Chapter 11 Alouatta Microsatellite

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. 369-394.
Chapter 11
MICROSATELLITE DNA ANALYSES OF

FOUR ALOUATTA SPECIES (ATELIDAE, PRIMATES):
EVOLUTIONARY MICROSATELLITE DYNAMICS
Manuel Ruiz-García1,*, Pablo Escobar-Armel1,

Marta Mudry2, Marina Ascunce2,
Gustavo Gutierrez-Espeleta3 and Joseph Mark Shostell4
1
Unidad de Genética (Grupo de Genética de Poblaciones-Biología Evolutiva).
Departamento de Biología. Facultad de Ciencias. Pontificia Universidad Javeriana. Cra 7ª
No 43-82. Bogotá DC., Colombia
2
GIBE. Departamento de Biología. Facultad de Ciencias Exactas y Naturales.
Universidad de Buenos Aires. 1428. Buenos Aires, Argentina
3
Escuela de Biología. Universidad de Costa Rica. San José, Costa Rica
4
Department of Math Science and Technology, University of Minnesota Crookston,
Crookston, MN, US
ABSTRACT
We discuss the evolution of nine microsatellite DNA markers in four Alouatta species
(howler monkeys; A. palliata, A. seniculus, A. macconnelli, and A. caraya) as well as
evolutionary population parameters. There are five main findings from this study:
1. Four microsatellite central moments (mean, variance, skewness, and kurtosis)

exhibited different distributions among species, with A. palliata being the most
differentiated. This may suggest that central moments provide important
phylogenetic signals.
*
Corresponding author: E-mail-mruizgar@yahoo.es, mruiz@javeriana.edu.co.
2 Manuel Ruiz-García, Pablo Escobar-Armel, Marta Mudry et al.
2. The “As is” bayesian method had the best percentage of classification (86.15%) of
the assignment analyses. A. seniculus was the most incorrectly classified of all
Alouatta species, which may suggest that some of their multigenotypes are original
and that this species may be in the origin of other Alouatta species.
3. A. seniculus and A. caraya showed the highest and lowest effective numbers
respectively.
4. Only 9% of the detected microsatellite mutations were multi-step and, therefore, the
majority of the mutations were of uni-step origin (91%). D5S117 was the unique
microsatellite that clearly showed a multi-step mutation model. Different
microsatellites frequently had different mutation rates per generation. An average
estimate value of 7 x 10-5 did not support Quaternary refuges as sufficiently
important for the molecular evolution of Alouatta.
5. All but two of the microsatellites (D14S51, diversifying selection and D8S165,
constrictive selection) behaved neutrally.
Keywords: Alouatta, central and south America, DNA microsatellites, molecular evolution,
mutation models, natural selection
INTRODUCTION
The Alouatta genus (howler monkeys) has the widest distribution of all Neotropical
primates with a distribution that extends from Southern Veracruz (Mexico) to Northern
Argentina. This genus, along with Cebus and Saimiri (Ayres 1986) consists of generalist
species that can occupy poor quality forest habitats and thus permits them to inhabit a wide
range of environments (Eisenberg, 1979). Alouatta is the first genus to colonize new islands
in the Amazon Basin followed by other genera such as Cebus and Saimiri. In addition, they
can live at sea level to elevations greater than 3,300 meters above sea level in the Andes.
Together with the genera Ateles, Lagothrix and Brachyteles, they are the largest Neotropical
primates (Milton, 1982). We herein studied four Alouatta species (A. palliata, A. seniculus, A.
macconnelli and A. caraya). A. palliata ( = villosa), the mantled howler, has a distribution
that extends from southern and eastern Mexico, southern Guatemala and south through the
remainder of Central America to the Pacific area of Colombia, Ecuador and Northern Perú.
We studied animals from México, Costa Rica and Colombia. A. seniculus, the red howler, has
a geographic distribution that extends from Northern Colombia crossing the majority of
Northern South America including north of the Amazon River in the east and extending south
into Northern Bolivia in the west. Although many different subspecies of A. seniculus have
been defined (among others, A. s. insulanus, A. s. stramineus, A. s. amazonica, A. s. juara, A.
s. puruensis, A. s. seniculus, see Rylands et al., 1997), all the samples we analyzed came from
Colombia, where “a priori” only the A. s. seniculus subspecies lives. A. macconnelli is
distributed in Surinam, Guyana, French Guiana and the northern-eastern Amazonian area of
Brazil. Although traditionally, A. macconnelli has been considered a subspecies of A.
seniculus, the microsatellite differences we observed (shown elsewhere), together with the
nucleotide sequence differences reported by Cortés-Ortiz et al., (2003) and the karyotypic
differences previously found by Lima and Seuánez (1991) and Vassart et al. (1996), in our
opinion, support the existence of a real and differentiated species. For example, while A.
seniculus has a diploid chromosome number of 44 (males) and 45 (females), including 4
Microsatellite DNA Analyses of Four Alouatta Species (Atelidae, Primates) 3
microchromosomes, A. macconnelli presents a diploid chromosome number of 47, 48 and 49.

In fact, de Oliveira et al. (2002) revealed that howlers represent the genus with the most
extensive karyotype diversity within Platyrrhini so far analyzed with high levels of
intraspecific chromosomal variability. We also studied A. caraya (black howler) which has a
habitat that includes Southern Brazil through Eastern Bolivia, Paraguay and Northern
Argentina. The samples we analyzed were from Bolivia and Northern Argentina.
All four of the species we analyzed were found in essentially allopatric ranges, with the
exception of a small area in Northwestern Colombia (Sinú Valley), where both A. palliata and
A. seniculus are found in sympatry (Hernández-Camacho and Cooper, 1976). The analyses of
DNA G6PD gene sequences by Von Dornum and Ruvolo (1999) supported that the split
between Alouatta and the Atelini (Ateles, Lagothrix, Brachyteles) occurred about 15.1 million
of years ago (MYA).
The development over the last few years of molecular procedures based upon the advent
of the polymerase chain reaction (PCR), has enabled population geneticists to analyze and
determine the genetic structure and the levels of genetic variability of many wild species from
small tissue fragments (blood, bones, epithelial). Among the most remarkable molecular
markers for these tasks are the STRPs (Short Tandem Repeat Polymorphisms, microsatellites)
(Weber and May, 1989). These kinds of markers are composed of short sequences of
nucleotides (one to six nucleotide base pairs) repeated in tandem arrays. These markers are
frequently inside the eukaryotic genomes, are randomly distributed, and are highly
polymorphic. Additionally, one determinant property of these markers is that the DNA
amount needed to carry out these molecular analyses is very small, which permit the
investigator to use non-invasive procedures to sample wild animals and to successfully
examine population biology dynamics on a molecular genetic level (Bruford and Wayne,
1993).
The main aims of the present work are as follows: 1- To determine the central moments
of nine STRPs studied in four Alouatta species and to investigate their degree of similarity
among the species analyzed. Diverse central moments could have some phylogenetic signal
information. 2- To investigate the degree of microsatellite divergence among the four species
of Alouatta studied and to determine possible original multigenotypes for these species. 3- To
determine effective and total numbers of these species by means of maximum likelihood
estimates using the  (= 4Ne) parameter. 4- To determine the percentages of uni-step and
multi-step mutations affecting the evolution of the microsatellites analyzed. 5- To determine
an approximate global value of  (mutation rate per generation) for the set of microsatellites
employed and to provide insights that support the existence of different mutation rates for
each of the microsatellites studied. 6- To determine if there is evidence of natural selection
that affects some of the markers using the Beaumont and Nichols (1996) method.
MATERIALS AND METHODS

Samples and Molecular Procedures
Twenty (20) Alouatta caraya individuals were caught in Isla Brasilera at the Argentinean
Chaco and in Southeastern Bolivia. Eighty four (84) A. seniculus individuals were sampled
from Colombia in the Departments of La Guajira, Meta, Antioquia, Chocó, Amazonas,
Vichada, Putumayo, Magdalena and Atlántico. A total of forty eight (48) A. palliata
individuals were sampled. Two samples were from México (A. palliata mexicana; States of
Costuxtla and Tabasco), three samples were from the Colombian Chocó (A. palliata
aequatorialis) and the remaining samples were from Costa Rica (A. palliata palliata). Seven
(7) A. macconnelli samples were obtained in French Guiana at the Carnopi River. In total, 159
Alouatta samples were collected and analyzed (Table 1).
Table 1. Sample sizes, morphological subspecies, countries, specific geographic origins,

type of samples and sources of the 159 Alouatta samples from four different species (A.
seniculus, A. palliata, A. caraya and A. macconnellii) studied herein
Subspecies and Geographic origin, type of samples and

