Kinship Analysis of Offspring of the Giant South American River Turtle

(Podocnemis expansa) Using Microsatellite DNA Markers

Author(s): Cleiton Fantin, Jorge Ferreira, Mara Magalhães, Thais da Silva Damasseno, Dorothy Ivila de
Melo Pereira, and Richard Carl Vogt
Source: Chelonian Conservation and Biology, 16(2):123-127.
Published By: Chelonian Research Foundation
https://doi.org/10.2744/CCB-1233.1
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 Chelonian Conservation and Biology, 2017, 16(2): 123–127
doi:10.2744/CCB-1233.1
Ó 2017 Chelonian Research Foundation

Kinship Analysis of Offspring of the Giant South American River Turtle

(Podocnemis expansa) Using Microsatellite DNA Markers

CLEITON FANTIN1,*, JORGE FERREIRA1, MARA MAGALHÃES1, THAIS DA SILVA DAMASSENO2,

DOROTHY IVILA DE MELO PEREIRA1, AND RICHARD CARL VOGT2
1
Laboratório de Proteômica e Genômica, Universidade do Estado do Amazonas, Manaus, Brasil
[cleitonfantin@hotmail.com; biologojorge@hotmail.com; mara_jhs@hotmail.com; dorothyivila@hotmail.com];
2
Instituto Nacional de Pesquisas da Amazônia, Manaus, Brasil [thaisdamasseno@gmail.com; vogt@inpa.gov.br]
*Corresponding author

ABSTRACT. – The giant South American river turtle (Podocnemis expansa) is the largest extant
member of the suborder Pleurodira and the largest freshwater chelonian in South America.
Owing to its size, its meat is sought for consumption and trade in the Amazon region. The aim of
the present study was to investigate the reproductive behavior of 2 different populations of P.
expansa. Allelic frequency variation was studied in 6 DNA microsatellite loci of recently hatched
offspring from 8 nests on a beach in the municipality of Oriximiná in the state of Pará, Brazil.
Multiple paternity was identified in all nests studied. Between 5 and 10 fathers contributed to each
nest. Compared with previous studies, a greater frequency of multiple paternity and a greater
number of fathers contributing to each nest were found in the present investigation. The results
suggest that populations from different locations may exhibit different paternity patterns due to
ecological or biological differences. By comparison with previous studies, we suggest that the
differences in the number of nests, proportion of offspring per nest, and number of loci analyzed
in each study may influence the frequency of multiple paternity detected.
KEY WORDS. – reproduction; polyandry; multiple paternity; chelonians; Brazilian Amazon

The giant South American river turtle (Podocnemis
expansa) is the largest member of the suborder Pleurodira
and the largest freshwater chelonian in South America
(Pritchard and Trebbau 1984). This turtle has a wide,
smooth carapace with black and gray coloration (Vogt
2008) and occurs in the Amazon, Orinoco, and Essequibo
river basins (Pritchard and Trebbau 1984). It has a
relatively prolonged sexual maturity and the replacement
of individuals in populations is low because of the high
mortality rate of offspring (Pritchard and Trebbau 1984).
Due to its large size, which exceeds 90 kg in body
weight and 80 cm in carapace length (Pritchard and
Trebbau 1984), P. expansa is heavily exploited for food
and trade for the consumption and direct sale of its meat,
viscera, and eggs, or for the use of its carapace in
craftwork (Cantarelli 2006). These factors contribute to the
listing of P. expansa as Endangered (International Union
for Conservation of Nature 2016). Although some aspects
of the reproductive behavior of P. expansa have previously
been described (Valenzuela 2000; Pearse et al. 2006b),
studies on this species are lacking from throughout most of
its range. Thus, there is a need to identify and understand
the ecological factors linked to its reproductive behavior to
provide information that can be used for the adequate
management of the species.
We present comparative findings on 2 different
populations of P. expansa using larger and more
homogenous samples sizes among nests, compared with
previous studies. The aim of the study was to investigate
the reproductive behavior of P. expansa determined by the
degree of kinship of recently emerged hatchlings from a
nesting beach in the municipality of Oriximiná (state of
Pará, northern Brazil), as inferred from hypervariable
microsatellite loci.
METHODS
Blood samples were obtained from recently hatched
specimens of P. expansa at Jacaú beach on the Trombetas
River, the municipality of Oriximiná, the state of Pará,
Brazil. A total of 221 individuals distributed among 8
nests were evaluated. Approximately 50 l of blood was
collected from each individual through the venous
puncture and stored in 500 ll of ethanol at 48C (Avery
and Vitt 1984). All individuals were then released at their
original capture sites.
DNA extraction was performed using 2% CTAB
(cetyl trimethylammonium bromide) extraction buffer
(Doyle and Doyle 1987). Extraction efficacies and DNA
concentrations were determined by electrophoresis in 1%
agarose gel, which was examined under an ultraviolet light
transilluminator. We followed the protocol described by
Schuelke (2000) and used nested polymerase chain
reaction (PCR) to amplify hypervariable regions for 6
pairs of oligonucleotides. Two of these were developed for
Podocnemis unifilis (Fantin et al. 2007) and 4 others were
developed for P. expansa (Sites et al. 1999; Valenzuela
124 CHELONIAN CONSERVATION AND BIOLOGY, Volume 16, Number 2 – 2017

