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Population genetics of the endangered Wattled

Curassow (Crax globulosa, Cracidae, Aves) of the
Colombian–Peruvian Amazon using DNA microsatellites
and ND2 mitochondrial sequences
a a
Diana Alvarez-Prada & Manuel Ruiz-García
a
Laboratorio de Genética de Poblaciones Molecular y Biología Evolutiva, Departamento de
Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá DC, Colombia
Published online: 26 Jun 2015.

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To cite this article: Diana Alvarez-Prada & Manuel Ruiz-García (2015): Population genetics of the endangered Wattled
Curassow (Crax globulosa, Cracidae, Aves) of the Colombian–Peruvian Amazon using DNA microsatellites and ND2
mitochondrial sequences, Studies on Neotropical Fauna and Environment, DOI: 10.1080/01650521.2015.1048615

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 Studies on Neotropical Fauna and Environment, 2015
http://dx.doi.org/10.1080/01650521.2015.1048615

Population genetics of the endangered Wattled Curassow (Crax globulosa, Cracidae, Aves)
of the Colombian–Peruvian Amazon using DNA microsatellites and ND2 mitochondrial
sequences
Diana Alvarez-Prada & Manuel Ruiz-García*
Laboratorio de Genética de Poblaciones Molecular y Biología Evolutiva, Departamento de Biología, Facultad de Ciencias,
Pontificia Universidad Javeriana, Bogotá DC, Colombia
(Received 14 January 2014; accepted 20 April 2015)

The Wattled Curassow (Crax globulosa, Cracidae, Aves) is a large bird living in the Western Amazon basin and a
critically endangered species in the Colombian and in the Peruvian Amazon. We carried out the first population
genetics analysis of this species employing six nuclear microsatellite markers and sequences of the mtND2 gene.
The main results are as follows. (1) The levels of gene diversity were high for the overall population as well as for
Downloaded by [Manuel Ruiz-Garcia] at 16:03 26 June 2015

each of the three islands for both microsatellites and mtDNA. (2) A small amount of genetic differentiation
among populations was found with both types of markers (FST = 0.027 for microsatellites and NST = 0.17 for
mitochondrial sequences). (3) Using microsatellites, the Geneclass 2.0 software detected a low correct assignment
of individuals to their respective populations. The Structure software only detected one gene pool for the entire
area studied. These results are relevant for conservation efforts of this critically endangered species.
Keywords: Crax globulosa; DNA microsatellites; mitochondrial ND2 gene; genetic structure; Colombian and
Peruvian Amazon

Introduction southern Colombian Amazon Basin, a small area of

The Galliformes is one of the oldest orders of birds in
the Neotropics, and the family Cracidae (11 genera,
50 species and near 60 subspecies) is one of the oldest
within this order. This family originated around 76
million years ago (MYA) and the diversification of
the current genera began around 41 MYA, coinciding
with drastic climatic changes in that epoch (Sick 1993;
Pereira et al. 2004). Its geographical distribution
ranges from southern USA to Argentina (Silva &
Strahl 1991). This family is the most threatened of
all bird families in Central and South America
because they are targeted by the local population for
their exquisite meat (Begazo & Bodmer 1998; Silva
1999). In Colombia, 25 species of Cracidae occur,
including five species of Crax (C. alberti, C. alector,
C. daubentoni, C. globulosa, and C. rubra), while Peru
has 15, including one species of Crax (C. globulosa)
(Rodríguez-Mahecha et al. 2005).
The Wattled Curassow (C. globulosa) is more
arboreal than other species of this genus (Santos
1998; Bennett 2000), having a relatively short tarsus,
which seems to be an adaptation to arboreal dwellings
in a habitat highly linked to forest flooded by white
water rivers (“várzea”) (Begazo 1997; Bennett 2000,
2003). Its geographic distribution includes the
the Ecuadorian Amazon Basin (although BirdLife
International [2013] stated that no recent reports
exist for this species in Ecuador), the Andean pied-
mont, the Peruvian Amazon, northern Bolivia and the
western Brazilian Amazon at altitudes lower than
300 m asl. The overall population size for this species
is estimated to be 2500 to 10,000 individuals (BirdLife
International 2013). IUCN globally classified this spe-
cies as Endangered and IUCN-Colombia and IUCN-
Ecuador classified it as Critically Endangered (CR)
(BirdLife International 2013). This species has only
been confirmed recently in two locations in Colombia:
Mirití Island in the Caquetá River (Alarcón-Nieto &
Palacios 2005), and Mocagua Island and neighboring
areas in the Amazon River (Bennett 2000, 2003). The
Mocagua population has nearly 150 individuals
(Bennett 2000, 2003) that are protected by the con-
servation efforts of the Piurí project led by three
Ticuna communities and the independent researcher
Sara Bennett. Since 2000, no individuals have been
hunted on Mocagua Island.
Populations of C. globulosa have higher densities
and are more stable along the Amazon and Marin
rivers in the Peruvian Amazon, where they are more
distant from human populations (Begazo 1997). The

