A. nigriceps and
A. azarae. Our mitochondrial result agrees quite well with Galbreath’s (1983) assumption, as
well as the chromosomal study of Pieczarka and Nagamachi (1988). These authors showed
that in the A. infulatus karyotype, except for pair B12, all the autosomic chromosomes and the
X chromosome are identical on G- and C-banding levels to the corresponding chromosomes
of A. a. boliviensis described by Ma et al., (1976a). The only differences between A. infulatus
and A. a. boliviensis are on the G-banding pattern of B12 and on the G- and C-banding
patterns of the Y/autosome. Pieczarka and Nagamachi (1988) tentatively explained that the
origin of the Y/autosome of A. infulatus and A. a. boliviensis could be related with an
ancestral population with a karyotype similar to that of A. nigriceps (2n = 51m/52f), which
gave rise to a group of animals that fixed three autosomal rearrangements, originating the
2n=49m/50f karyotype and maintaining the Y/autosome chromosome of the ancestral
population. This newly derived population was further divided, with a pericentric inversion of
the Y/autosome becoming fixed in one group (A. infulatus), and another peri centric inversion
of the Y/autosome, becoming fixed on the other group (A. a. boliviensis). They concluded that
there are insufficient differences between A. infulatus and A. a. boliviensis to support two
distinct species. Also, our results agree quite well with Babb et al., (2011), who suggested
that the Southern expansion of Aotus was gradual (only one chromosomal fission event and
the maintenance of the Y-autosomal fusion event in Southern males), with taxa diversifying
steadily at different points in time, not through multiple splits or population bottlenecks
(Pieczarka et al., 1993, 1998; Torres et al., 1998).
However, we obtained two samples in Rondonia (Brazil) from two specimens with some
morphological resemblance to specimens of A. a. boliviensis. Pieczarka et al., (1993)
established that these two morphologically similar A. a. boliviensis individuals were
karyotypically intermediate between A. a. boliviensis and A. infulatus. These authors assumed
that the Rondonia individuals, A. a. boliviensis and A. infulatus formed only one species. Our
mitochondrial results placed the two Rondonia specimens we sequenced within the A.
infulatus cluster, thus agreeing with the karyotype study of Pieczarka et al., (1993).
Regrettably, we don’t know if the relationship among the three animals we sampled in
Bolivia and amplified for numts is similar to the morphotypes from Rondonia. This too must
be investigated. Plautz et al., (2009) stated that A. nigriceps was clearly divergent from all
other species suggesting that it is closest to the ancestral form, contrary to the hypothesis of
Ruiz-Herrera et al., (2005). They proposed that the most divergent species is a karyotypically
distinct northern form. Clearly this statement of Plautz et al., (2009) is incorrect because their
alleged A. nigriceps sequence is from an A. trivirgatus. Menezes et al., (2010) used a
maximum likelihood tree from SRY data (nuclear gen) to show three collapsed lineages, one
leading to A. vociferans, a second one leading to A. l. griseimembra and A. l. lemurinus, and a
third one leading to A. trivirgatus, A. nigriceps, A. azarae boliviensis and A. infulatus. Indeed,
some SRY haplotypes differed by only one nucleotide such as the case of A. infulatus when
compared to A. azarae boliviensis and A. nigriceps. The karyotypic similarity between A.
infulatus and A. azarae suggested a close proximity and recent common ancestry, a finding
coincident with their low genetic distance estimates and by the recent time of their
evolutionary divergence (0.53 MYA) following Menezes et al., (2010). Thus, this Southern
lineage agrees quite well with what we detected with the mt COII gene.
Another relevant question is if the Northern and Southern Aotus groups (sensu
Hershkovitz, 1983) are real. Three of our analyses, AMOVA and Structure with
microsatellites and the mitochondrial trees, clearly revealed that one Northern form, A.
trivirgatus, is more related to the Southern group than to the remaining Northern Aotus forms.
For instance, the Structure analysis with microsatellites revealed a strong relationship
between A. trivirgatus and A. a. boliviensis. Similarly, the Southern forms, A. nancymaae and
A. miconax, are not placed together with the rest of the Southern taxa group. In our
mitochondrial analysis, we detected A. nancymaae-A. miconax as the sister clade of all the
Northern Aotus forms, excluding A. trivirgatus. However, Menezes et al., (2010), studying
three mitochondrial genes (COI, COII and Cyt-b) but only with 18 individuals (versus 190
individuals herein reported), detected that the ancestor of A. nancymaae should be in the
origin of all the other Aotus species. Their maximum likelihood and Bayesian reconstructions
used these three mitochondrial genes and showed two sister lineages, one leading to the most
basal offshoot represented by A. nancymaae and another to a clade grouping seven other
Aotus taxa. This clade split into two sister clades, one leading to A. vociferans and the other
one further splitting in A. l. griseimembra and A. l. lemurinus and to a more derived clade (A.
trivirgatus (A. nigriceps (A. infulatus, A. azarae))). Indeed, A. nancymaae has 2n = 54, which
was suggested as the original karyotype in Aotus, such as we previously commented (Ma,
1981a and Galbreath, 1983). However, in contrast to this view, Ruiz-Herrera et al., (2005),
combined G-banding comparisons and molecular cytogenetic techniques, and described the
most likely pattern of chromosome evolution and phylogenetic position of two Aotus
karyomorphs from Venezuela. They indicated that the homologies between these two Aotus
karyomorphs and human chromosomes agree well with a karyotype of 2n = 50 (they
classified this karyomorph as A. sp.; this specimen could be A. trivirgatus or A. brumbacki).
They are closer to the ancestral Platyrrhini karyotype, whereas a specimen of A. nancymaae
(2n = 54) presented a more derived karyotype with respect to the ancestral Platyrrhini
karyotype. It’s intriguing that this last exemplar was present as part of the Venezuelan fauna
because this Aotus taxon is not reported for this country. A clear result is that, although A.
nancymaae could be in the origin of the Northern group, there is a complete reproductive
barrier between this taxon and A. vociferans (2n = 46). Pieczarka et al., (1992) studied the
karyotypes of 11 A. vociferans and 11 A. nancymaae using G- and C-banding and NOR-
staining. Five A. nancymaae and five A. vociferans were sympatric in the Aramaza Island in
Brazil (not in Colombia as cited the authors), three A. nancymaae and one A. vociferans were
sympatric in Yahuma in Peru and three A. nancymaae and one A. vociferans were sympatric
in Terezinha in Brazil. In no case, were signs of hybridization detected showing clear
chromosomal reproductive barriers between both taxa. The real position of A. nancymaae in
the Aotus phylogeny must be carefully studied.
Therefore, with the results obtained here and taking into consideration the previous
karyotype studies, we propose the existence of four superspecies within the Aotus genus:
1. A. vociferans (Spix 1823). We agree with the viewpoint of Ford (1994) and the taxa
named vociferans, brumbacki, lemurinus, jorgehernandezi, zonalis and griseimembra
should be treated as a superspecies (very noteworthy chromosomal differences but
with very limited molecular and morphological differences). This superspecies
should be named A. vociferans because it is the oldest name (brumbacki, Hershkovitz
1983; lemurinus, Geoffroy 1843; jorgehernandezi, Defler and Bueno 2007; zonalis,
Goldman 1914; griseimembra, Elliot 1912). This superspecies, A. vociferans, could
contain six subspecies: A. v. vociferans, A. v. brumbacki, A. v. lemurinus, A. v.
jorgehernandezi, A. v. zonalis and A. v. griseimembra or better yet, this species
contains six karyomorphms (1, 2, 6, 7, 8 and 9) following the nomenclature of De
Boer and Reumer (1978), Reumer and De Boer (1980) and Torres et al., (1998).
Menezes et al., (2010) claimed that A. lemurinus, A. griseimembra and A. vociferans