Species N
Origin sources
Alouatta 84 A. s. seniculus La Guajira (20 samples, all blood drop),
seniculus COLOMBIA Meta (7 samples, blood, hair and teeth),
Antioquia (16 samples, all blood) Chocó (7
samples, blood and teeth), Amazonas (17
samples, blood, hair, teeth and bones),
Vichada (3 samples, teeth), Putumayo (3
samples, teeth and bones), Magdalena (2
samples, teeth), Atlántico (2 samples, teeth),
Caquetá (2 samples, hair and teeth), Arauca
(1 sample, teeth), Bolivar (4 samples teeth
and bones).
M. Ruiz-García, P. Escobar-Armel,
D. Alvarez, Indian tribes across all
Colombia, F. Nassar, J. Gardeazábal,
L. M. Borrero, D. M. Ramírez,
Instituto Von Humboldt (4 skin samples).
Alouatta 48 A. p. palliata Across all Costa Rica (43 samples, blood
palliata COSTA RICA and hairs).
G. Gutierrez-Espeleta, M. Ruiz-García
D. Alvarez, P. Escobar-Armel
A. p. mexicana Costuxtla (1 sample, DNA), Tabasco (1
MEXICO sample, DNA).
L. Cortés-Ortiz
A. p. aequatorialis Chocó (3 samples, teeth and bones).
COLOMBIA M. Ruiz-García
Alouatta 20 A.caraya Isla Brasilera and other three geographical
caraya ARGENTINA points, Argentinan Chaco (14 samples, blood
A.caraya drops).
BOLIVIA M. Mudry, M. Ascunce
Santa Cruz Department, and diverse
localities at the Mamoré River (6 samples,
hairs).
M. Ruiz-García, D. Alvarez
Alouatta 7 A. macconnellii Carnopi River (4 samples of muscle tissue

macconnelli FRENCH and 3 samples of hairs from three localities).
GUIANA F. Catzeflis, M. Ruiz-García.
Table 2. Microsatellites, length size in base pairs and forward

and reverse primer sequences of the nine DNA microsatellite markers
applied to four Alouatta species
Length
Microsatelite Forward Primer Reverse Primer
size
AP40 168 5'-CCACGGTGGCA 5'-AGAGGCACGAA
GAGGAGATTT-3' GACAAGGACA-3'
AP68 190 5'-TGTTGGTATAAT 5'-ACATACACCTT
CTTTCCTA-3' TGAGTTTCT-3'
AP74 154 5'-TGCACCTCATC 5'-CATCTTTGTTT
TCTTTCTCTG-3' TCCTCATAGC-3'
D5S111 165 5’-GGCATCATTTT 5’-ACATTTGTTC
AGAAGGAAAT-3’ AGGACCAAAG-3’
D5S117 157 5’-TGTCTCCTGAGA 5’-TAATATCCAAA
ATAG-3’ CCACAAAGGT-3’
D6S260 170 5’-TTTTCACTATCA 5’-TTCATTTTCAGC
ATGGCAGC-3’ AGCAATTT-3’
D8S165 142 5’-ACAAGAGCACA 5’-AGCTTCATTTT
TTTAGTCAG-3’ TCCCTCTAG-3’
D14S51 175 5’-GATTCTGCACCC 5’-ATGCTCAATGA
CTAAATCC-3” ACAGCCTGA-3’
D17S804 181 5’-GCCTGTGCTGC 5’-CACTGTGATG
TGATAACC-3’ AGATGTCATTCC-3’
DNA, from blood, hair (with roots), skin, bones and teeth samples, was extracted using
the phenol-chloroform procedure (Sambrock et al., 1989). The Chelex 10% method (Walsh et
al., 1991) was also used to extract DNA from blood droplets and hairs. Nine microsatellites
molecular markers (AP40, AP68, AP74, D5S111, D5S117, D6S260, D8S165, D14S51 and
D17S804) were used in the current study (Table 2). AP68 did not amplify in A. caraya
probably due to the poor quality of the DNA used. The final PCR volume of the STRPs for
DNA extracted from blood, skin, bone, and teeth was 25 l (3 l of MgCl2 3 mM, 2.5 l of
Buffer 10x, 1 l of dNTPs 1 mM, 10 pmol of forward and reverse primers, 13.5 l of H2O, 2
l of DNA (50-100 ng per l), and one Taq Polymerase unit. For the DNA extracted from
hair and blood (droplets), the overall volume of the PCR reactions was 50 l, with 20 l of
DNA and twofold amounts of MgCl2, Buffer, dNTPs, primers and Taq Polymerase. The PCR
reactions were carried out in a Geneamp PCR System 9600 Perkin Elmer thermocycler. The
temperatures used were as follows: 95°C for 5 minutes, 30 cycles of 1 minute at 95°C, 1
minute at the most accurate annealing temperature (57°C for AP40, 50°C for AP68 and 52°C
for the remaining markers), one minute at 72°C, and 5 minutes at 72°C. The amplification
products were kept at 4°C until used. The PCR amplification products were run in denaturant
6% polyacrilamide gels within a Hoefer SQ3 sequencer vertical chamber. Gels migrated for
2-3 hours depending on marker sizes, and were then stained with AgNO3 (silver nitrate).
Every sixth line in the gel contained molecular markers (174 cut with Hind III and Hinf I).
The PCR reactions were repeated three times for DNA extracted from hairs, teeth and bones
in order to confirm the genotypes obtained from these tissues. Therefore, allelic dropout was
highly improbable. The existence of null alleles cannot be totally excluded, which could
increase the number of false homozygous genotypes. Nevertheless, it is improbable that all
loci were affected in the same way.
Population Genetics Analyses
Microsatellite Central Moments