*Corresponding author. Email: mruizgar@yahoo.es

© 2015 Taylor & Francis

 2 D. Alvarez-Prada & M. Ruiz-García
same pattern was observed at the Mamirauá Reserve
at the central Brazilian Amazon (Santos 1998).
During the 1960s, within the Beni Department
(Bolivia), this species disappeared because of non-
regulated hunting (Bennett 2003). However, the popu-
lation of C. globulosa has currently reappeared
because hunting pressures have diminished
(Hennessey 1998), thus implying the significant influ-
ence of human pressure on the species’ survival.
Analysis of mitochondrial DNA revealed that
within the subfamily Cracinae the genus Crax is
monophyletic and is the sister genus to Nothocrax,
Mitu, and Pauxi clades (Pereira & Baker 2004).
Additionally, Pereira et al. (2004) sequenced the con-
trol region of 27 species representing the 11 Cracidae
genera and found that its evolutionary rate was higher
for ND2 (around 0.37–0.47%) than for others such as
ND5 (0.14–0.3%) and cytochrome b (0.29–0.35%), a
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reason why ND2 was selected in this work. The first
genetic study on a natural population of Cracidae was
that of Pereira and Wajntal (2001b) with C. fascio-
lata. They found that a natural population of this
species had similar levels of gene diversity to captive
Cracidae populations. They concluded that natural
populations of C. fasciolata lost gene diversity by
habitat reduction in previous decades. More recently,
Gonçalves et al. (2010) employed microsatellites to
compare gene diversity levels between captive and
natural populations of C. fasciolata. Of the six micro-
satellites employed, three were polymorphic with a
total of 27 alleles, with eight private alleles in the
wild population and seven private alleles in the cap-
tive population. They also claimed that these popula-
tions were genetically impoverished.
Herein we studied different aspects of the genetic
structure of C. globulosa in several islands and main-
land on the Colombian–Peruvian Amazon border,
where the number of animals is extremely small.
This is particularly important because small popula-
tions quickly lose genetic diversity mainly due to
inbreeding (Frankham et al. 2002). To carry out this
task, we employed six DNA microsatellites and
sequences of the mtND2 gene. The microsatellites or
STRPs (short tandem repeat polymorphisms) are
composed of tandem, repetitive units of 2–6 base
pairs in length (Weber & May 1989). STRPs are
randomly distributed, highly polymorphic and fre-
quently found inside eukaryotic genomes. The small
amount of DNA needed for these molecular analyses
has helped the investigator to use noninvasive proce-
dures (the use of feathers in this case) when sampling
wild animals (Bruford & Wayne 1993). Hughes and
Larson (2000) developed six microsatellites for
C. globulosa to study the genetic relationships
among individuals in captivity.
The main aims of the current study were: (1) to
determine the levels of gene diversity and the degree
of heterogeneity in C. globulosa populations from the
Colombian and Peruvian Amazon; (2) to determine
the mutation model that predominates in the micro-
satellites studied in C. globulosa and to estimate
possible historical effective numbers in the
Colombian-Peruvian Amazon area; (3) to evaluate
the assignment of individuals to their respective
population of origin; and (4) to determine possible
demographic changes along the evolutionary history
of this species.
Materials and methods
Study sites
Samples of Crax globulosa for DNA extraction were
obtained on three islands on the Amazon River
(Cacao and Yaumas, Peru, and Mocagua,
Colombia) and in Puerto Nariño (mainland
Colombia) (Figure 1, Table 1) These Amazonian
islands are situated between 70°40ʹ W–3°48ʹ S and
69°55ʹ W–4°15ʹ S. Mocagua Island is 10 km long
and 2.5 km wide (Prieto et al. 1995) and belongs to
three Ticuna communities (Mocagua, Macedonia,
and Vergel, all on the northern bank of the Amazon
River in front of the island). Yaumas Island has
similar dimensions as Mocagua Island and has two
villages located in its eastern area, close to the
Colombian city of Leticia. Cacao Island is the largest
of the three islands and it has three villages. In
Mocagua, the samples were obtained along transects
followed by local people responsible for the protec-
tion of fauna on this island. They work for the “Piurí
project” looking for molted feathers collected in wal-
lows or in self-made traps where the curassows are
captured live and some feathers are plucked. The
majority of the samples came from molted feathers
collected in wallows, but from one individual a blood
sample was obtained. In the other three sampling
areas, the samples were obtained from small pieces
of tissue (muscle) or feathers from hunted animals.
These communities were visited only once, all sample
donations were voluntary, and no financial or other
inducement was offered for supplying samples for
analysis. A total of 64 samples were obtained
(Table 1) representing around 1/3 of the estimated
population for this species in this area (Bennett
2000, 2003).
The microsatellite analysis showed that in
Mocagua, the feathers from three specimens were
sampled twice because the multi-genotypic profiles
were identical. A total of 47 specimens were used in
the microsatellite analyses for Mocagua (61 in total).
 Studies on Neotropical Fauna and Environment 3
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Figure 1. Locations of the four sites along the Amazon River from which the DNA samples of Crax globulosa were obtained
in the Colombian and Peruvian Amazon.