were valid species, arguing that genetic distance estimates between A. lemurinus and A.
griseimembra were higher than many other interspecific estimates and even higher when
comparing A. vociferans with A. lemurinus and A. griseimembra (Table 4). These findings
argue against the proposition that A. lemurinus and A. griseimembra are junior synonyms of
A. vociferans (Ford, 1994) and are in agreement with Defler and Bueno (2007) indicating that
these taxa are valid species. However, we disagree with this claim for three reasons. First,
within the genetic distances they published, the values between A. lemurinus and A.
griseimembra are among the lowest values between taxa considered “a priori” different
species (around 2% of differences which are values typical of subspecies). Second, they used
many sequences obtained in the GenBank and many of the Aotus sequences in GenBank are
erroneously classified. Third, the number of sequences they used for the Northern Aotus
group is very limited (five individuals). As we demonstrated in Ruiz-García et al., (2016a,
this volume, for Cebus), at the moment that we considerably augment the sample size of the
Northern group (we herein employed 87 Northern Aotus specimens, excluding A. trivirgatus
and the numt sequences), many haplotypes were intermixed and thus reducing the reciprocal
monophylia among the Northern Aotus taxa.
2. A. trivirgatus (Humboldt 1812). We again agree with Ford’s (1994) perspective only
distinguishing two northern species of Aotus, A. vociferans and A. trivirgatus. One
unclear question with this species (if it is a single unique species) is its karyotype
make-up. Menezes et al., (2010) analyzed an A. trivirgatus female (from Barcelos,
left side of the Negro River in the Amazonas State in Brazil) with 2n = 50. Its
chromosome complement contained 12 pairs of biarmed chromosomes varying in
size from large to small and 13 pairs of acrocentric chromosomes varying in size
from medium to small. A Maipures Aotus specimen (frontier between Colombia and
Venezuela) karyotyped by Monsalve (unpublished; see Defler and Bueno, 2007) also
presented 2n = 50 (13 pairs of biarmed chromosomes and 11 pairs of acrocentric
chromosomes). Hershkovitz examined color slides of Maipures specimens and stated
that he believed they were A. trivirgatus and that, therefore, A. trivirgatus had 2n =
50 (see Defler and Bueno, 2007). However, a renowned Colombian primatologist,
Jorge I. Hernández Camacho did not agree with this interpretation, believing that the
Maipures specimen was A. brumbacki or a new species. Defler and Bueno (2007)
were unable to distinguish if these Maipures specimens were A. trivirgatus or A.
brumbacki. Other authors showed other possible karyotypes for A. trivirgatus.
Santos-Mello and Thiago de Mello (1985, 1986) described a karyomorph 2n = 51 for
one male, 2n = 52 for one female and 2n = 51 or 52 for another male, for some Aotus
exemplars collected around Manaus (right side of the Negro River). According to the
authors, this is the true karyotype for A. trivirgatus. However, as the distribution of
the Aotus taxa is not clearly delimited, their identification is questionable. Indeed,
Manaus is located at the confluence of the distribution of A. vociferans, A. nigriceps
and A. trivirgatus. The karyotypes of the work of Santos-Mello and Thiago de Mello
(1986) coincided with that of A. nigriceps. Also, the karyotypes of Menezes et al.,
(2010) and Monsalve (unpublished) coincided with the karyotypes of A. brumbacki
(10-11 pairs of biarmed chromosomes and 13-14 pairs of acrocentric chromosomes
following Torres et al., 1998). Indeed, the question could be more complex. Torres et
al., (1998), citing Chu and Bender (1961) and Descailleux et al., (1990), showed 2n =
50-54 for A. trivirgatus. As we previously commented, Ruiz-Herrera et al., (2005)
reported a Venezuelan specimen with 2n = 54 and classified it as A. nancymaae, but
this taxon is not reported for Venezuela (Bodini and Pérez-Hernández, 1989). Should
this specimen with 2n = 54 represent the original A. trivirgatus, whereas the animals
with 2n = 50 are A. brumbacki and those with 2n = 51m/52f are A. nigriceps? We
don’t know. Thus, determining the sex chromosome system and the real karyotype of