inference of families of true siblings is possible even

Table 1. Characteristics of microsatellite loci used for paternity
analysis of P. expansa. without having knowledge of the parental genotype, as this
program uses information on shared alleles at multiple loci
Temperature among individuals from each nest.
Locus Repeat unit (8C) Source
Puni_1D12 (GA)10 55 Fantin et al. (2007) RESULTS
Puni_1E1 (CT)9TT(CT)7 64 Fantin et al. (2007)
PE344 (AG)13 50 Valenzuela (2000) Based on the minimum method allele counts, multiple
PE1075 (AC)11 54 Valenzuela (2000) paternity was found in all nests analyzed (Table 2). When
Pod1 (CA)32 56 Sites et al. (1999)
Pod91 G9(GT)17(GA)8 55 Sites et al. (1999) it was not possible to infer parental genotypes, we inferred
a minimum of 2 paternal contributions to the same nest
when at least 5 alleles were found for the same locus
2000; Table 1). Amplification reactions were adjusted to a among the offspring: 2 from the mother and 3 (or more)
total volume of 13.5 ll containing 1.5 ll of 103 from at least 2 contributing fathers. When one of the
(NH4)2SO4 buffer, 2.7 mM MgCl2, 0.2 lM reverse primer, maternal alleles could be identified, the occurrence of 4
0.14 lM forward primer, 0.14 lM TET-6 M13 primer, 0.2 different alleles of the same locus was considered
mM (each) dNTP, 0.4 U of Taq DNA polymerase, and indicative of multiple paternity. However, when both
approximately 100 ng of genomic DNA. The initial maternal and paternal alleles could be identified through
denaturation temperature was 948C for 1 min; followed by the examination of homozygous offspring, the presence of
25 cycles at 948C for 30 sec, 558C for 30 sec, and 688C for 3 alleles segregating at one locus confirmed multiple
30 sec; followed by 20 cycles at 948C for 30 sec, 508C for paternity.
30 sec, and 688C for 30 sec; with a final extension at 728C The number of alleles per locus ranged from 4 to 22,
for 15 min. with locus 91 (Nest 1) being the least polymorphic and
Amplified DNA fragments were separated by electro- locus 1 (Nest 5) the most polymorphic. Nest 4 had the
phoresis on a 1% agarose gel, after which all PCR fewest paternal contributors (5 fathers) and Nest 5 had the
products were then diluted in distilled water in a ratio of most (10 fathers). Table 2 summarizes the number of
1:100, followed by the addition of the size marker ROX alleles per nest, the inferred number of males that
pUC-19, as modified from the protocol described by contributed to the offspring of each nest, and the
DeWoody et al. (2004). Genotyping was performed in an combination of all loci used to infer sibling groups, as
ABI 3130XL automatic DNA sequencer and analyses of estimated using Kinalyzer.
allele frequencies for each locus were performed with the
aid of the GeneMarker V2.2.0 program (Applied Biosys- DISCUSSION
tems).
Paternity estimates were based on the minimum Polyandry and multiple paternity have been found in a
method of allele counts (Myers and Zamudio 2004), wide range of animal taxa, including mammals such as
which presupposes a Mendelian distribution of alleles in Mus musculus musculus (Thonhauser et al. 2014); insects
the offspring. However, this method does not estimate an of the order Hymenoptera (Jaffé et al. 2012); crocodilians
actual number of contributing parents to a single nest; it is such as Melanosuchus niger (Muniz et al. 2011),
conservative because it cannot distinguish among parents Crocodylus porosus (Lewis et al. 2013), and Caiman
sharing homologous alleles. Thus, multilocus combinato- crocodilus (Oliveira et al. 2014); amphibians such as
rial analyses were also performed using the program Chiromantis xerampelina (Byrne and Whiting 2008) and
Kinalyzer (Berger-Wolf et al. 2007), to estimate relation- Crinia georgiana (Roberts et al. 1999); and birds,
ships between true siblings and half-siblings of the same including Ficedula albicollis (Michl et al. 2002), Ember-
nest and to assign paternity and infer mating systems. The iza schoeniclus, and Malarus cyaneus (Griffith et al.