Table 1. Localities, number of samples employed for micro- Additionally, nine Great Curassow samples (Crax
satellites, types of tissues and number of samples employed rubra) (feathers) were analyzed for comparative pur-
for mitochondrial ND2 sequences in the molecular analysis poses. For the mtND2 gene sequences, 12 samples
of Crax globulosa and Crax rubra. showing the best DNA quality (six samples from
Number of Mocagua, four samples from Cacao and two from
Number of samples Yaumas) were selected and analyzed.
samples used employed for
Localities sampled for DNA Types of mitochondrial
for Crax globulosa microsatellites samples ND2 gene
Molecular analyses for microsatellites and for the
Mocagua Island 50 but 3 Feathers (49) 6 mtND2 gene
(Colombian individuals Blood (1)
Amazon) were The DNA was extracted by using a salting out pro-
sampled cedure (Sambrock et al. 1989) using 3 cm of the basal
twice calamus from the feathers. Six C. globulosa tri-nucleo-
Puerto Nariño 2 Feathers (2) — tide microsatellites (CgAAT11, CgAAT32, CgAAT62,
(Colombian
CgAAT82, CgAAT85, and CgAAT190, Hughes &
Amazon)
Cacao Island 8 Muscle (4) 4 Larson 2000) were amplified in our samples. A poly-
(Peruvian Feathers (4) merase chain reaction (PCR) was performed in a 25μl
Amazon) volume. In this case, PCR reaction mixtures included
Yaumas Island 4 Feathers (4) 2 3μl of 3 mM MgCl2, 2.5μl of 10× buffer, 1μl of 1 mM
(Peruvian
dNTPs, 1.5μl of 10 pmol of each primer, 14–16.5μl of
Amazon)
H2O, 2.5μl of DNA (50–100 ngμl–1) if it was extracted
Locality sampled
from blood or muscle and 5μl of DNA if it was
for Crax rubra
Peten (Guatemala) 9 Feathers (9) — extracted from the feathers and 0.5 units of Taq
polymerase. PCR reactions were carried out in a MJ
 4 D. Alvarez-Prada & M. Ruiz-García
Research-P 100 thermocycler. The temperatures were
set as follows: 94°C for 5 min, 45 cycles (feathers; 30
cycles for tissues and muscle) of 1 min at 94°C, 1 min
at 55°C for CgAAT32, CgAAT62, and CgAAT190,
and at 60°C for CgAAT11, CgAAT82, and
CgAAT85 and 1 min at 72°C. A final extension for
5 min at 72°C was used. Amplification products were
kept at 4°C until use. PCR products were electro-
phoresed in denaturing 6% polyacrylamide gels and
visualized in a Hoefer SQ3 sequencer vertical cam-
era. Allele sizes were obtained by comparison with
the molecular weight f174 DNA digested with Hind
III and Hinf I. A molecular weight marker was
loaded every four lanes. The PCR amplifications
were performed in triplicate to ensure the accuracy
of the genotypes obtained.
We used primers L: GGCCCAQTACCCCGR
AAATG and H: GGATGAGAAGGCTAGGATT
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TTKCG to amplify the mtND2 gene (Sorenson et al.
1999; Sorenson 2003). The PCR amounts were the same
used for the microsatellites, and an iCycler Thermal
Cycler (Bio-Rad, Hercules, CA, USA) was employed
for the PCRs. A touch-down procedure was used: 94°C
for 5 min, 30 cycles of 1 min at 94°C, 1 min at 57°C, and
1 min at 72°C. After the first three cycles the annealing
temperature was reduced by 0.5°C in each cycle to reach
52°C. A final extension for 5 min at 72°C was used.
All amplifications, including positive and negative
controls, were checked in 2% agarose gels. Those
samples that amplified were purified using the kit
Qiaquick (Qiagen Inc, Valencia, CA, USA). The PCR
products were sequenced in both directions using the
Big Dye™ kit (Qiagen Inc) in an ABI 377A automated
DNA sequencer (Qiagen Inc). A consensus of the for-
ward and reverse sequences was determined using the
Sequencher program (www.genecodes.com/).
Microsatellite analyses
Gene diversity, Hardy–Weinberg, genetic heterogeneity
and gene flow analyses
The arcsine transformed expected heterozygosity (H,
Nei 1973) values among populations were compared
with t-tests, as proposed by Archie (1985). The
Hardy–Weinberg equilibrium (HWE) was estimated
using the F statistic of Weir and Cockerham (1984).
This procedure was used to calculate the excess degree
of homo- and heterozygous genotypes within each
C. globulosa population. We used the Markov chain
method (with 10,000 dememorization, 100 batches
and 5000 iterations per batch) (Raymond & Rousset
1995) to measure the probabilities and the Fisher
method to analyze the global HWE estimates (by
loci and by populations). To estimate possible
amounts of gametic disequilibrium among the micro-
satellites we employed the Markov chain method with
the same parameters as it was described for the HWE.
Genepop V. 4.0 (http://kimura.univ-montp2.fr/~rous
set/Genepop.htm) was used for these tasks.
Three strategies were used to calculate genetic
heterogeneity among species populations. First, the
gene and genotypic frequencies of the five microsa-
tellites, which showed good reproducibility, were
tested with Markov chains (10,000 dememorizations,
100 batches at 5000 iterations per batch). Second,
Wright’s F statistics (1951), with the procedure of
Michalakis and Excoffier (1996), were applied. The
standard deviations of the F statistics and the con-
fidence intervals (95 and 99%) were calculated with
jackknifing and bootstrapping over loci, respectively.
The significance of FST (genetic heterogeneity of the
subpopulation with regard to total population) was
calculated with the G test (10,000 allele randomiza-
tions, random mating assumed) and with the log-
likelihood G test (10,000 genotype randomizations,
random mating not assumed) for the populations
studied (Goudet et al. 1996). The significances of
FIS and FIT were similarly calculated (10,000 rando-
mizations of alleles within and overall for the popu-
lation analyzed). Third, the RST statistic (Slatkin
1995), designed for microsatellites by using the num-
ber of tandem repeats of the alleles, was also applied.
The programs Genepop V. 4.0 and Fstat 2.9.3.2
(www2.unil.ch/popgen/softwares/fstat.htm) were
used.
Estimates of theoretical gene flow among popula-
tions were obtained by means of the allele private
method (Slatkin 1985; Barton & Slatkin 1986) and
by means of the FST and RST statistics (Slatkin &
Barton 1989).
Mutation models and effective sizes
A maximum likelihood procedure with a Markov
chain recursion method (Nielsen 1997) was used to
estimate the θ statistic(= 4Neμ, where Ne is the effec-
tive population size and μ the mutation rate per gen-
eration) to determine the microsatellite mutation
model (one-step versus multi-step mutations) and to
calculate historical effective numbers for C. globulosa
assuming a mutation rate oscillating between
5.6 × 10–3, and 5.6 × 10–4. These are typical mutation
rates per generation for vertebrates (Serikawa 1992;
Weber & Wong 1993; Ellegren 1995, 2000; Schlötterer
2000). The MISAT program (Nielsen 1997) was used
to estimate the likelihood surfaces for θ. A grid size of
40 in a mutation one-step model with 1,000,000
Markov chains was used. The θ value with the least
negative log likelihood is the estimate of the θ

maximum likelihood. From this value, Ne was calcu-
lated for our C. globulosa population. In addition, the
largest possible multi-step mutation percentages (ran-
ging from 0 to 0.5) were calculated through the max-
imum likelihood of θ by means of 3,000,000 Markov
chains. Likewise, we measured whether the multi-step
mutation models’ estimates were significant improve-
ments over the uni-step mutation models. The like-
lihood ratio of −2 log [L(θ, p = 0)/L(θ, p)] was applied
to the maximum likelihood obtained multi-step p
percentage. Large samples have a value of χ2 with
one degree of freedom with the null hypothesis
p = 0. A probability lower than α = 0.05 indicates
that the multi-step mutation percentage is signifi-
cantly different from the uni-step mutation model
and the last model is then rejected.
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Possible bottlenecks
The difference between observed and expected hetero-
zygosity was used to determine the signature of bottle-
necks in microsatellite data using the software
BOTTLENECK 3.2.02 (Cornuet & Luikart 1996;
Luikart et al. 1998). The analysis was carried out
under the infinite allele model (IAM), the stepwise
mutation model (SMM) and the two-phased mutation
model (TPM).
Genetic assignment
We used the Geneclass 2 program (Piry et al. 2004) to
develop diverse assignment analyses for the C. globu-
losa populations. Different strategies were performed
by using a Bayesian procedure (Rannala & Mountain
1997), one frequency procedure (Paetkau et al. 1995),
several genetic distance methods (Cavalli-Sforza &
Edwards 1967; Nei 1972) and the Distance Allele
Shared (DAS) (Chakraborty & Jin 1993). The assign-
ment analyses were conducted without simulations
and estimating probabilities of belonging, or exclu-
sion, of the individuals to the original populations,
where they were a priori assigned (p < 0.05). Also,
some assignment analyses were performed with
10,000 resampling simulations by means of the
Monte Carlo technique and with the Paetkau et al.
(2004), and Rannala and Mountain (1997)
procedures.
Determination of the gene pool numbers
We used the theory described by Pritchard et al.
(2000) to determine the number of different possible
populations in our group of C. globulosa. This
Studies on Neotropical Fauna and Environment 5
theory was also extended by Falush et al. (2003a,
2003b, 2007), and developed in the Structure pro-
gram v 2.3. This method employs a Markov Chain
Monte Carlo (MCMC) procedure and the Gibbs
sampler. The method also uses the different geno-
types obtained for the microsatellites employed to
infer population assignment of the individuals ana-
lyzed. The model considers K populations, where K
may be unknown as we employed in this case, and
the individuals are assigned tentatively to one popu-
lation or jointly to two or more populations if their
genotypes are considered to be admixed. The theory
considers HWE and linkage equilibrium in a real
population. Departures from both of these assump-
tions led to the global sample being split into differ-
ent populations. The posterior K probabilities were
calculated assuming uniform prior values of K, in
our case, between 1 and 5 (USEPOPINFO = 0).
Once the most likely number of populations was
found, the analysis was repeated, but introducing
the model with prior geographic information
(USEPOPINFO = 1). We employed the admixture
model, because possible hybridization could occur
among different populations of C. globulosa (with
initial alpha value of 1 and maximum alpha value
of 10). Also, the correlation of allele frequencies
among populations varied with different values of
FST and different migration rates (m = 0.01, 0.05
and 0.2, respectively). The analysis shown here was
carried out with 1,000,000 iterations, following a
burn-period of 100,000 iterations. The analysis was
performed twice and provided completely convergent
results.
mtDNA analyses
Genetic diversity and heterogeneity analyses
The statistics used to determine the genetic diversity
within the three C. globulosa Island populations were:
the number of polymorphic sites (S), the haplotypic
diversity (Hd), the nucleotide diversity (π), the average
number of nucleotide differences (k) and the θ statistic
per sequence. For the mtDNA analyses, the two sam-
ples from the Puerto Nariño population were not
employed.
We also obtained genetic heterogeneity and theo-
retical gene flow estimates, among the populations.
Some statistics were applied to haplotypic frequencies
(GST statistic) and to nucleotide sequences (γST, NST
and FST Dxy and Da statistics; Hudson et al. 1992).
To determine possible differences among populations,
we employed exact tests (Raymond & Rousset 1995)
and FST statistics (Weir & Hill 2002). Gene diversity
 6 D. Alvarez-Prada & M. Ruiz-García