The central moments of the microsatellite distributions were calculated in order to
analyze interspecific and intraspecific distribution dynamics. We used the analytical
equations for moments up to the fourth order within a locus and the between-locus variance at
mutation-drift equilibrium (Zhivotovsky and Feldman, 1995). The main power of these
statistics is to test the effectiveness of the one-step mutation model as well as to detect
between-locus variation in the mutation rate per generation. A description of the analytical
expressions used to accomplish this follows. The total mutation rate for the one and two-step
stepwise models is w = c vc c2 = v 2m, (v = c different to 0 vc; c = j – i, the change in repeat
number due to mutation of the allele carrying j repeats to the allele with i repeats; vc is the
probability of a mutational change by c with the repeat number not depended on the mutated
allele; 2m is the variance of changes in repeats among the new mutations). Similarly, the first
four central sample moments of the allele frequency distributions were calculated as r = i i pi
(mean of repeat tandems), V = i pi (i – r)2 (variance of the repeat tandems), S = i pi (i – r)3
(skewness of repeat tandems), K = i pi (i – r)4 (kurtosis of repeat tandems), where pi is the
frequency of allele Ai which carries i repeats. Those microsatellites affected by negative
selection, or mutation constriction, will show statistical values that are clearly differentiated
compared to microsatellites with more neutral dynamics.
The GENECLASS program (Cornuet et al., 1999) was used to determine the capacity of
the microsatellite markers to differentiate Alouatta species. This method of analysis could
also be indirectly useful to determine the degree of molecular evolution (rates of evolution
and selective constrictions) of microsatellites. The frequency (Paetkau et al., 1995), Bayesian
(Rannala and Mountain, 1997) and genetic distance-based methods were applied. The
distance-based methods assign individuals to the “closest” species and requires the defining
of a distance between the individuals and the species considered. For this, the following
genetic distances were employed: Nei (1978), Cavalli-Sforza and Edwards (1967), DAS
(shared allele distance; Chakraborty and Jin, 1993) and the 2 genetic distance (Goldstein et
al., 1995). Distance methods have an advantage over other methods because they work
without meeting the Hardy-Weinberg equilibrium and linkage equilibrium assumptions. The
“leave one out” and “as is” statistical methods were applied to the above assigning analysis.
Exclusion methods without any prior information were also employed (Cornuet et al., 1999).
For each one of the Alouatta species studied, the historical effective number was
calculated by using a maximum likelihood procedure with a Markov chain recursion method
(Nielsen, 1997), to estimate the probable  (= 4Ne) values, where Ne is the historical
effective number of the species studied and  is the mutation rate per generation. Once the 
value is known, Ne can be obtained which indicates the historical reproductive population
sizes of the four Alouatta species. Estimations of mutation values in humans, pigs, and rats
are 5.6 x 10-4 (Weber and Wong, 1993), 7 x 10-5 (Ellegren, 1995) and 1.5 x 10-4 (Serikawa,
1992) respectively. Therefore, to obtain a wide range of feasibly effective numbers in the
Alouatta species analyzed, the mutation rates employed in this work ranged from 5.6 x 10-4 to
7 x 10-5. The Nielsen (1997)’s model is based on the likelihood function of , calculated as
L() = P(), where  is a vector with the observed data in the obtained samples. A one-
step mutation model typical of the microsatellites was adopted. Nielsen’s model is also based
on the recursivity of the coalescence theory to obtain the likelihood functions of  for samples
of a determined size. The coalescence time between two alleles is exponentially distributed
with a mean equal to 1 and the conditional number of mutations in each lineage follows a
Poisson distribution with a mean of t/2. It is feasible to calculate the probabilities to observe
an allele sample determined in the previous generations by means of recursion considering
the allele genealogies of the sample and summing up all the possible previous states in the
time. This result is obtained by conditioning the last event that occurs prior to the present, by
mutation or coalescence and following a symmetric random walk with k-allele states
reflecting some types of barriers. The probability q () of the sample is determined by the
addition of all the possible states that are previous to the probability of being in a determined
state, multiplied by the transition probability of these states to the current state. With the
chosen mutation model (uni-step), this probability is
q () = (n +  - 1))  (ni + 1/(n)) 1/2 q ( + i – j) + (n – 1)/(n +  -1)  (nj – 1)/(n – 1)
q ( - j),
where i is a unity vector which adds values equal to 1 to the entry of i in . This recursive
procedure is easy to obtain if we have the capacity to obtain the values of ( /(n +  - 1)),
which is the probability that the last event before the present moment is a mutation and that a
mutational or a coalescence event of (n-1)/(n +  -1) had previously occurred. This event is
the probability of a coalescence event when considering that a previous mutation or
coalescence of (ni – 1)/(n –1) occurred. The equation ((ni – 1)/(n –1)) represents the
probability that a mutation occurs in an i allele, given that a mutation of (nj – 1)/(n –1)
occurred. This is the probability that two alleles belonging to the j state will be coalescent,
given that a coalescence event occurred. There is a 0.50 probability that a mutation occurs
from an i state to a j state given that a mutation in the i state occurred. We calculated q()
with the Monte Carlo method after an evaluation of the likelihood functions (Griffiths and
Tavaré, 1994, based on the recursion of the expression) were evaluated. The likelihood
surfaces for  were estimated with the MISAT program (Nielsen, 1997) and the 5 %
confidence interval was calculated by multiplying the log likelihood of the maximum
likelihood value by 2. We used a grid size of 40, a previously calculated  (calculated with
the method of the moments, 0) and a mutation one-step model with a 1,000,000 Markov
chains. The estimate of the  maximum likelihood (the  value with the least negative log
likelihood) was used to estimate Ne for each Alouatta species studied.
In addition, the greatest possible multi-step mutation percentages (ranging from 0 to 0.5)
were calculated through the maximum likelihood of  by means of 3,000,000 Markov chains.
We analyzed the different possible mutation rates affecting each one of the microsatellites for
each Alouatta species studied. For this, we started from the hypothesis 1 = 2 =  (the values
of  for two different microsatellites) and we tested it by a likelihood ratio test with the
expression -2 log [L1(L2()]/[L(,2)], which follows a 2 with one degree of freedom. A
probability lower than  = 0.05 indicates that both microsatellites have different mutation
rates. Likewise, we measured if the estimated multi-step mutation models were significantly
better than the uni-step mutation models within each Alouatta species. We applied the
likelihood ratio of -2 log [L(, p = 0)/L(, p)], which for large samples is similar to 2 with
one degree of freedom under the null hypothesis that p = 0, to the obtained maximum
likelihood multi-step p percentage. A probability lower than  = 0.05 indicates that the multi-
step mutation percentage is significantly different from the uni-step mutation model and this
last model is rejected.
Lastly, the coalescence theory generated by Beaumont and Nichols (1996) was used to
detect whether DNA-microsatellites were effected by constrictive or diversifying natural
selection within the Alouatta 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 and the step-wise 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 conformed by the median and the quartiles. Values that are outside of this
theoretical distribution obtained indicate that the microsatellites in question are being affected
by natural selection.
RESULTS
The central moments of the microsatellite distributions for the four Alouatta species
depicted dissimilar intraspecific trends among different microsatellites as well as interspecific
differences among the same markers (Table 3). For example, within A. caraya, we can
observe that all microsatellites had a moment of order 1 (mean, r) and were relatively similar
with the exception of AP40, which had an r-value lower than the remaining markers. The