A. trivirgatus might be essential in determining the status of this species. However, at
the molecular (this work) and morphometrics (Ford, 1994) levels, A. trivirgatus
seems to be differentiated. Ford (1994) showed that whorls, crests or tufts and the
head stripes do not unite posteriorly. The dorsum is usually grayish, sometimes with
buffy agouti and with a narrow and strongly contrasting orange middorsal band.
Morphometrically this species is also easily distinguishable from the rest of the
Northern Aotus with a canonical variate from cranial measurements separating A.
trivirgatus completely from the other Northern group.
3. A. miconax (Thomas 1927). This superspecies should be named A. miconax, because
this name is older than A. nancymaae (Hershkovitz 1983). Both Hershkovitz (1983)
and Ford (1994) also suspected that both forms could be conspecific. As we don’t
know the karyotype of miconax, we could define two different taxa as A. miconax
miconax and A. miconax nancymaee.
4. A. azarae (Humboldt 1812). Within this superspecies we could include azarae,
boliviensis, infulatus and nigriceps. This superspecies should be named A. azarae
because it is the oldest name (boliviensis, Elliot 1907; infulatus, Kuhl 1820;
nigriceps, Dollman 1909). The superspecies, A. azarae, could contain four
subspecies: A. a. azarae, A. a. boliviensis, A. a. infulatus and A. a. nigriceps. Or,
even better, this species could contain two or three karyomorphms (4, 5 and possibly
10) following the nomenclature of De Boer and Reumer (1978), Reumer and De
Boer (1980) and Torres et al., (1998). The monophyly of this superspecies was in
agreement with karyologic data showing that they shared the same
X1X1X2X2/X1X2Y sex chromosome system, contrary to other Aotus taxa with an
XX/XY sex chromosome system (Torres et al., 1998). Menezes et al., (2010) claimed
that A. a. boliviensis is a full species because the insertion of one cytosine in position
59 of MT-TS1. This is exclusive to this taxon. This difference as well as the presence
of different SRY haplotypes was claimed by these authors as a fundamental fact to
consider A. infulatus as a different species from A. azarae. However, these very
limited differences don’t necessarily mean that they are different species. They
probably don’t translate into any reproductive isolation mechanism. Thus, we believe
that these four Southern Aotus taxa are a valid superspecies.
A variety of molecular markers should be applied to the Aotus taxa to validate the
phylogenetic inferences we described.
ACKOWLEDGMENTS
Thanks go to the SDA (Secretaria Distrital Ambiental of Bogota DC, Colombia) for the
project entitled “Fortalecimiento del control y prevención del tráfico ilegal de fauna silvestre,
especialmente de Primates, a través de la determinación de zonas sometidas a extracción
ilegal utilizando pruebas de genética molecular de poblaciones,” and to Corpoamazonía
(Leticia-Colombia), which allowed us to obtain the necessary financial resources to carry out
the current study. Additional thanks to P. Escobar-Armel, L. Castellanos-Mora and N.
Lichilín for their respective help in obtaining Aotus samples during the last 18 years. Many
thanks go to the Bolivian and Peruvian Ministry of Environment, to the Dirección General de
Biodiversidad, to the Wildlife Conservation Society and CITES from Bolivia, to the
PRODUCE, Dirección Nacional de Extracción and Procesamiento Pesquero from Peru, the
Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales (INRENA)
and to the Ministerio del Ambiente (permission HJK-9788) in Coca (Ecuador) for their role in
facilitating the obtainment of the collection permits. Special thanks goes to the Colección
Boliviana de Fauna (Dr. Julieta Vargas) in La Paz (Bolivia). We also thank the following
Indian communities for helping to obtain monkey samples: Ticuna, Yucuna, Yaguas, Witoto
and Cocama in the Colombian Amazon, Bora, Ocaina, Shipibo-Comibo, Capanahua,
Angoteros, Orejón, Cocama, Kishuarana and Alama in the Peruvian Amazon, the Movima,
Moxeño, Sirionó, Canichana, Cayubaba and Chacobo in Bolivia and to the Kichwa,
Huaorani, Shuar and Achuar in Ecuador.
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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.