and genetic heterogeneity statistics were measured by Table 2. Allele frequencies at five microsatellite loci
means of the programs DNAsp 4.56 and Arlequin (CgAAT11, CgAAT32, CgAAT62, CgAAT82, and
CgAAT85) for Crax globulosa studied in three Amazonian
3.1. Mitochondrial gene flow estimates were obtained
islands (Mocagua, Colombia and Cacao and Yaumas,
from these statistics. Peru).
Marker Alleles (bp) Mocagua Cacao Yaumas
Phylogenetic analyses CgAAT11 210 0.013 0.000 0.000
The mtND2 sequence alignments were carried out 216 0.000 0.000 0.125
manually and with the DNA Alignment program 222 0.013 0.188 0.000
225 0.026 0.063 0.000
(Fluxus Technology, Charlotte, NC, USA). 228 0.192 0.313 0.250
Phylogenetic trees were obtained by means of genetic 231 0.244 0.063 0.000
distances (Kimura 2-P: Kimura 1980; Tamura 3-P: 234 0.295 0.063 0.050
Tamura 1992), maximum likelihood and maximum 237 0.205 0.313 0.125
parsimony with tree bisection and reconnection) 240 0.013 0.000 0.000
CgAAT32 159 0.429 0.200 0.000
with the programs Mega 5.05 (http://megasoftware. 162 0.000 0.100 0.000
net/) and PAUP*4.0b8 (www.paup.csit.fsu.edu). 165 0.143 0.200 0.000
171 0.429 0.300 0.500
Downloaded by [Manuel Ruiz-Garcia] at 16:03 26 June 2015

174 0.000 0.100 0.000

Demographic changes 177 0.000 0.100 0.500
CgAAT62 106 0.263 0.100 0.000
We used two procedures to determine possible histor- 109 0.026 0.000 0.000
ical population changes. (1) The mismatch distribu- 115 0.263 0.200 0.500
tion (pairwise sequence differences) was obtained 118 0.000 0.100 0.250
121 0.263 0.000 0.000
following the method of Rogers and Harpending
124 0.026 0.100 0.250
(1992) and Rogers et al. (1996) where two theoretical 127 0.000 0.100 0.000
curves were obtained (one for constant size and the 130 0.158 0.300 0.000
other considering population growth). We compared 136 0.000 0.100 0.000
these curves to the observed empirical distribution. CgAAT82 101 0.231 0.200 0.000
104 0.115 0.000 0.000
We used the raggedness rg statistic (Harpending
107 0.115 0.100 0.000
et al. 1993; Harpending 1994) to determine the simi- 110 0.231 0.200 0.000
larity between the observed and the theoretical curves. 113 0.077 0.200 0.000
(2) We used the Fu and Li D* and F* tests (Fu & Li 119 0.115 0.000 0.000
1993), the Fu FS statistic (Fu 1997), the Tajima D test 122 0.115 0.000 0.500
125 0.000 0.000 0.500
(Tajima 1989) and the R2 statistic of Ramos-Onsins
CgAAT85 187 0.016 0.000 0.000
and Rozas (2002) to determine possible population 190 0.032 0.125 0.125
size changes (Simonsen et al. 1995). 193 0.258 0.250 0.000
196 0.065 0.125 0.500
199 0.258 0.063 0.125
202 0.032 0.063 0.000
Results
205 0.097 0.250 0.000
Microsatellites 208 0.113 0.000 0.250
211 0.065 0.125 0.000
The marker CgAAT190 was excluded from the cur-
214 0.065 0.000 0.000
rent analysis because of its low reproducibility.
CgAAT85 showed 10 alleles for C. globulosa (187– bp = base pairs.
214 base pairs (bp), and also the C. rubra individuals
showed a similar range (193–205 bp). CgAAT11
showed nine alleles (210–240 bp for C. globulosa, populations are shown in Table 2. No microsatellite
234–249 bp for C. rubra). CgAAT62 had also nine pair showed gametic disequilibrium, which means
alleles (106–136 bp). For C. rubra they were 148–166 that each marker is independent from the others.
bp with alleles clearly larger than in C. globulosa. The mean H for the overall population of
CgAAT82 showed eight alleles (101–125 bp) and C. globulosa was 0.838 ± 0.051. This elevated gene
CgAAT32 yielded six alleles (159–177 bp), although diversity was extended for all the microsatellites stu-
the amplification rate of this last marker was lower died. CgAAT32 had the lowest value (H = 0.781),
than the other microsatellites. The gene frequencies of whereas CgAAT62 and CgAAT85 yielded the highest
the different alleles found in the five microsatellites values (H = 0.887 and 0.886, respectively). By sub-
analyzed for the Mocagua, Cacao and Yaumas populations, the largest sample (Mocagua) had the
 Studies on Neotropical Fauna and Environment 7