variances (moment of order 2, V) were relatively similar for AP40, AP74 and D5S111,
considerably higher for D8S165 (11 times higher) and considerably lower for D14S51 (6
times lower). The asymmetry coefficient (moment of order 3, S) was small (therefore there is
symmetry) for all microsatellites studied with the exception of D8S165 which yielded clear
evidence of positive asymmetry in its distribution. The kurtosis coefficient (moment of order
4, K) showed a platykurtic distribution for this species for AP40, AP74, D5S111 and D14S51,
while D5S117 and, especially, D8S165 showed a well developed leptokurtic trend. Therefore,
it seems probable that the microsatellites studied in A. caraya have different mutation rates
and different selective constrictions. No microsatellite studied in this species showed a
perfectly normal distribution, which would be expected if no mutation or selective
constrictions were present. The comparative analysis of A. seniculus revealed that the
dynamics of some microsatellites were different to that determined in the first species. For
example, in A. caraya, AP40 showed the lowest value of r (also present in A. macconelli),
lower than in A. seniculus. All the other microsatellites in A. seniculus yielded similar r
values. The r value of DS5111 was seven times higher in A. caraya compared to A. seniculus.
The markers AP40, AP68, AP74 and D5S111 showed the lowest variance (V) values for A.
seniculus, whereas D6S260 and especially D5S117 yielded more pronounced variances.
D5S117’s variance in A. seniculus was 24 times greater than in A. caraya, showing a clear
difference in mutation rate or selective constriction for both species. A conspicuous
difference in the variance of allele repeats between these two species was recorded for
D14S51, which denotes the possibility of a different selective constraint in both species. The
allele distribution for AP40, AP68, AP74 and D5S111 in A. seniculus was nearly symmetric
whereas D14S51 and mainly D5S117 had a negative asymmetric trend. In contrast, D8S165
and D17S804 manifested a clear positive asymmetric trend. Only two microsatellites (AP40
and AP68) showed platykurtic distributions in A. seniculus. All other markers in this species
showed noteworthy leptokurtic levels, especially D5S117, D6S260, D17S804 and D14S51.
These levels of leptokurtic distributions are noticeably greater in this species than in A.
caraya. The r values for A. macconnelli were more heterogeneous than for the other two
species commented probably as a consequence of the smaller sample size used for this taxon.
Nonetheless, as it was previously said, AP40 showed the same dynamics in this species as in
the two previous ones and was the marker with the lowest r value. D5S117 showed a clear
lower r value in A. macconnelli than in A. caraya and in A. seniculus (a reduction of five and
four times respectively). Similar to a trend observed in A. seniculus (but not in A. caraya) the
V value for AP40 was exceedingly low relative to the V values reported for the other
microsatellites. Nonetheless, the V values for several markers such as AP74 and D5S111
(variances clearly higher in A. macconnelli than in A. caraya and in A. seniculus although the
sample size of the first species was smaller compared to the others), D5S117 and D6S260
revealed marked differences between the three Alouatta species. No symmetrical distributions
were determined in A. macconnelli (with the exception of AP40) and this species yielded
relevant differences from the other Alouatta species studied. AP68, AP74 and D5S111
showed negative asymmetric distributions, while D8S165, D17S804 and, especially, D5S117
and D6S260 presented conspicuously positive asymmetries. For example, D6S260 showed
astonishing asymmetric differences between A. macconnelli and A. seniculus. In A.
macconnelli, only AP40 showed a platykurtic distribution whereas all the other microsatellites
were clearly leptokurtic. Some markers, such as AP74, D5S111, D5S117 and D6S260
(especially the two last), were extremely leptokurtic, which denotes possible selective or
mutation constrictions in this species as well. No other Alouatta species presented such
extreme leptokurtic values, with the exception of A. seniculus for D17S804 and especially
D5S117. A. palliata yielded values of r relatively similar to those observed in the other three
Alouatta species. The two loci which showed the greatest heterogeneity of r among the four
Alouatta species studied were D5S111 and D5S117. AP68 showed the highest V value in A.
palliata while D5S111, D6S260 and D17S804 yielded the smallest variances of all species
studied. D14S51 and D17S804 practically presented symmetric distributions, which is in
contrast to that found in the other species with the exception of D14S51 in A. caraya. AP68,
AP74 and D5S117 showed negative asymmetry, whereas D5S111 and D6S260 presented
positive asymmetries. D5S117 and D6S260 markers yielded the greatest heterogeneity for S
among the four Alouatta species studied. D5S111 was the only marker that had a normal
distribution for A. palliata. AP68 showed the highest leptokurtic distribution in A. palliata
among the four species analyzed. The only two microsatellites presenting platykurtic
distributions were D14S51 and D17S804 in contrast with the findings determined for the
other Alouatta species with the exception of D14S51 in A. caraya. Therefore, this provides
more evidence that supports different microsatellite dynamics for the same markers in
different Alouatta species.
Table 3. Four central moments of DNA microsatellites studied in four Alouatta species
A.seniculus r v s k
AP40 2.000 0.560 0.240 2.000
AP68 20.706 0.706 0.106 1.042
AP74 24.064 1.336 0.217 5.341
D5S111 23.293 1.678 1.613 7.463
D5S117 12.206 40.320 -276.001 4212.200
D6S260 30.100 17.090 -9.828 550.406
D8S165 18.960 2.746 4.125 28.940
D14S51 18.946 5.681 -17.938 112.621
D17S804 18.812 5.277 24.162 177.570
A.caraya
AP40 1.750 0.437 -0.656 1.176
AP74 23.750 0.437 -0.656 1.176
D5S111 24.334 0.888 -0.583 1.186
D5S117 15.830 1.693 -0.965 8.102
D8S165 20.600 4.940 4.692 51.879
D14S51 20.070 0.066 0.056 0.053
A.palliata
AP68 15.613 6.698 -3.174 127.903
AP74 18.812 2.277 -3.025 16.812
D5S111 21.364 0.698 1.352 3.483
D5S117 16.094 8.460 -3.036 566.411
D6S260 29.660 6.053 11.867 72.065
D14S51 23.750 0.312 -0.375 0.629
D17S804 17.315 0.554 0.768 1.346
A.macconnelli
AP40 1.5 0.25 0 0.062
AP68 17.7 4.61 -5.124 52.453
AP74 16.75 8.187 -3.093 86.082
D5S111 28.3 8.01 -1.536 153.210
D5S117 3.1 20.09 172.872 2078.5957
D6S260 35.3228 124.913 3355.5088 91199.854
D8S165 23.100 1.999 2.000 6.000
D17S804 17.75 4.437 6.093 2078.596
r = Mean of repeat tandems; v = Variance of the repeat tandems; S = skewness of repeat tandems; K =
kurtosis of repeat tandems
Table 4 shows the assignment analyses for all the markers within the 65 Alouatta
individuals with complete multi-genotypes (nearly 41% of the animals studied). The Bayesian
procedure with the “As is” technique (86.15%) yielded the best assignment percentages.
Other methods which offered elevated percentages of correct assignment included the DA “as
is” (84.62%) and Cavalli “as is” (84.62%) (Cavalli-Sforza and Edwards, 1967). The worst
results (lowest percentages) were obtained by means of the standard and minimum Nei’s
genetic distance with the “leave-one-out” technique (73.85%) and the Cavalli-Sforza &
Edwards’s genetic distance with the “leave one out” method (72.31%). We provide comments
about the results of two methods. The Bayesian procedure with “as is” incorrectly classified 9
out of 65 animals: one A. caraya was classified as A. seniculus, four A. seniculus were
assigned as A. caraya, two as A. macconnelli and two as A. palliata. The DA genetic distance
with “as is” incorrectly assigned 10 individuals out of 65 studied: one A. caraya like A.
seniculus, three A. seniculus as A. caraya, four as A. palliata and two as A. macconnelli.
Therefore, A. seniculus had many characteristics that were also dispersed in the other species.
Neither A. macconnelli nor A. palliata were incorrectly classified, which complements the
fact that both species have peculiar gene pools well differentiated from the other Alouatta
species. Conversely, some A. seniculus and A. caraya were similar enough to be misidentified
in all analyses. This means that both species are relatively similar from a genetic standpoint at
least in consideration of the microsatellites used in this study.
Table 4. Population assignment results for the four Alouatta species studied (A. palliata,
A. seniculus, A. macconnelli and A.caraya) using nine DNA Microsatellite markers
Method procedure % of correct assignment