Table 2. Paternity analyses of P. expansa nests examined in the present study, with minimum number of fathers inferred through simple
allele counts and number of sibling groups inferred by Kinalyzer.

Microsatellite alleles per locus

Total no. Minimum no. No. of sibling
Nest of offspring 1 91 1E1 344 1075 1D12 of fathers groups
N1 21 13 4 9 15 13 11 7 9
N2 30 15 5 16 14 10 13 7 13
N3 26 12 5 14 19 14 12 9 12
N4 24 7 9 4 12 7 — 5 9
N5 30 22 6 8 16 7 — 10 12
N6 30 17 7 8 10 7 — 8 12
N7 30 15 6 13 13 — 11 7 12
N8 30 15 13 10 20 — 11 9 13
 FANTIN ET AL. — Kinship Analysis of Podocnemis expansa Offspring
Table 3. Multiple paternity in the genus Podocnemis.
No. (range)
Species (source) of hatchlings
P. unifilis (Fantin et al. 2008) 66 (9 12)
P. erythrocephala (Fantin et al. 2010) 37 (5 8)
P. sextuberculata (Fantin et al. 2015) 80 (8 12)
P. expansa (Valenzuela 2000) 65 (19 46)
Pearse et al. (2006b) 885 (9 76)
Present study 221 (21 30)
2002). In chelonians, high levels of extra-pair paternity
have been found in multiple species: Chrysemys picta
(Pearse et al. 2001, 2002), Caretta caretta (Moore and
Ball 2002), Chelonia mydas (Lee and Hays 2004;
Ekanayake et al. 2013), and Emys blandingii (Refsnider
2009), and in other species of the genus Podocnemis,
namely P. unifilis (Fantin et al. 2008), Podocnemis
sextuberculata (Fantin et al. 2008), and Podocnemis
erythrocephala (Fantin et al. 2010).
Different hypotheses have been suggested to explain
the polyandrous behavior exhibited by species represent-
ing different phyla of animals. Different costs and benefits
are derived from polyandry, so many factors may
contribute to the evolution of this type of reproduction.
Researchers suggest that polyandry and multiple paternity
increase the odds of fertilization, the acquisition of more
competitive gametes, and the viability of the offspring
through increased genetic diversity (FitzSimmons 1998;
Jennions and Petrie 2000; Uller and Olsson 2008;
Thonhauser et al. 2014).
According to Yasui (1997), the probability of
acquiring genes that increase offspring fitness is higher
when females are promiscuous (the ‘‘good genes’’ and
‘‘compatibility of genes’’ hypotheses). Myers and Zamudio
(2004) suggest that polyandry could have evolved in
environments in which optimal environmental conditions
are only intermittently available, thus favoring promiscu-
ous females. According to Slatyer et al. (2012), polyandry
is favored by natural selection when mating with multiple
males provides females with more nutrients contained in
sperm. The same authors also suggest that such substances
may increase in number and/or quality of eggs, which then
provides an effective increase in the clutch size.
Multiple paternity within P. expansa. — The first
evidence of multiple paternity in P. expansa was
demonstrated by Valenzuela (2000) in nests on the Caquetá
River in Colombia. Subsequently, Pearse et al. (2006b)
found evidence multiple paternity in P. expansa nests on
the Orinoco River in Venezuela (Table 3). Sample sizes
between these studies likely account for the considerable
differences in frequency of multiple paternity of P.
expansa. Valenzuela (2000) studied 2 nests with 19 and
46 offspring and found multiple paternity in both, while
Pearse et al. (2006a) analyzed 32 nests containing 9 to 76
offspring per nest and found multiple paternity in only
10%. These differences in frequency of multiple paternity
125
Clutches multiply sired
(no. of contributing males) No. of DNA markers
6 of 6 (at least 2) 8