Table 3. Probability tests to estimate possible Hardy–Weinberg equilibrium individually and globally for the Crax globulosa
populations and five microsatellites.
Markers

Populations CgAAT11 CgAAT32 CgAAT62 CgAAT82 CgAAT85 Fisher’s test by populations

Mocagua 0.0014* 0.1608 0.0362* 0.1484 0.4378 0.0007* χ2 = 30.5, 10 df

Cacao 0.0976 0.6183 1 0.7010 0.5400 0.2713 χ2 = 12.2, 10 df
Yaumas 0.0286* 0.4327 0.3333 0.3589 1 0.1570 χ2 = 9.30, 10 df
Fisher’s test by markers 0.0030* 0.3278 0.1754 0.3334 0.2049 0.0050* χ2 = 50.8, 26 df
χ2 = 25.4 χ2 = 4.6 χ2 = 9.0 χ2 = 4.6 χ2 = 8.4
6 df 6 df 6 df 6 df 6 df

*Significant probability.

Table 4. Genetic diversity analysis for the populations of Crax globulosa using Nei’s and Wright’s F statistics.
Loci HO HS HT DST GST GST′ FIS FST FIT RST

AAT11 0.379 0.739 0.788 0.049 0.062 0.081 0.240** 0.044** 0.274** 0.077
Downloaded by [Manuel Ruiz-Garcia] at 16:03 26 June 2015

AAT32 0.933 0.776 0.779 0.003 0.004 0.007 −0.247 0.015 −0.229 0.240
AAT62 0.533 0.776 0.859 0.083 0.097 0.125 0.174 0.048* 0.214* 0.054
AAT82 0.942 0.895 0.883 −0.012 −0.013 −0.018 0.032 −0.018 0.015 0.229
AAT85 0.795 0.867 0.881 0.014 0.016 0.021 0.087 0.046 0.129* −0.05
Overall 0.717 0.810 0.838 0.027 0.033 0.043 0.061 0.027** 0.087* 0.117
Jackknife 0.062 ± 0.077 0.027 ± 0.013 0.088 ± 0.082
Bootstrap 99% confidence interval −0.157, 0.213 −0.006, 0.047 −0.131, 0.249

Ho = observed gene diversity in the total Crax globulosa population; HS = average gene diversity within the Crax globulosa subpopulations;
HT = expected gene diversity in the total Crax globulosa population; DST = absolute gene differentiation among subpopulations; GST = relative
genetic differentiation among subpopulations with regard to the total gene diversity; GST′ = the same as the previous statistics but corrected by
sample size; FIS – FST – FIT = Wright’s F-statistics; RST = Slatkin (1995)’s genetic heterogeneity statistic measured by allele variances.
*p < 0.05; **p < 0.01.

lowest gene diversity (H = 0.785 ± 0.0093), whereas Table 5. Overall genotypic and genic differentiation for the
Cacao and Yaumas showed the highest levels total population of Crax globulosa at five microsatellites.
(H = 0.873 ± 0.0356 and H = 0.861 ± 0.1273, respec-
Genotypic differentiation Genic differentiation
tively). Nevertheless, there was no significant differ-
ence among these three mean gene diversity values. Loci Probability SD Probability SD
Mocagua did not show HWE when all five mar-
CgAAT11 0.0525 0.0075 0.0131* 0.0014
kers were simultaneously taken into account CgAAT32 0.2254 0.0070 0.3426 0.0386
(p = 0.0007) due mainly to the influence of two mar- CgAAT62 0.0351* 0.0054 0.0082* 0.0009
kers which were not at HWE, CgAAT11 (p = 0.0014) CgAAT82 0.2878 0.0122 0.3465 0.0054
and CgAAT62 (p = 0.0362). Cacao and Yaumas did CgAAT85 0.0497* 0.0067 0.0181* 0.0014
not deviate from HWE globally (p = 0.2713 and *Significant probability.
p = 0.157, respectively) (Table 3). All the subpopula-
tions taken as a whole population significantly
deviated from HWE for all the microsatellites 0.062 ± 0.077 (99% confidence interval: −0.157–
(p = 0.005). For all of the subpopulations, 0.213; p = 0.0930), but it was not significant. Only
CgAAT11 was the only marker which significantly CgAAT11 showed a significant FIS value by homo-
deviated from HWE (p = 0.003). The deviations for zygote excess within the subpopulations (p = 0.0014–
all the cases were due to homozygote excess. 0.0002). The genetic heterogeneity measured using
The Wright’s F statistics (Table 4) showed a FST was of small but significant magnitude – assum-
significant excess of homozygotes (FIT = ing random mating (FST = 0.027 ± 0.013; 99, %
0.088 ± 0.082; 99% confidence interval: −0.131 – confidence interval: −0.006–0.047; p = 0.0002), but
0.249; p = 0.0286) at the total population level. not if non-random mating was assumed (p = 0.610–
The markers CgAAT11, CgAAT62, and CgAAT85 0.563) in which case no marker showed significant
showed significant values of FIT by homozygote heterogeneity. However, if random mating was
excess. The mean FIS yielded a value of assumed, the markers CgAAT11, CgAAT62, and
 8 D. Alvarez-Prada & M. Ruiz-García
Table 6. Microsatellite mutation model analysis by using the maximum likelihood procedure of Nielsen (1997).
Uni-step mutation model
Microsatellites θO θu log likelihood
CgAAT11 4.972 9.967 −29.8325
CgAAT32 9.341 8.968 −19.1657
CgAAT62 15.163 8.794 −27.4811
CgAAT82 13.093 17.544 −22.1219
CgAAT85 11.061 15.873 −28.0096
Mean 10.726 12.229
± 3.886 ± 4.155
θO = 4Neμ estimated by the moment method; θu and θm were estimated assuming a uni-step and a multi-step mutation model, respectively; %
multi-step (MSM) = most probable percentage of multi-step mutations; χ2 = test to measure if the percentage of multi-step mutations is
significantly better than the uni-step mutation model, 1 df; NS = non-significant values.
*p < 0.05, **p < 0.01.
CgAAT85 showed significant values of FST. The exact
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tests with genotypic and genic frequencies also yielded
that CgAAT11, CgAAT62, and CgAAT85 presented
significant genetic heterogeneity (Table 5). The mean
RST value showed a value slightly higher than the
mean FST value (RST = 0.1168 ± 0.109). The theoretical
gene flow values in all cases were higher than 1. The
gene flow estimates from FST and RST were 9 and 1.9,
respectively. The gene flow estimate from the private
allele method was 1.2. The gene flow estimates by popu-
lation pairs were Mocagua-Cacao (Nm = 21.5),
Mocagua-Yaumas (Nm = 4.7), and Cacao-
Yaumas (Nm = 8).
The results on the mutation models and the possi-
ble effective numbers are shown in Table 6. The mean
θ values for the uni-step mutation model
(θ = 12.229 ± 4.155) and for the multi-step mutation
model (θ = 14.094 ± 6.155) were relatively similar. In
other words, estimating the possible effective numbers,
the choice of the particular mutational model was
unimportant. With a mutation rate of 5.6 × 10–3, we
obtained an effective number of 545 individuals,
whereas we yielded 5459 individuals if a mutation
rate of 5.6 × 10–4 was selected. The mean percentage
of multi-step mutations was 3.5% and two markers,
CgAAT11 (2.5%; χ2 = 5.13, 1 df, p < 0.05) and
CgAAT62 (2.5%; χ2 = 9.16, 1 df, p < 0.01) presented
significant multi-step mutations models.
The analysis did not show clear evidence of bottle-
necks. Two mutation models were evaluated. For
IAM, CgAAT82 and CgAAT85 showed some evi-
dence of bottleneck (p = 0.0061 and p = 0.0279).
The standardized difference test and the Wilcoxon’s
signed rank test showed significant results in agree-
ment with a recent bottleneck (p = 0.014 and 0.0312,
respectively). However, for the SMM, no marker or
test showed any evidence of a bottleneck. As the
microsatellites follow more clearly a SMM than an
Multi-step mutation model
θm log likelihood % MSM χ2
10.441 −27.2656 2.50% 5.13*
7.193 −18.7716 0% NS
11.676 −32.0594 2.50% 9.16**
20.032 −21.0957 2.50% NS
21.127 −29.5162 10.0% NS
14.094 3.5%
± 6.155 ± 3.79%
IAM, our data did not reveal evidence of a recent
bottleneck for this very small population of C. globu-
losa. The same was obtained for the population of
Mocagua. For the IAM, CgAAT82 (p = 0.0096), the
standardized difference test and the Wilcoxon’s signed
rank test showed significant results in agreement with
a recent bottleneck (p = 0.008 and 0.0156, respec-
tively). But no test showed evidence of bottleneck
for the SMM. For the Cacao population, the situation
was the same than in the previous island. For Yaumas
and Puerto Nariño, the sample sizes were not large
enough to carry out this analysis.
The assignment analyses without simulations
showed that the worst procedure for determining the
genetic distance was DAS with “leave one out”, with
46.15% of the individuals correctly assigned to their
respective populations, and the best procedure was
the Bayesian method with “as is”, with 81% of the
individuals correctly assigned to their respective
populations. However, the assignment analyses with
simulations showed that the major part of the indivi-
duals could simultaneously belong to any of the popu-
lations analyzed. The two best procedures, which
classified the individuals to only one population,
were the DAS and Cavalli-Sforza and Edwards
(1967) genetic distances with the method “as is”
(31.37% and 29.41%, respectively). Thus, there was
no evidence of exclusive multi-genotypes within each
one of the populations.
According to the results of the Structure program,
if no geographic information was given, the allele
frequencies were presumed not to be correlated to
the assumed migration rates (m = 0.01, 0.05. 0.2);
and only one population (K = 1) was identified
(Table 7). If the allele frequencies were considered
correlated, K = 1 if m = 0.2, but K = 2 for
m = 0.01 and K = 4 if m = 0.05. When geographic
information was included into the program, the most
 Studies on Neotropical Fauna and Environment 9