Bayesian – “leave one out” 75.38
Bayesian – “as is” 86.15
Nei – “leave one out” 73.85
Nei – “as is” 81.54
Nei minimun – “leave one out” 73.85
Nei minimun – “as is” 81.54
DA – “leave one out” 80.00
DA – “as is” 84.62
Cavalli – “leave one out” 72.31
Cavalli – “as is” 84.62
Table 5. Effective number estimates by using uni-step and a multi-step mutation models
by means of two extreme mutation rates per generation (5.6 x 10-4 and 7 x 10-5)
throughout maximum likelihood estimates of (= 4Ne). Historical total population
sizes were calculated by means of the Ne/N ratio = 0.4. XA =Average means. Xh =
harmonic means
Effective numbers Total numbers

Uni-step model Ne/N = 0.4
Mutation rates Mutation rates
5.6  10-4 7  10-5 5.6  10-4 7  10-5
Alouatta palliata
= 8.811 ± 5.772 XA = 3,934 31,470 9,835 78,675
Xh = 1,060 8,482 2,650 21,205
Alouatta seniculus
= 19.803 ± 12.662 XA = 8,841 70,726 22,103 176,815

Xh = 2,342 18,740 5,855 46,850
Table 5. (Continued)
Effective numbers Total numbers

Uni-step model Ne/N = 0.4
Mutation rates Mutation rates
5.6  10-4 7  10-5 5.6  10-4 7  10-5
Alouatta macconnelli
= 16.121 ± 6.983 XA = 7,197 57,574 17,993 143,935
Xh = 2,114 16,907 5,285 42,267
Alouatta caraya
= 5.268 ± 4.631 XA = 2,352 18,818 5,880 47,045
Xh = 395 3,163 988 7,908
Multi-step model
5.6  10-4 7  10-5 5.6  10-4 7  10-5
Alouatta palliata
= 7.063 ± 3.556 XA = 3,153 25,227 7,883 63,068
Xh = 954 7,640 2,385 19,100
Alouatta seniculus
= 15.872 ± 11.334 XA = 7,086 56,687 17,715 141,717
Xh = 2,118 16,941 5,295 42,352
= 16.218 ± 7.537 XA = 7,241 57,925 18,102 144,812
Xh = 1,537 16,941 3,843 30,730
Alouatta caraya
= 4.551 ± 3.882 XA = 2,032 16,254 5,080 40,635
Xh = 285 2,280 712 5,700
Table 5 shows effective number estimates by using uni-step and a multi-step mutation
models by means of two extreme mutation rates per generation (5.6 x 10-4 and 7 x 10-5)
throughout maximum likelihood estimates of (= 4Ne). Historical total population sizes
were calculated by means of the Ne/N ratio = 0.4.
A. seniculus presented the highest  and Ne values (8,841 and 70,726, for the one-step
mutation model, arithmetic mean and depending on the selected mutation rate). A.
macconnelli showed the second highest effective numbers (7,197 and 57,574). Considerably
smaller effective numbers occurred in A. palliata and A. caraya. The first species showed a
mean Ne range of 3,934-31,470. The historical effective sizes of A. seniculus and A.
macconnelli were approximately between 1.8 and 2.2 times higher than that of A. palliata.
Indisputably, A. caraya had the lowest historically effective size and total numbers (2,352 and
18,818, respectively). The Ne for A. caraya was 3.76 times lower than for A. seniculus and
3.01 times lower than for A. macconnelli. Therefore, A. caraya and A. seniculus seem to have
the (historically) smallest and largest of the Alouatta populations studied respectively.
Although the average multi-step mutation percentages were different for each Alouatta
species studied, we detected no significant difference among the averages. A. caraya (p =
0.158) presented the highest multi-step mutation percentage and A. palliata presented the
lowest (p = 0.039). Both, A. seniculus and A. macconnelli, yielded very similar multi-step
mutation percentages (0.087 and 0.066, respectively). This mean percentage oscillated for the
genus Alouatta from p = 0.0879 to p = 0.0841 depending as the average was calculated.
Therefore, on average, 91-92 % of the microsatellite mutations studied were uni-step and only
8-9 % were multi-step. The Ne values were extremely similar when they were calculated with
either the  multi-step or uni-step mutation models. For example, when only the 5.6 x 10-4
mutation rate is considered, the values for the four Alouatta species were 3,153 vs. 3,934 (A.
palliata), 7,086 vs 8,841 (A. seniculus), 7,242 vs. 7,197 (A. macconnelli) and 2,032 vs. 2,352
(A. caraya). Therefore, there is no obvious bias when effective numbers are estimated with a
uni-step mutation model instead of the more accurate multi-step mutation model, at least not
in the Alouatta of this study. The historical total number of individuals in each species
generated with the uni-step model oscillated from 4,711 to 44,255 (A. palliata), 10,409 to
99,459 (A. seniculus), 9,395 to 80,964 (A. macconnelli) and 1,757 to 26,462 (A. caraya).
These values were only slightly different from those generated with the multi-step model. As
expected, the harmonic averages calculated from all the obtained estimates for each species
showed a trend in favor of the smallest values. Table 6 displays the significant multi-step
mutation percentages in regard to the uni-step model. Nonetheless, very few cases of multi-
step percentages were significant, with one in A. palliata (D5S117: 7.5%, 2 = 3.427, P <
0.05), two in A. seniculus, (D5S117: 10%, 2= 4.208, P < 0.05 and D14S51: 32.5%, 2 =
7.802, P < 0.01), one in A. macconnelli (D5S117: 20%, 2 = 6.91, P < 0.01) and none in A.
caraya. Even though, A. seniculus showed the highest significant multi-step percentage (2/9 =
22.22%, 2 = 1.135, 1 df, NS), no percentage was significantly greater than the type I error of
5%. Consequently, the overall trends do not favor a generalized multi-step mutation model
over a uni-step mutation model in the Alouatta species studied. Only D5S117 presented a
noteworthy trend in support of a multi-step mutation model inside of this genus. However,
there is incontrovertible evidence in favor of generalized differences in the mutation rates at
the microsatellites analyzed within each one of the four species studied. The percentages of
significantly different mutation rate pairs were 86.66% (A. caraya, for both uni-step and
multi-step models), 80.85% (A. palliata for both models), 82.14-78.57% (A. macconnelli for
both models, respectively) and 77.77-75% (A. seniculus, for both models, respectively). There
was no significant difference in percentage pairs between A. caraya and A. seniculus (2 =
2.257, 1 df, NS). Additionally, all of these percentages were significantly greater than the 5%
type I error. This means that the four species have similar percentage of mutation rate
differences among the microsatellite pairs studied and that these different mutation rates are
systematic and not due to chance. As the majority of comparison pairs were significant, we
have focused our comments on non-significant pairs. For A. caraya, only the D5S111-
D14S51 and the AP40-AP74 pairs did not have significantly different mutation rates
(considering both uni-step and multi-step models). For A. palliata, the pairs AP74-D6S260,
D5S111-AP74, D6S260-D5S111, and D14S51-D17S804 showed similar mutation rates. In A.
macconnelli, the microsatellite pairs with non significant mutation rates were D5S111-AP68,
D5S111-D17S804 (uni-step model only), AP68-D17S804, AP74-D6S260, AP74-D8S165 and
D8S165-D6S260; while in A. seniculus, these pairs were D5S111-D17S804, D5S111-AP40,
D5S111-AP74, D14S51-D8S165 (uni-step model only), D17S804-D8S165, D17S804-AP74,

D8S165-D6S260, AP40-AP74, and AP74-D6S260. Only a few pairs were repeated among
several species combinations: AP74-D6S260 (A. palliata, A.macconnelli and A. seniculus),
D5S111-AP74 (A. palliata and A. seniculus) and D5S111-D17S804 and D8S165-D6S260 (A.
macconnelli and A. seniculus). This could mean that these microsatellites have similar
mutation rates for these groups of species and that A. macconnelli and A. seniculus were the
species with the greatest number of microsatellites suffering similar mutation rates. These
analyses could also provide an interesting phylogenetic signal. Nevertheless, in general the
majority of the microsatellites studied inside each species have significantly different
mutation rates. Furthermore, with the exceptions aforementioned, the same microsatellites
have different mutation rates in each species analyzed. For instance, for A. palliata the order
of mutation rates was (from higher to lower): D5S117 > AP68 > (AP74 = D5S111 = D6S260)
> (D14S51 = D17S804), meanwhile in A. seniculus this order was D5S117 > (D6S260 =
D17S804 = D14S51 = D8S165) > (AP74 = D5S111 = AP40) > AP68. D5S117 provided the
only similarity of mutation rates among the four Alouatta species studied. This was always
the microsatellite with the highest mutation rate per generation in all four species.
The last analysis carried out tried to detect possible natural selection affecting the nine
DNA microsatellites studied. Both mutation models (infinite allele and step-wise) offered the
same results with two out of nine microsatellites showing evidence of natural selection within
the Alouatta genus. Diversifying selection was detected at D14S51 and constrictive selection
was detected at D8S165. The remaining microsatellites were within the limits of a neutral
behavior.
DISCUSSION
The diverse central moments for the markers and for the species revealed noteworthy
intraspecific differences between different markers and interspecific differences of a
determined marker. For example, the first moment was extremely different for the four
species considered at D5S111 and D5S117, and this trend was independent of the sample size
studied. The D14S51 and D17S804 markers showed symmetrical distributions for the third
and fourth central moment in A. palliata, while the opposite trend was determined for the
three other South American Alouatta species analyzed. In fact, A. palliata was the species
with the most divergent central moments compared to the other species studied, which
concurs with its phylogenetic position as demonstrated by Cortés-Ortiz et al., (2003). There
were similar differences (in quantity) of the central moments among the three South
American species. Relevant differences were recorded between A. seniculus and A.
macconnelli at D5S117 and between A. seniculus and A. caraya at D5S111 for the first central
moment and there were conspicuous differences for several central moments at D5S117 and
D6S260.
Table 6. Estimates of maximum likelihood of  with uni-step and a multi-step mutation models. This analysis was carried for the
detection of DNA microsatellites that had significantly evolved following a multi-step model. Also the multi-step mutation proportions
were calculated for each species
Uni-step Multi-step
Species and markers 0  Log likelihood  Log likelihood Multi-step mutation proportion
Alouatta palliata
AP68 13.9320 6.7572 -22.1792 10.7279 -22.0 778 P = 0.025 2 = 0.202 NS
AP74 4.8580 11.1256 -10.9069 10.2025 -10.8703 P = 0.000 2 = 0.073 NS
D5S111 1.4460 1.9376 -10.7159 0.8387 -11.3512 P = 0.000 2 = 1.270 NS
D5S117 17.4660 33.3595 -29.7882 20.0856 -28.0743 P = 0.075 2 = 3.427 SIG
D6S260 13.2120 6.4079 -12.0983 5.1527 -11.8396 P = 0.175 2 = 0.517 NS
D14S51 0.6670 1.2733 -5.9213 1.2733 -5.9037 P = 0.000 2 = 0.035 NS
D17S804 1.2120 0.8182 -5.5851 1.1636 -5.6529 P = 0.000 2 = 0.134 NS
P = 0.0392 ± 0.0659
Alouatta seniculus
AP40 1.1430 1.8571 -13.2 689 1.9657 -13.1854 P = 0.000 2 = 0.012 NS
AP68 1.4750 3.0967 -7.9361 3.0967 -7.9301 P = 0.000 2 = 2.111 NS
AP74 2.7350 7.0418 -13.99 51 3.1449 -15.0506 P = 0.000 2 = 0.167 NS
D5S111 3.4580 2.9913 -13.2 204 3.3198 -13.2881 P = 0.000 2 = 0.135 NS
D5S117 88.5760 68.2036 -42.7 391 68.2036 -44.8431 P = 0.100 2 = 4.208 SIG
D6S260 37.9780 58.1060 -15.6 952 36.4587 -16.7864 P = 0.100 2 = 2.182 NS
D8S165 5.6560 14.0258 -17.7 441 10.8021 -18.5809 P = 0.025 2 = 1.673 NS
D14S51 11.6700 4.5513 -23.6 177 4.5513 -19.7163 P = 0.325 2 = 7.802 SIG
D17S804 11.2580 18.2948 -14.8 482 10.8080 -14.6334 P = 0.050 2 = 0.429 NS
P = 0.0666
+ 0.1053
AP40 0.6670 1.1467 -2.0 428 1.1467 -2.0473 P = 0.000 2 = 0.009 NS
Table 6. (Continued)
Uni-step Multi-step
Species and markers 0  Log likelihood  Log likelihood Multi-step mutation proportion
AP68 10.2440 15.6740 -10.3 792 23.4597 -10.3052 P = 0.000 2 = 0.148 NS
AP74 21.8330 39.6275 -5.86 51 29.2567 -6.2323 P = 0.025 2 = 0.734 NS
D5S111 17.8000 33.9980 -12.1 119 44.1440 -10.7584 P = 0.000 2 = 2.706 NS
D5S117 44.6440 13.1701 -17.3 892 8.9289 -13.9341 P = 0.200 2 = 6.910 SIG
D6S260 6.1330 15.2107 -6.0369 14.0453 -6.1174 P = 0.000 2 = 0.161 NS
D8S165 4.8000 2.3280 -5.6465 0.9600 -5.3214 P = 0.475 2 = 0.650 NS
D17S804 10.1430 7.8100 -8.9352 7.8100 -8.8559 P = 0.000 2 = 0.158 NS
P = 0.0875 ± 0.1711
Alouatta caraya
AP40 0.9330 0.7187 -6.0118 0.3640 -5.9931 P = 0.475 2 = 0.037 NS
AP74 0.9330 0.7187 -6.0111 0.3640 -5.9931 P = 0.475 2 = 0.037 NS
D5S111 0.5330 0.8160 -2.5250 0.8160 -2.5184 P = 0.000 2 = 0.013 NS
D5S117 3.5880 5.1491 -11.5713 5.4900 -11.5259 P = 0.000 2 = 0.091 NS
D14S51 0.1430 0.3950 -2.6979 0.4086 -2.6961 P = 0.000 2 = 0.004 NS
P = 0.1583 ± 0.2452
Total Average of P for the four Alouatta species studied
(P = 0.0879 ± 0.0509)
o = Initial value to undertake the  maximum likelihood estimates. SIG = microsatellites which significantly departure from a uni-step mutation model. P =
Average multi-step mutation proportions affecting the markers and the species studied. NS = No significant multi-step mutation proportion
18 Microsatellite DNA Analyses of Four Alouatta Species (Atelidae, Primates)
It is fundamentally important both from evolutionary and genetic conservation points of