5 of 6 (at least 2) 4
11 of 12 (at least 2) 6
2 of 2 (at least 2) 8
3 of 32 (at least 2) 7
8 of 8 (at least 5) 6
might be explained by any combination of different factors,
including perhaps the dramatic decline in nesting females
on the single nesting beach sampled in Venezuela.
First, both nests studied by Valenzuela (2000) in the
Rio Caqueta basin had moderate to large clutch sizes,
while Pearse et al. (2006b) showed that in the Rio Orinoco
basin, nests with smaller clutch sizes had single paternity
and multiple paternity was documented only in the larger
clutches. Low fecundity of many of the Orinoco female P.
expansa may be an artifact of the low statistical power of
the combination of loci used in this study; rare alleles
would likely be missed.
A second factor that may contribute to the differences
between these river basins would be impacts of human
exploitation. Historically in the Orinoco basin, the
numbers of P. expansa nesting females decreased
drastically from 330,000 (Humboldt 1820) to 13,800
(Ojasti 1967). Although P. expansa nesting is protected by
the Ministry of the Environment on a single beach on the
Orinoco River with only ~ 1000 females (Pearse et al.
2006b), Mogollones et al. (2010) affirmed that this turtle
population seemed to be stable by the time captive rearing
and reintroduction programs were initiated in 1989.
Despite these conservation efforts, the earlier estimates
of the severe population decline of nesting P. expansa on
this single beach constitutes a severe bottleneck and if it
was sufficient to eliminate low-frequency alleles, then
many males would share only high-frequency alleles at
most loci and the markers used by Pearse et al. (2006b)
likely would not have sufficient resolution to identify low-
frequency cases of multiple paternity.
Regarding estimates of the number of paternal
contributors to P. expansa nests, Valenzuela (2000) found
a minimum of 2 fathers in one nest and 3 in the other, with
19 and 46 hatchlings, respectively. Pearse et al. (2006b)
found only 3 of 32 multiple-sired clutches, with a
minimum of 3 fathers contributing to clutches of 20
hatchlings, 71 hatchlings, and 70 hatchlings. In our study,
all clutches had multiple fathers, with a minimum
contribution of 5 males in Nest 4 and a maximum of 10
males in Nest 5. In part, some of the differences among
these 3 studies likely reflect differences in sampling
efforts, including variation in 1) the number of nests
sampled, 2) clutch size per nest, 3) number of loci
sampled, and 4) number of alleles per locus. Some
combination of these variables will influence paternity
126 CHELONIAN CONSERVATION AND BIOLOGY, Volume 16, Number 2 – 2017
estimates; for example, in a demographic study of the
North American turtle Chrysemys picta, Pearse et al.
(2002) showed that the likelihood of detection of multiple
paternity increased with the number of nests and number
of offspring per nest sampled.
Among biological variables, genotypic diversity of
males in a population will exert a direct influence on the
calculation of the paternal contribution to offspring.
When any particular locus with few alleles is included,
different individuals within the breeding population in
question are likely to sometimes share the same
genotype, which can lead to an underestimation of the
number of males that contribute to the production of
offspring. This error can be minimized by sampling many
nests, many hatchlings per nest, and more variable loci;