Table 7. Number of possible different gene pools for the Crax globulosa populations analyzed using the Structure
program.
No correlated AF Correlated AF

Migration rates

Population m = 0.01 m = 0.05 m = 0.2 m = 0.01 m = 0.05 m = 0.2

Without information about the populations

K = 1 −607.4* −607.4* −607.4* −606.1 −601.1 −604.7*
K = 2 −612.5 −697.2 −618.1 −600.6* −605.1 −663.6
K = 3 −612.8 −766.9 −631.1 −619.6 −600.8* −619.1
K = 4 −612.8 −823.0 −631.4 −619.6 −600.8* −619.1
With information about the populations
K = 1 −607.4 −607.4 −607.4* −604.9 −607.4 −601.9*
K = 2 −606.0 −607.2 −610.3 −604.3* −603.0* −631.0
K = 3 −603.4* −604.6* −608.4 −614.1 −617.6 −614.9
K = 4 −605.3 −609.0 −622.2 −615.5 −611.0 −615.4

AF = allele frequencies. * = Most probable number of populations. K = number of populations.

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probable number of populations was K = 1 for mtDNA

m = 0.2 whether or not the allele frequencies were
correlated. When m was 0.01 and 0.5 and when there
were no correlated allele frequencies, K was equal to
3. However, when m was 0.01 and 0.05 and there
were correlated allele frequencies then K was equal
to 2. The historical value of m is probably quite high
since the population underwent fragmentation fairly
recently. Under this condition, a value of 1 for K is
highly probably and agrees quite well with the fact
that the Geneclass 2 program did not show very high
levels of correctly assigning individuals.
The mtND2 gene was 564 bp long. A total of 34
sites showed polymorphisms (S) and the total num-
ber of mutations was 44. Each of the 12 indivi-
duals sequenced showed a different haplotype.
Thus, the haplotype diversity was the maximum
possible (Hd = 1 ± 0.034). Also, other statistics,
e.g. π (=0.0228 ± 0.0021), k (=12.864), and θ per
sequence (=11.259), presented elevated levels of
gene diversity. Therefore, both microsatellites and
mitochondrial gene sequences showed very high
levels of gene diversity in the very small

Table 8. A, Global genetic heterogeneity at the mtND2 gene for the three Crax globulosa populations studied. B, Genetic
heterogeneity statistics (Kxy, Gst, Δst, γst, Nst, Fst and Da) of three Crax globulosa population pairs at the mtND2 gene.
(A)

Statistics Value Probability Statistics Nm

χ2 24.0, 22 df 0.3472 Gst = 0.0182 27.00

Hst 0.0 1.0000 γst = 0.2261 1.71
Kst 0.08787 0.0390* Nst = 0.1724 2.40
Kst* −0.00769 0.6030 Fst = 0.1709 2.43
Z 32.57778 0.4890
Z* 3.31871 0.6500
Snn 0.54167 0.1280

(B)

Pop 1 Pop 2 Kxy Gst Δst γst Nst Fst Da

Moc Cac 13.000 0.004 0.0025 0.119 0.027 0.026 0.0006

Moc Yau 14.250 0.040 0.0046 0.219 0.308 0.305 0.0077
Cac Yau 10.500 0.020 0.0037 0.236 0.167 0.166 0.0031

*p < 0.05. Moc = Mocagua; Cac = Cacao; Yau = Yaumas.