view, to have a thorough understanding of the possible differential dynamics concerning
microsatellite central moments used in order to better highlight evolutionary entities that
might support other postulated phylogenetic frameworks of the Alouatta genus (Cortés-Ortiz
et al., 2003 or Ruiz-García et al., 2016a, in the current book). The molecular evolution of
microsatellites and other molecular markers seems to be a better tool for the determination of
evolutionary genetics relationships among Neotropical primates compared to past methods
that relied on differences in pelage traits (such as color). For instance, Shedd and Macedonia
(1991) and Jacobs et al., (1995) have cautioned against using metachronism inferences to
determine evolutionary relationships among taxa of Neotropical primates due to varying
evolutionary pathways that occur once gene flow is interrupted. Evidence suggest that there is
no agreement between pelage-based and DNA marker-based taxonomy studies for Ateles
(Collins, 2001 or Ruiz-García et al., 2016b, in the current book). Therefore the molecular
results indicate that an Alouatta species’ definition based only on pelage color may lead to
erroneous conclusions. This was further ratified by Schneider et al., (1991)’s study that found
differences between two Alouatta belzebul populations living on the east and west banks of
the Tocantins River in Brazil. These populations showed opposite patterns when
morphological and molecular traits were compared. The color diversity on the east bank was
the highest and the expected heterozygosity was lowest whereas the opposite was found on
the west bank. In addition, some red howler monkeys were captured in the east bank of the
Tocantins River that were similar to A. seniculus, not previously documented in that area, and
living together in troops with A. belzebul. These rare animals presented no distinguishable
difference in karyotype from that of A. belzebul (Lima and Seuánez, 1989). All of these
studies emphasize that color descriptions are not sufficient to describe the evolutionary
relationships among the Alouatta taxa. Therefore, microsatellite central moments (evolution
and differences) may be a strong tool in the analysis of different Alouatta taxa as well as other
species taxonomy problems.
Assignation Differentiation Power in the Microsatellite Evolution of Alouatta