all of these variables will improve estimates of genetic
variability among the males within a breeding population,
which would improve estimates of male contributions to
single clutches.
Multiple paternity in the genus Podocnemis. —
Paternity studies of different species of the genus
Podocnemis also report the existence of polyandry. Fantin
et al. (2008) analyzed the reproductive behavior of P.
unifilis on a beach of the Amazon River (Brazil) and found
that at least 2 males contributed to all of the clutches
tested. These same authors also reported multiple paternity
in P. sextuberculata nests on the Amazon River in the
municipality of Barreirinha, Brazil; at least 3 males
contributed to the offspring in a single nest, and at least
2 males sired the offspring of all other nests. Fantin et al.
(2010) found that P. erythrocephala is also promiscuous,
reporting multiple paternity in 83% of nests analyzed in 2
locations of the Brazilian Amazon. Single paternity was
found in one nest of this species, demonstrating the
possibility of differences in the paternity pattern among
populations of the species (Table 3).
Our study underscores the importance of considering
ecological differences among populations. The scarcity of
resources is related to a reduction in population density
and the number of copulations (Engqvist 2011). The
higher the population density, the more likely the chances
of encounters that could result in mating (Taylor et al.
2014). According to Crim et al. (2002), sex ratio is also an
important factor to be considered, given that populations
with male-biased sex ratios are more likely to exhibit
multiple paternity.
In kinship analysis, we suggest that larger and more
homogenous sample sizes among nests be considered in
order to minimize among-locality ecological or biological
variation when comparing different populations of P.
expansa, given that such variation could influence the rate
of multiple paternity found.
ACKNOWLEDGMENTS
The authors are grateful to the ‘‘Pé de Pincha’’
Chelonian Management Project for assistance in the
collection of the samples. Permission to collect tissue
samples was granted by RAN/IBAMA, no. 113/2006. This
study was funded by a grant from the State of Amazonas
Research Assistance Foundation (FAPEAM).
LITERATURE CITED
AVERY, H.W. AND VITT, L.J. 1984. How to get blood from a turtle.
Copeia 1984:209–210.
BERGER-WOLF, T.Y., SHEIKH, S.I., DASGUPTA, B., AND ASHLEY,
M.V. 2007. Reconstructing sibling relationships in wild
populations. Bioinformatics 23:49–55.
BYRNE, P.G. AND WHITING, M.J. 2008. Simultaneous polyandry
increases fertilization success in an African foam-nesting
treefrog. Animal Behavior 76:1157–1164.
CANTARELLI, V.H. 2006. Alometria reprodutiva da tartaruga-da-
Amazônia (Podocnemis expansa): bases biológicas para o
manejo. MS Thesis, Universidade de São Paulo, São Paulo,
Brazil.
CRIM, J.L., SPOTILA, L.D., SPOTILA, J.R., AND O’CONNOR, M. 2002.
The leatherback turtle, Dermochelys coriacea, exhibits both
polyandry and polygyny. Molecular Ecology 11:2097–2106.
DEWOODY, J.A., SCHUPP, J., KENEFIC, L., AND BUSCH, J. 2004.
Universal method for producing rox-labeled size standards
suitable for automated genotyping. Biotechniques 37:348–
352.
DOYLE, J.J. AND DOYLE, J.L. 1987. Isolation of plant DNA from
fresh tissue. Focus 12:13–15.
EKANAYAKE, E.M.L., KAPUPUSHINGUE, T., SAMAN, M.M., RATHNA-
KUMARA, D.S., SAMARAWEERA, P., RANAWANA, K.B., AND
RAJAKARUNA, R.S. 2013. Paternity of green turtle (Chelonia
mydas) clutches laid at Kosgoda, Sri Lanka. Herpetological