 10 D. Alvarez-Prada & M. Ruiz-García
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Figure 2. Neighbor-joining tree (unrooted) with the Tamura 3P genetic distance with 12 Crax globulosa individuals sampled in
three Colombian and Peruvian Amazon islands.
population of C. globulosa inhabiting the
Amazonian Colombian–Peruvian frontier.
The major part of all the genetic heterogeneity
statistics employed (Table 8) showed low levels of
genetic fragmentation. The theoretical female gene
flow was high, ranging from 1.71 to 27. When these
genetic heterogeneity estimates were obtained by
population pairs, the pair Mocagua–Yaumas pre-
sented elevated levels of genetic fragmentation
(NST = 0.308 and FST = 0.305) with Yaumas
being the geographically most distant population.
This may indicate that some isolation by distance
should exist among different C. globulosa popula-
tions in wider Amazon areas than those studied
here.
The genetic relationship among the 12 indivi-
duals sequenced is shown in a neighbor-joining
tree with the Tamura 3P genetic distance
(Figure 2). All the trees showed the same results.
The individuals from the three islands were inter-
mixed in the different branches of the tree, which
agree with the results of Geneclass 2 and Structure.
The different tests applied to detect some
population changes revealed negative values
(D = −0.533; D* = −0.113; F* = −0.255,
p > 0.1), which is in agreement with a population
expansion, but the sample size is too small to obtain
statistically significant results. Nevertheless, the mis-
match distribution (pairwise sequence differences)
showed that the observed distribution fitted very
well with an expected distribution typical of a popu-
lation expansion and not with a distribution of a
constant population size (Figure 3).
Discussion
General findings
The main findings of this work, with a potential bio-
logical conservation interest, were the high levels of
gene diversity found in the studied area, although the
population size of C. globulosa in these Colombian–
Peruvian islands is extremely small and there is a
limited genetic heterogeneity found for both microsa-
tellites and mtDNA. Additionally, the low assignment
power of the multi-genotypes to exclusive populations
 Studies on Neotropical Fauna and Environment 11
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Figure 3. Mismatch distribution (pairwise sequence differences) for the mtND2 gene for the Crax globulosa population
analyzed. A, Assuming a constant population size; B, assuming a population expansion. The analysis showed a clear and
significant population expansion.
 12 D. Alvarez-Prada & M. Ruiz-García
being this compatible with a unique gene pool in the
studied area, the lack of evidence of recent bottle-
necks for the microsatellites and some evidence of
historical population expansion with the mtDNA
were other outstanding findings.
A potential problem when obtaining DNA from
feathers is that it can be of poor quality, which may
result in preferential amplification so that heterozy-
gote individuals could amplify as homozygote ones
(Taberlet et al. 1999). However, the use of tri-nucleo-
tide microsatellites, as well as high levels of gene
diversity, decreases the risk of preferential amplifica-
tion (Taberlet et al. 1999). Both conditions are present
in the current study.
This study showed that, although some microsa-
tellites could evolve following a multi-step mutation
model, the results are very similar to the results we
would have obtained if we had analyzed the data
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assuming a general uni-step mutation model, because
the average θ values were very similar in both muta-
tion models and the percentage of multi-step mutation
was lower than 4%.
Gene diversity
Our study determined the largest number of alleles in
the five microsatellites analyzed relative to the pre-
vious study of Hughes and Larson (2000) with captive
stocks of C. globulosa in the US (23 individuals). This
means that these captive birds had lost some of the
gene diversity present in the wild populations studied
by us. The levels of expected H we found were sig-
nificantly higher than those for captive C. globulosa in
the USA (H = 0.71; Hughes & Larson 2000), for C.
alberti (H = 0.65; Diana Ramirez-Arias, pers. comm.)
and for captive and wild populations of C. fasciolata
from Brazil (H = 0.68, both populations, Gonçalves
et al. 2010). For the wild population of C. fasciolata
studied by Pereira and Wajntal (2001a) Gonçalves
et al. (2010) found levels of genetic diversity similar
to those of two reintroduced Guan (Penelope spp.)
populations established from a small number of cap-
tive founders. This evidence suggests that the wild C.
fasciolata population may have lost some of its origi-
nal genetic variation as a result of habitat loss and
population decline over the past few decades. This
does not seem to be the case for the Colombian–
Peruvian Amazon C. globulosa population.
Therefore, although the populations were extremely
small, their nuclear gene diversity was high. This is
good news for the conservation of the species because,
even with a small number of breeders, the evolution-
ary potential of the Colombian–Peruvian Amazon
populations is still very high.
The same was discovered for mtDNA. The values
of Hd and π were substantially elevated (1 and 0.023,
respectively). For example, Zink et al. (2002) studied
60 samples of Dendrocopus major (Picidae) from 17
localities within Eurasia for three mitochondrial
genes. They estimated Hd = 0.81 and π = 0.0056 – a
substantially lower mitochondrial gene diversity than
that found for three Amazonian islands separated by
around 55 km. This means that the small C. globulosa
population analyzed has more mitochondrial gene
diversity than other bird species with wide continental
distribution, which is extremely positive for the evolu-
tionary potential of C. globulosa in the Amazon.
Even though C. globulosa populations have
decreased in the Amazonian area in recent decades
(Bennett 2003), no clear evidences of recent bottle-
necks were detected in the populations probably
because the genetic diversity has traditionally been
very high, so the recent demographic trend has not
yet affected the genetic variability. The same trend
was discovered for other Amazonian species, such as
the jaguar, Panthera onca, by Ruiz-García et al.
(2006, 2007, 2013). This species was intensively
hunted during the 1960s and 1970s. In only two
years (1968–1970) 2000 jaguar skins were removed
from the Peruvian Department of Loreto. However,
the level of microsatellite gene diversity for the jaguar
population of the Peruvian Amazon, was above 0.80
– revealing a very high value. Thus, although some
population sizes have significantly decreased, they still
conserve elevated gene diversities and elevated evolu-
tionary potential. This seems to be the case for the
Colombian–Peruvian population of C. globulosa.
One explanation for the extreme gene diversity
suggested by Pereira et al. (2002) is the old age of
the family Cracidae. It is one of the oldest bird
families (64–90 MYA, with an average of 76 MYA),
and the genus Crax seems to have diverged on aver-
age around 10.0 MYA, with an interval of 8.4–11.9
MYA, during the Late Miocene (Pereira et al. 2002).
As one of the first bird families to appear, its popula-
tion size could have quickly expanded and, through-
out a long evolutionary history, could have
accumulated many mutations and therefore main-
tained high levels of gene diversity until today as has
been shown in human populations (Shriver et al.
1997). This hypothesis could be tested in future stu-
dies because other Cracidae genera are even older
than Crax (Ortalis, 30.9 MYA, with interval of
25.8–36.5 MYA, or Oreophasis, 31.1 MYA, with an
interval of 26.6–36.1 MYA) and they might have
greater gene diversity levels than Crax, whereas
other genera younger than Crax (divergence between
the current Aburria and Pipile, 3.8 MYA, with an
interval of 3.2–4.5 MYA; Pereira et al. 2002; Grau