The assignment analyses revealed that the best procedure to correctly classify all the
animals genotypified in their respective species was the Bayesian method with the “as is”
technique (86.15%). In contrast, other procedures that relied on standard and the minimum
Nei genetic distance (73.85%) and the Cavalli-Sforza & Edwards chord genetic distance
(72.31%) with the “leave-one-out” technique had lower classification percentages. This is one
indication that the classical genetic distances based on allele frequencies probably do not take
into consideration the most primordial information of the microsatellite evolution, unlike
some other techniques. Some of the most frequent incorrectly classified cases involve the A.
seniculus-A. caraya pair, classifying animals of the first species in the second one. These
taxonomic errors reveal a close genetic relationship between A. seniculus and A. caraya not
previously found in other traditional studies. For example, Herkovitz (1949) used hyoid bone
morphology to separate A. caraya in a different clade than A. seniculus. A. caraya has a
smaller, more angular and less inflated hyoid bone compared to that within A. seniculus. The
same separation result was also reported by Meireles et al. (1999), but based on globin
pseudogene sequences. However, Cortés-Ortiz et al. (2003) sequenced five genes (three
mitochondrial and two nuclear) and revealed that A. caraya was within the same clade as A.
seniculus, which is the same as we determined with microsatellites. Some A. seniculus
individuals were also incorrectly classified within A. macconnelli and A. palliata. This
supports that the genetic variability found within other Alouatta species and their ancestors is
contained within A. seniculus and that this species (or some direct ancestor) could be the
origin of the other Alouatta species studied. It agrees quite well with the fact that A. seniculus
was the species with the highest levels of heterozygosity and mean number of alleles per
locus (shown elsewhere), although all of the animals in that study came from Colombia.
Recall that the species, or populations, with the highest levels of gene diversity are frequently
considered the original geographical centers of dispersion (Dobzhansky, 1971). In agreement
with this idea, recall that the oldest Alouttinae fossil registered is Stirtonia tatacoensis from
La Venta in Colombia, a form allied with Alouatta and dated to 12-16 million years (MY)
(Feagle, 1988). Therefore, Alouatta could have originated in some part of the current
Colombian territory, where A. seniculus lives today. In contrast, A. macconnelli and A.
palliata individuals have probably not been misclassified because they have well delimited
and particular gene pools which were likely derived from A. seniculus. In agreement with
Cortés-Ortiz et al., (2003), our microsatellite data differentiated A. macconnelli from A.
seniculus. Cortés-Ortiz et al., (2003) used mtDNA-based phylogenetic analyses to show that
A. macconnelli and A. sara were phylogenetically distinct forms from A. seniculus. Some of
our microsatellite results (unpublished) also strongly differentiated A. sara (from Bolivia)
from A. seniculus, such as we observed with A. macconnelli.
Effective and Total Numbers, the Mutation Dynamics and Natural Selection of DNA
Microsatellites in Howler Monkeys
Several interesting features can be obtained from an analysis of effective and total
numbers and mutation dynamics of the microsatellites studied in the four Alouatta species
analyzed. First, the multi-step mutation model that was globally determined for the four
species did not significantly differ from the uni-step mutation model. D5S117 was the unique
marker which systematically presented a significant departure from the uni-step mutation
model in three of the four species studied (A. seniculus, A. macconnelli, and A. palliata) and
yielded the highest mutation rate in all four species. A. seniculus was the species, with the
most cases of significant multi-step mutation percentages (22.22%). Nevertheless, this
percentage was not significantly different from the 5% type I error. As it was commented
before, the two species with the most divergent multi-step mutation proportion average
percentages were A. caraya (15.8%-the highest) and A. palliata (3.9%- the lowest), whereas
the two northern South American species presented similar percentages (A. seniculus – 8.7%-
and A. macconnelli – 6.7%-). Therefore, the two most peripheral species studied showed the
highest degree of deviation in regard to the two northern South-American species, which
revealed the highest degrees of gene diversity (not shown here) and the highest effective and
historical numbers. It is possible that the speciation events with relevant founder effects could
have influenced the levels of multi-step mutation percentages and therefore provide an
explanation of why A. caraya and A. palliata are the species with the extreme values. The
overall multi-step percentage averages for the four species was around 8%. Therefore, from
an evolutionary point of view, the uni-step is more remarkable than the multistep mutation
model, at least for the microsatellites we studied in Alouatta, in contradiction to that affirmed
for humans by DiRienzo et al., (1994). Second, through the application of maximum
likelihood tests we determined that most of the microsatellites analyzed presented different
mutation rates within each one of the Alouatta species considered. A. macconnelli /A.
seniculus was identified as the species pair with the greatest number of microsatellites that
suffered from similar mutation rates. As previously mentioned, this could be of use as a
phylogenetic signal. Third, as a consequence of no conspicuous differences among the uni-
step and the multi-step mutation models, the effective and the total historical numbers were
very similar for both mutation models. Consequently, the use of the uni-step mutation model,
when the maximum likelihood multi-step mutation model is not estimated, does not introduce
an excessive bias, at least, for the howler monkeys with the microsatellites used in this study.
Fourth, the arithmetic and harmonic effective and total numbers clearly revealed that A.
seniculus was the species which presented the largest historical demographic populations.
This agrees quite well with the fact that this is the species with the largest distribution range.
Its total numbers ranged from 22,100 to 176,800 animals (arithmetic mean) or from 5,900 to
46,900 individuals (harmonic mean). The large effective numbers probably indicate that until
recently, the gene flow has been elevated within populations of this species. On the other
hand, the values obtained from the most peripheral Alouatta species were clearly lower (A.
palliata and A. caraya), especially for A. caraya, which ranged from 5,900 to 47,000
(arithmetic mean) or from 990 to 7,900 (harmonic mean). The fact that A. macconnelli
presented a gene diversity (not shown here),  estimates, historical effective and total
numbers almost as high as A. seniculus is quite interesting, even though its sample size and its
distribution range were small in comparison to A. seniculus. Indeed, the distribution range of
A. macconnelli is considerable smaller than that of either A. palliata or A. caraya, but its
estimates of effective and total numbers are higher than either of these two species. It could
mean that the origin of the current Alouatta species studied was in northern South America, as
we previously speculated. A. macconnelli could be a direct descendent of A. seniculus without
loss of genetic diversity during speciation, while a relevant founder effect was effective in the
appearance of A. palliata and A. caraya. A. belzebul was not included in this study, but it has
been shown to have extremely high levels of protein gene diversity. It could be another good
original-species candidate from which other current Alouatta species derived. For this reason
microsatellites should be applied to this species to support or not support the proposed
hypothesis. As we will demonstrate, some of our microsatellite mutation rates agree quite
well with a divergence between A. seniculus and A. macconnelli around 3.3 MYA (Cortés-
Ortiz et al., 2003). This suggests that they were isolated by the separation of the Guyana
region from the Amazon basin and the emergence of the current Amazon River as the major
drainage of this river basin more than 3 MYA. We believe that the pathway of speciation was
from A. seniculus to A. macconnelli because several A. seniculus individuals were classified
in the A. macconnelli group with the assignment analysis as previously quoted. This could
mean that the original microsatellite genotypes of A. macconnelli were derived from
individuals that resembled the multi-genotype of A. seniculus. Furthermore, this would
demonstrate that A. macconnelli not only derived from A. seniculus but that no extreme
founder effect occurred when both forms were isolated because A. macconnelli retained the
major part of the original gene diversity of A. seniculus. The isolation of the Guyana region
also affected other Atelidae primates such as Ateles paniscus (Collins and Dubach, 2000b).
Ateles paniscus separated from the other Ateles species around 3.27 MYA, which correlates
with this time of separation for A. macconnelli and A. seniculus. Thus, the same
biogeographic event simultaneously affected both Ateles and Alouatta in that region of South-
America. The trans-Andean Alouatta species (A. palliata) separated from the South American
Alouatta species around 6.8 MYA. Meanwhile the Pleistocene refugia, with the Magdalena
river valley fluctuations, could provide a relevant explanation to the appearance (by isolation)
of several Ateles species, such as Ateles hybridus (1.41 MYA) or the Central American Ateles
geoffroyi (2.0 MYA) (Collins and Dubach, 2000b), this explanation is not satisfactory for
Alouatta. The South and the Central American Alouatta species had diverged previously and
the climatic Pleistocene fluctuations seemed to have no noticeable impact on the
microsatellite gene dynamics of Alouatta. Therefore the use of microsatellite evolutionary
dynamics may therefore allow for better recreations of past biogeographic events mainly
responsible of speciation by measuring levels of gene diversity in isolated populations and
providing approximate dates for species ancestors that can be associated with previous
climatic and geological changes. Molecular evolution studies of microsatellites in Neotropical
primates may also lead to an increase of our evolutionary knowledge and to the development
of new conservation ideas. Fifth, several fundamental questions however must be resolved in
order to have more confidence in the obtained results. To calculate total historical numbers
from the effective numbers estimated, we need to determine the ratio Ne/N from demographic
data. By using the ecological and demographical studies of Baldwin and Baldwin (1976),
Heltne et al., (1976), Milton (1982) and Estrada (1982) for A. palliata, of Neville (1972),
Eisenberg (1979), Braza et al., (1981), Defler (1981), Terborgh (1983), Crockett (1984, 1985)
and Crockett and Eisenberg (1987) for A. seniculus and of Thorington et al., (1984) for A.
caraya, we calculated this ratio considering the mean number of adult males, the mean
number of adult females, the mean troop size and the fraction of adult males that did not
reproduce within their troops. This value was around 0.4 for the three species indicated. We
assumed that this ratio was also feasible for A. macconnelli. With this in mind, we will try to
answer three questions as follows: (1) From an evolutionary perspective what is the likely
average microsatellite mutation rate per generation (5.6 x 10-4 or 7 x 10-5) for Alouatta? This
is interesting not only from a microsatellite mutation evolution perspective, but also because
it is important to know the most feasible effective and total numbers throughout the history of
the species in question. Additionally, this is of interest to conservation geneticists. For
example, the conservation measures to be implemented for the conservation of A. seniculus
will not be the same if we consider the lowest estimate of the total number obtained, 5,855
animals (5.6 x 10-4 and harmonic averages), or the highest total number estimated of 176,815
animals (7 x 10-5 and arithmetic averages). The same could be mentioned for A. caraya, the
species which showed the lowest number estimates. There is a difference in the conservation
of 988 compared to 47,045 animals. (2) Which of the two methods used to obtain means
(arithmetic or harmonic) fits more adequately with reality? (3) Several genetic heterogeneity
statistics (FST and GST) could be employed to measure the degree of differences among the
Alouatta species. One relevant question is, which of these two statistics is more consistent
with the molecular evolution of the microsatellites? Herein, we can answer these three
questions. Cortés-Ortiz et al., (2003) obtained a maximum likelihood tree of three
mitochondrial genes (ATPase 8 and 6 and Cyt b) with an enforced molecular clock for
different Alouatta species and estimated the divergence time between the Central and the
South American species to be 6.8 MYA. By means of Slatkin (1995), we can employ the
following equations:
F = 4Ne (FST/(1 – FST)) and R = 4Ne (RST/(1 – RST)),