Conservation and Biology 8:27–36.
ENGQVIST, L. 2011. Male attractiveness is negatively genetically
associated with investment in copulations. Behavioral Ecology
22:345–349.
FANTIN, C., CARVALHO, C.F., HRBEK, T., SITES, J.W., JR., MONJELÓ,
L.A.S., ASTOLFI-FILHO, S., AND FARIAS, I.P. 2007. Microsatellite
DNA markers for Podocnemis unifilis, the endangered yellow-
spotted Amazon River turtle. Molecular Ecology Resources 7:
1235–1238.
FANTIN, C., FARIAS, I.P., MONJELÓ, L.A.S., AND T. HRBEK. 2010.
Polyandry in the redheaded river turtle Podocnemis eryth-
rocephala (Testudines, Podocnemididae) in the Brazilian
Amazon. Genetics and Molecular Research 9:435–440.
FANTIN, C., PEREIRA, D.I.M., FERREIRA, J.F., MACHADO, P.C.,
MONJELÓ, L.A., AND FARIAS, I.P. 2015. Evidence of multiple
paternal contribution in Podocnemis sextuberculata (Testu-
dines: Podocnemididade) detected by microsatellite markers.
Phyllomedusa 14:89–97.
FANTIN, C., VIANA, L.S., AND FARIAS, I.P. 2008. Polyandry in
Podocnemis unifilis (Pleurodira; Podocnemididae), the vul-
nerable yellow-spotted Amazon River turtle. Amphibia-
Reptilia 29:479–486.
FITZSIMMONS, N. 1998. Single paternity of clutches and sperm
storage in the promiscuous green turtle (Chelonia mydas).
Molecular Ecology 7:575–584.
GRIFFITH, S.C., OWENS, I.P.F., AND THUMAN, K.A. 2002. Extra pair
paternity in birds: a review of interspecific variation and
adaptive function. Molecular Ecology 11:2195–2212.
HUMBOLDT, A. VON. 1820. Voyages aux Regions Équinoxiales du
Nouveau Continent Fait en 1799, 1800, 1801, 1802, 1803 et
1804 par A. von Humboldt et A. Bonpland, Tôme IV. Paris:
Librairie Grecque-Latine.Allemagne, 413 pp.
 FANTIN ET AL. — Kinship Analysis of Podocnemis expansa Offspring
INTERNATIONAL UNION FOR CONSERVATION OF NATURE (IUCN).
2016. IUCN Red List of Threatened Species. Version 2016.
http://www.iucnredlist.org/details/summary/17822/0 (15 April
2017).
JAFFÉ, R., GARCÍA-GONZÁLEZ, F., DEN BOER, S.P.A., SIMMONS,
L.W., AND BAER, B. 2012. Patterns of paternity skew among
polyandrous social insects: what can they tell us about the
potential for sexual selection? Evolution 66:3778–3788.
JENNIONS, M. AND PETRIE, M. 2000. Why do females mate
multiply? A review of the genetic benefits. Biological Review
of the Cambridge Philosophical Society 75:21–64.
LEE, P.L.M. AND HAYS, G.C. 2004. Polyandry in a marine turtle:
females make the best of a bad job. Proceedings of the
National Academy of Sciences of the United States of America
101:6530–6535.
LEWIS, J.L., FITZSIMMONS, N.N., JAMERLAN, M.L., BUCHAN, J.C.,
AND GRIGG, G.C. 2013. Mating systems and multiple paternity
in the estuarine crocodile (Crocodylus porosus). Journal of
Herpetology 47:24–33.
MICHL, G., TÖRÖK, J., GRIFFITH, S.C., AND SHELDON, B.C. 2002.
An experimental analysis of sperm competition mechanisms in
a wild bird population. Proceedings of the National Academy
of Sciences of the United States of America 99:5466–5470.
MOGOLLONES, S.C., RODRÍGUEZ, D.J., HERNÁNDEZ, O., AND
BARRETO, G.R.A. 2010. Demographic study of the Arrau
turtle (Podocnemis expansa) in the Middle Orinoco River,
Venezuela. Chelonian Conservation and Biology 9:79–89.
MOORE, M.K. AND BALL, R.M., JR. 2002. Multiple paternity in
loggerhead turtle (Caretta caretta) nests on Melbourne Beach,
Florida: a microsatellite analysis. Molecular Ecology 11:281–
288.
MUNIZ, F.L., SILVEIRA, R., CAMPOS, Z., MAGNUSSON, W.E., HRBEK,
T., AND FARIAS, I.P. 2011. Multiple paternity in the black