et al. 2005) should have lower gene diversity than
Crax if this hypothesis is supported.
A second explanation, which could contribute to
an elevated gene diversity within this species, is that
it could be polygamous. Traditionally, Cracidae has
been considered monogamous (Delacour & Amadon
2004), but some species (C. daubentoni) might be
polygamous as demonstrated by Strahl et al. (1997)
and Buchholz (1991). In monogamous species, males
and females have non-important secondary sexual
differences, whereas in polygamous species there is
considerable sexual dimorphism. The case of C. glo-
bulosa is similar to C. daubentoni and thus a poly-
gamous pattern is probable (Sara Bennett, pers.
comm.). Polygamous males could have a greater
capacity to migrate looking for females. The posses-
sion of such a reproductive system over the evolu-
tionary long term could maintain an elevated
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gene diversity in extremely small demographic
populations.
Genetic pools and effective population sizes
The populations analyzed seem to belong to one
unique genetic pool, although the area has been frag-
mented in recent decades. This agrees quite well with
the fact that the Geneclass program did not have
elevated percentages for the assignation of individuals
to each population. Additionally, the theoretical esti-
mations of Nm were always higher than 1. Similarly,
mtDNA showed intermixed haplotypes in the tree
independent of the geographic origins of the indivi-
duals analyzed and elevated values of gene flow esti-
mates among the populations studied, with the
exception of the Mocagua–Yaumas pair, which is
the most distant one (this could be a preliminary
evidence of some isolation by distance). Evidence of
incipient gene drift could be the non-HWE of some
markers by homozygote excess. This is especially true
when all the populations are taken as a unique popu-
lation (Wahlund effect with a slight genetic sub-struc-
turation) as well as when the value of FST, although
very low (0.027), is significant. This contrasted with
that observed in C. alberti in northern Colombia,
where no genetic structure was observed (Diana
Ramirez-Arias, pers. comm.). Conversely, Gonçalves
et al. (2010) found that wild C. fasciolata populations
from Brazil were genetically impoverished and
structured.
The effective population sizes obtained for two
possible microsatellite mutation rates (5.6 × 10–3 and
5.6 x 10–4) varied from 545 to 5460 breeders.
Brohede et al. (2004) estimated a range of microsa-
tellite mutation rates from 1.1 × 10–2 to 3.2 × 10–2,
with an average of 1.81 × 10–2 for four populations
Studies on Neotropical Fauna and Environment 13
of Hirundo rustica. Ortego et al. (2008) determined a
mutation rate of 2.96 × 10–3 for a raptor bird (Falco
naumanni) and Anmarkrud et al. (2011) estimated,
for six species of Hirundinidae (Delichon, Hirundo,
Petrochelidon, Progne, Riparia, and Tachycineta),
microsatellite mutation rates ranging from
1.01 × 10–1 to 6 × 10–3. Therefore, the first mutation
rate is probably optimal for birds such as Crax. The
historic effective size could then be closer to 545, so
the effects of gene drift could be more considerable.
Henceforth, from a conservation point of view, a
larger area inclusive of a metapopulation could be
managed, and translocations of individuals among
different islands could preserve unique smaller popu-
lations from the future effects of gene drift. The fact
that all of the specimens of this area of the
Colombian and Peruvian Amazon River could
belong to a unique gene pool is important. Any
translocation could help to increase gene flow and
to avoid the negative effects of gene drift (Slatkin
1987). Probably, in this case, translocations did not
cause a reduction in fitness related to outbreeding
depression, which could cause lost or alteration of
coadapted gene complexes (Shields 1982; Templeton
1986).
Species of the Crax genus are not strong fliers.
However, C. globulosa might have a greater flying
ability than other Crax species because it is more
arboreal (Sara Bennett, pers. comm.). No migration
among islands has been observed, although some C.
globulosa might be able to fly between Mocagua and
Cacao during the low water period of the Amazon
River. Therefore, the elevated gene flow estimates we
obtained were not a consequence of the very recent
fragmentation, but rather due to past gene flow. For
this reason, translocation of some individuals among
islands (after pathogen analyses) could be an effective
measure in maintaining a high gene diversity, and
could help to avoid future negative consequences of
inbreeding and gene drift in these very small
populations.
There may be two interesting conservation
aspects for this species in Colombia. First, it may
be noteworthy to carry out an ex situ conservation
program of C. globulosa. Although the Mocagua
population presented high gene diversity, its census
size has been small (around 150 animals). Thus,
natural or anthropogenic disasters could easily elim-
inate this population. Second, it is very important to
analyze the other C. globulosa population on Mirití
Island in the Caquetá River (in the Colombian
Amazon). It would be of value to know if this
second very small population is an extension of the
gene pool already studied or if it represents another
different gene pool which needs a different in situ
 14 D. Alvarez-Prada & M. Ruiz-García
and ex situ conservation program from the Mocagua
population.
Furthermore, we suggest that future studies ana-
lyze the possible genetic structure of this species in
other Peruvian, Bolivian and Western Brazilian
Amazon localities to have a complete picture of the
genetics and evolution of this species and to determine
if all the populations of this species have elevated gene
diversity levels.
Acknowledgments
Thanks to Dr. Sara Bennett and the participants of the “Piurí”
project for their help in obtaining samples. Thanks to Dr. C.
Hughes for sending primers of the microsatellites. Thanks to Kelly
Luengas for helping with the figures. Dr. Joseph Shostell helped with
the English syntax. We also thank Dr. Anne Zillikens, Dr. Angela
Schmitz Ornés, and two anonymous reviewers for the comments and
Downloaded by [Manuel Ruiz-Garcia] at 16:03 26 June 2015
corrections on an early version of the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors thank Tropenbos Internacional Colombia and
Conservación Internacional (IEA; grants [110785-2006] in memor-
iam of Jorge Ignacio Hernández) for their financial support.
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