where  is the number of generations elapsed from the separation of the taxa analyzed and Ne
is the effective number. We obtained average FST values ranging from 0.189 to 0.259 and
average RST values from 0.545 to 0.696, depending on the procedure used. To simplify we
used the mean values of these ranges (FST = 0.224 and RST = 0.621, respectively) jointly with
the effective numbers summed for all the species analyzed estimated throughout the two
extreme mutation rates (5.6 x 10-4 and 7 x 10-5). Moreover, 6.6-6.8 MYA approximately
represents 1,100,000 to 1,133,333 generations for Alouatta. Most red howler females first
give birth at about age 5 whereas males actively reproduce through age 7 (Crockett and
Eisenberg, 1987). Therefore, one generation in this genus is approximately 6 years. The
combination of data (mutation rate of 7 x 10-5, the mean numbers obtained with the arithmetic
procedure and the mean value of RST [= 0.621]) used to estimate the number of generation (
of 1,170,481) agrees quite well with the data of Cortes-Ortiz et al., (2003). Other
combinations values were not compatible with this number of generations. For example, only
6,825 (41,000 years ago) elapsed using the 5.6 x 10-4 mutation rate, the harmonic effective
number means, and FST (= 0.224). This is completely incompatible with the divergence times
obtained by Cortés-Ortiz et al., (2003). These values are compatible with the fact that the
diversification of the current howler monkey species was parallel to the formation of the
northern Andes (Lundberg et al., 1998) approximately 6.8 MYA. The posterior diversification
for the cis-Andean howler monkeys (5.1 MYA) is roughly parallel to the origin of the modern
Amazon River. Also, our estimates (using microsatellites) with mutation rates of 7 x 10-5,
arithmetic means, and the heterogeneity RST statistic offer a divergence time strikingly similar
to that determined with mtDNA (Cortés-Ortiz et al., 2003). Thus, future works with these
microsatellites and with Alouatta should use the 7 x 10-5 mutation rate, the arithmetic means
to calculate overall effective and total numbers, and RST to more accurately reconstruct
evolutionary relationships. It is interesting to note that the present results for RST disagree
with that claimed by Gaggiotti et al., (1999). Another interesting conclusion is that these
nuclear DNA microsatellites provided a good phylogenetic resolution with these parameters
comparable to mtDNA genes. Other nuclear regions such as the sequences of the Calmodulin
(CAL) and the Recombination activating (RAG1) genes did not because they presented very
low levels of sequence heterogeneity, even between Alouatta and Ateles (Cortés-Ortiz et al.,
2003).
Hence, with the average mutation rate of 7 x 105, we were able to address theories of
biogeographic mechanisms responsible for the production of Alouatta species patterns. We
can affirm that the majority of the speciation events among the Alouatta species studied
occurred during the middle to late Pliocene and very early Pleistocene. They were primarily
the result of major events such as the final uplift of the Andes Cordillera and development of
savannas in northern and middle-southern South America. There is little evidence to support
the importance of Pleistocene refugia formation or current riverine barriers as primary
mechanisms for the formation of the Alouatta species analyzed herein. Recall that the
Pleistocene refugia has been used by many authors to try to explain speciation among many
Neotropical animals (Bush, 1994; Haffer, 1997). Although some Neotropical primate species
(and other species) were probably affected by the existence of smaller and less stable
Pleistocene refugia among large and extensive primary rain forests, these smaller and less
stable Pleistocene refugia were probably not an obstacle for the Alouatta dispersion. For
example, Estrada and Coates-Estrada (1988) determined that Mexican Ateles populations
have been absent from forest fragments as large as 250 ha for many decades and that howler
monkeys are extremely efficient colonizers of any size Neotropical ecosystem. Perhaps only
A. macconnelli, of the four Alouatta species that we studied, supports the riverine barrier
hypothesis as a cause of speciation, because this species’ distribution is limited by the
Amazon in southern Guyana and in northern Brazil and by several black water rivers in the
northwest. Its distribution coincides within an area that houses multiple endemic species of
vertebrates (Haffer, 1992), including A. paniscus (Collins and Dubach, 2000a). This last
species diverged from the other Ateles lineages approximately 3.3-3.6 MYA, a value range
extremely similar to the estimate of Cortés-Ortiz et al. (2003) and our estimation of around 3
MYA using microsatellites. These divergence times are probably too large to be explained by
the original Pleistocene Refuge model (Haffer, 1969). Other authors such as Suemitsu et al.,
(2000) have claimed that several Alouatta populations (A. fusca clamitans) have specific
cytotypes associated with geographical patterns in Brazil that suggests a north-south division
due to possible reproductive isolation during the existence of quaternary refuges. However,
our microsatellite data and mitochondrial data of Cortés-Ortiz et al.’s (2003) do not support
quaternary refuges as important to the molecular gene evolution of the four Alouatta species
studied herein.
Lastly, the Beaumont and Nichols (1996) test, independent of the infinite allele or the
step-wise mutation models, revealed two microsatellites (D14S51 and D8S165) inside the
Alouatta genus that were outside neutral behavior. D14S51 seems to be affected by
diversifying natural selection while D8S165 revealed the impact of constrictive natural
selection. Although all the other microsatellite seem to be neutrally regulated, D14S51 could
be impacted by hitchhiking of advantageous mutations (selective sweeps), whereas D8S165
could be affected by the continued removal of deleterious mutations by means of background
selection (Schug et al., 1998). Therefore, the use of microsatellites in the genome of diverse
Neotropical primates may reveal relevant selective patterns.
The analysis of molecular microsatellite evolution is straightforward and interesting
because it helps to determine the mutation mechanisms that underlie these repetitive segments
of DNA. Furthermore, by coalescence this analysis helps to determine fundamental
parameters (such as effective numbers) in the evolution of species as well as compares
microsatellite phylogenetic signals with those of other molecular markers. Similar studies to
that presented herein are ongoing in our laboratory with other Neotropical primate genera
such as Ateles, Lagothrix, Cebus, Saimiri, Aotus, Callicebus, Pithecia and Saguinus.
ACKNOWLEDGMENTS
We thank the Dean of the Faculty of Sciences and the Academic Vicerectory of the
Pontificia Universidad Javeriana at Bogotá, Colombia for their financial support. Thanks also
goes to the Instituto von Humboldt (IVH) at Villa de Leyva for the use of its facilities for
providing the opportunity to sample integumentary tissues from its howler pelt collection for
the extraction of DNA. These acknowledgments are mainly directed to the ex-curator of the
mammalogy collection, Miss. Yaneth Muñoz-Sabas and the directors of IVH, Drs. Cristian
Samper and Fernando Gast. Likely, we are indebted to many people throughout Colombia,
who provided samples (hair) of howlers maintained in captivity or provided samples (teeth,
bone or skin) from howler that had been hunted. Indeed, many thanks go to Luz Mercedes
Borrero and Marcela Ramirez from Santa Fe zoo at Medellín who provided samples of red
howler from the Antioquia region and to Dr. Francois Catzeflis who provided four of the
seven DNA samples used of A. macconnelli from French Guiana. Similarly, many thanks to
Dr. Liliana Cortés-Ortiz, who provided two DNA samples of A. palliata mexicana. Thanks go
to the Huitoto, Ticuna and Jaguas Indian communities throughout the Colombian Amazon,
and the Movima, Moxeño, Sirionó, Canichana, Cayubaba and Chacobo Indian communities
in Bolivia.
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Chapter 11 Alouatta Microsatellite

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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.

MICROSATELLITE DNA ANALYSES OF

Manuel Ruiz-García1,*, Pablo Escobar-Armel1,

1. Four microsatellite central moments (mean, variance, skewness, and kurtosis)

microchromosomes, A. macconnelli presents a diploid chromosome number of 47, 48 and 49.

MATERIALS AND METHODS

Table 1. Sample sizes, morphological subspecies, countries, specific geographic origins,

Subspecies and Geographic origin, type of samples and

Alouatta 7 A. macconnellii Carnopi River (4 samples of muscle tissue

Table 2. Microsatellites, length size in base pairs and forward

Population Genetics Analyses

Microsatellite Central Moments

Method procedure % of correct assignment

Effective numbers Total numbers

= 19.803 ± 12.662 XA = 8,841 70,726 22,103 176,815

Effective numbers Total numbers

species studied, we detected no significant difference among the averages. A. caraya (p =

D8S165-D6S260; while in A. seniculus, these pairs were D5S111-D17S804, D5S111-AP40,

D5S111-AP74, D14S51-D8S165 (uni-step model only), D17S804-D8S165, D17S804-AP74,

It is fundamentally important both from evolutionary and genetic conservation points of

Assignation Differentiation Power in the Microsatellite Evolution of Alouatta

F = 4Ne (FST/(1 – FST)) and R = 4Ne (RST/(1 – RST)),

Population Genetics, Evolutionary Biology and Conservation of the Neotropical

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