CAN MITOCHONDRIAL DNA, NUCLEAR

Manuel Ruiz-García1,, Adriana Vallejo1, Emily Camargo1,

Keywords: Aotus, skull morphometrics, nuclear DNA microsatellites, mitochondrial genes,

subspecies, A. l. lemurinus and A. l. griseimembra], A. brumbacki, A. trivirgatus and A.

l. griseimembra; the sample of an alleged A. nigriceps of Plautz et al., (2009) is really A.

MATERIAL AND METHODS

We applied different multivariate techniques to analyze relationships among 80 Aotus

Table 1. Cranial, mandible and dental measurements (38) analyzed in 80 skulls

1-Maximum Transversal Width

Molecular and Sample Procedures

We used 12 microsatellite markers (AP40, AP68, AP74, D5S111, D5S117, D6S260,

Mitochondrial Phylogenetics Procedures

Microsatellite Statistical Analyses

Table 3. Genic differentiation at 12 nuclear DNA microsatellites taken simultaneously

Locus Probability Standard Error

Locus Ho HS HT DST DST’ H T’ GST GST’

Figure 11. (Continued).

Figure 12. (Continued).

were enclosed in A. l. griseimembra, nine individuals of other taxa in A. l. zonalis, four

Table 9. Application of the Bottleneck Software to detect possible recent bottleneck

Aotus lemurinus griseimembra

different chromosome numbers (from 2n = 46, 47, 48 of A. vociferans to 2n = 58 of A. l.

Phylogenetics and Systematics of Aotus

paniscus (which is morphologically, karyotypically and molecularly different), are

However, if large samples of A. jorgehernandezi, A. l. lemurinus and A. l. zonalis will be

Menezes et al., (2010) claimed that A. lemurinus, A. griseimembra and A. vociferans

Figueiredo, W. B., Carvalho-Filho, N. M., Schneider, H., Sampaio, I. (1998). Mitochondrial

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