caiman (Melanosuchus niger) population in the Anavilhanas
National Park, Brazilian Amazonia. Amphibia-Reptilia 32:
428–434.
MYERS, E.M. AND ZAMUDIO, K.R. 2004. Multiple paternity in an
aggregate breeding amphibian: the effect of reproductive skew
on estimates of male reproductive success. Molecular Ecology
13:1951–1963.
OJASTI, J. 1967. Consideraciones sobre la ecologı́a y la
conservación de la tortuga Podocnemis expansa. Atas do
Simpósio sobre a Biota Amazônica 7:201–206.
OLIVEIRA, D.P., MARIONI, B., FARIAS, I.P., AND HRBEK, T. 2014.
Genetic evidence for polygamy as a mating system in Caiman
crocodilus. Journal of Heredity 105:485–492.
PEARSE, D.E., ARNDT, A.D., VALENZUELA, N., MILLER, B.A.,
CANTARELLI, V., AND SITES, J.W., JR. 2006a. Estimating
population structure under nonequilibrium conditions in a
conservation context: continent-wide population genetics of
the giant Amazon river turtle, Podocnemis expansa (Chelonia;
Podocnemididae). Molecular Ecology 15:985–1006.
PEARSE, D.E., DASTRUP, R.B., HERNANDEZ, O., AND SITES, J.W., JR.
2006b. Paternity in an Orinoco population of endangered
127
Arrau River turtles, Podocnemis expansa (Pleurodira; Podoc-
nemididae), from Venezuela. Chelonian Conservation and
Biology 5:232–238.
PEARSE, D.E., JANZEN, F.J., AND AVISE, J.C. 2001. Genetic markers
substantiate long-term storage and utilization of sperm by
female painted turtle. Heredity 86:378–384.
PEARSE, D.E., JANZEN, F.J., AND AVISE, J.C. 2002. Multiple
paternity, sperm storage, and reproductive success of female
and male painted turtle (Chrysemys picta) in nature.
Behavioral Ecology and Sociobiology 51:164–171.
PRITCHARD, P.C.H. AND TREBBAU, P. 1984. The Turtles of
Venezuela. Oxford, OH: Society for the Study of Amphibians
and Reptiles, 414 pp.
REFSNIDER, J.M. 2009. High frequency of multiple paternity in
Blanding’s turtle (Emys blandingii). Journal of Herpetology
43:74–81.
ROBERTS, J.D., STANDISH, R.J., BYRNE, P.G., AND DOUGHTY, P.
1999. Synchronous polyandry and multiple paternity in the
frog Crinia georgiana (Anura: Myobatrachidae). Animal
Behavior 57:721–726.
SCHUELKE, M. 2000. An economic method for the fluorescent
labeling of PCR fragments. Nature Biotechnology 18:233–
234.
SITES, J.W., JR., FITZSIMMONS, N.N., SILVA, N.J., AND CANTARELLI,
V.H. 1999. Conservation genetics of the giant Amazon River
turtle (Podocnemis expansa; Pelomedusidae)—inferences
from two classes of molecular markers. Chelonian Conserva-
tion and Biology 3:454–463.
SLATYER, R., MAUTZ, B., BACKWELL, P., AND JENNIONS, M. 2012.
Estimating genetic benefits from experimental studies: a meta-
analysis. Biological Reviews 87:1–33.
TAYLOR, M., PRICE, T., AND WENDELL, N. 2014. Polyandry in
nature: a global analysis. Trends in Ecology and Evolution 29:
376–383.
THONHAUSER, K.E., RAVEH, S., AND PENN D.J. 2014. Multiple
paternity does not depend on male genetic diversity. Animal
Behaviour 93:135–141.
ULLER, T. AND OLSSON, M. 2008. Multiple paternity in reptiles:
patterns and processes. Molecular Ecology 17:2566–2580.
VALENZUELA, N. 2000. Multiple paternity in side-neck turtles
Podocnemis expansa: evidence from microsatellite DNA data.
Molecular Ecology 9:99–105.
VOGT, R.C. 2008. Tartarugas da Amazônia. First edition. Lima,
Peru: Gráfica Biblos, 104 pp.
YASUI, Y. 1997. A ‘‘good sperm’’ model can explain the evolution
of costly multiple mating by females. The American Naturalist
149:573–584.
Received: 13 September 2016
Revised and Accepted: 10 April 2017
Published Online: 23 August 2017
Handling Editor: Peter V. Lindeman