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versión impresa ISSN 0188-8897

Hidrobiológica vol.20 no.1 Ciudad de México abr. 2010




Detecting patterns of fertilization and frequency of multiple paternity in Chelonia mydas of Colola (Michoacán, Mexico)


Detección de patrones de fertilización y frecuencia de paternidad múltiple en Chelonia mydas de Colola (Michoacán, México)


Libny Ingrid Lara–De La Cruz1, Ken Oyama Nakagawa2, Horacio Cano–Camacho3, María Guadalupe Zavala–Paramo3, Gerardo Vázquez–Marrufo3 y Omar Chassin–Noria1,*


1 Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo. Av. Francisco J. Mújica S/N, Morelia Michoacán, 58030. México. *E–mail:

2 Centro de Investigaciones en Ecosistemas, Universidad Nacional Autónoma de México. Antigua carretera a Pátzcuaro 8701 Colonia San José de la Huerta, Morelia, Michoacán, 58089. México.

3 Centro Multidisciplinario de Estudios en Biotecnología, Universidad Michoacana de San Nicolás de Hidalgo. Km 9.5 Carretera Morelia–Zinapécuaro. Morelia, Michoacán, México.


Recibido: 1 de febrero de 2010.
Aceptado: 15 de marzo de 2010.



We present a microsatellite analysis that allows an indirect determination of the process of fertilization in the genital tract of Chelonia mydas. The strategy was based in that the order of oviposition is related to fertilization order. Once the genotype of the offspring was obtained through microsatellite analyses, it was possible to determine the frequency of multiple paternity and to infer the presence of anatomical structures or physiological mechanisms that allow females to undergo cryptic choice processes, which allowed us to hypothesize the existence of post–copula factors that determine reproductive success in a polyandrous system. This paper does not show the presence of physiological mechanisms that allow control of fertilization order in polyandrous females of Chelonia mydas.

Key words: Multiple paternity, microsatellite, genetics.



Presentamos un análisis con microsatélites para determinar de manera indirecta si existe un mecanismo de control del proceso de fertilización en el tracto genital de Chelonia mydas. La estrategia utilizada se basa en que el orden de oviposición está relacionado con el orden de fertilización. Determinando el genotipo de la progenie, fue posible determinar la frecuencia de paternidad múltiple e inferir la presencia de estructurasanatómicas o mecanismos fisiológicos, que actúen como factores post–copulatorios para regular los eventos reproductivos en un sistema poliándrico. El presente trabajo no muestra la presencia de mecanismos fisiológicos que permitan el control del orden de fertilización en hembras poliándricas de Chelonia mydas.

Palabras claves: Paternidad múltiple, microsatélites, genética.


Five species of marine turtles have polyandrous mating systems (Uller & Olsson, 2008) where the percentage of clutches with multiple paternity (MP) can vary from 0 to 100% across species. There is no evident pattern that explains this variation, which may have implications on the genetic diversity and effective population size (Jennions & Petrie, 2000; Karl, 2008). It has been suggested that MP variation can be explained through different process. For Lepidochelys olivacea (Eschscholtz, 1829) Jensen et al. (2006) showed that in localities with massive nesting (arribadas) patterns, the frequency of multiple paternities, is higher than that of localities with solitary nesting females. This suggests that demographic conditions of a population affect MP frequency.

However, there may be other factors that determine MP frequency such as effects of the mating process, such as relative contribution of a male to the total pool of semen. In this scenario the amount of semen of each male in the genital tract of a female can be positively related with the time and/or number of couplings with a male as well as ejaculated semen volume per male (Otronen, 1997). Paternity bias can also be correlated to the structure of the genitals, corporal size of the male, or production of seminal chemicals (Jennions & Petrie, 2000) or sperm competition. It has been suggested that semen storage is a common event in reptiles (Girling, 2002) for up to seven years, as has been reported for the snake Acrochordus javanicus (Hornstedt, 1787) (Mangusson, 1979). The capacity to store semen obtained during copulation has been detected in Chelonia mydas (Linneo, 1758) (Chaves et al., 2000), even though structures that facilitate sperm storage have not been described in marine turtles (Wyneken, 2001). Stored semen has been found inside the reproductive tract of the Green Turtle (C. mydas), Kemp's Ridley Turtle [Lepidochelys kempii (Garman, 1880)] and Olive or Pacific Ridley Turtle (L. olivacea), and it has been suggested that this storage is only present in short periods such as within one reproductive season (Miller, 1997).

Sperm competition might be present in turtles of the genus Chelonia, given the presence of a polyandrous system and semen storage within a nesting season. Birkhead & Pizzari (2002) suggested that females in polyandrous systems have the capacity to bias male participation in ovule fertilization after mating. It has been shown that females can identify the genotype of the sperm (Vaquier, 1998), allowing the possibility to choose the semen of a preferred male, and that there might even exist intracellular choice mechanisms.

We analyzed paternity keeping the order of egg arrangement in a nest, making inferences about how those eggs were fertilized in the genital tract of female marine turtles. We inferred the order of fertilization of two turtle's eggs nests assuming one of the two following possible scenarios: 1) egg fertilization by the semen of at least two males followed copulation order, or 2) egg fertilization by the semen of at least two males did not follow copulation order. To assess the multiple paternity, we used standard molecular tools, as has been done by other authors working with marine turtles (Uller & Olsson, 2008) and most importantly, we complemented this study by recording the oviposition order, that allowed us to describe for the first time, fine–grained aspects of egg fertilization in marine turtles.

We followed the oviposition process in two females of Eastern Pacific Green Turtle, known locally as Tortuga Prieta or Black turtle (C. mydas) in the beach of Colola, Michoacán, collecting the eggs directly from female cloaca and maintaining the order with a plastic extruded net. We transported and planted the eggs in a hatchery, maintaining the oviposition order until eclosion. Immediately after oviposition, we collected 500 ul of blood from adult females. We also collected 50ul of blood from all hatchlings after eclosion (54 and 55 days of incubation respectively for each nest). We used the protocol described by Owens & Ruiz (1980), resuspending samples in a lythic solution (Dutton, 1996). To comply with local regulations, we obtained a collecting permit from the General Direction for Wildlife–SEMARNAT (SGPA/SGVS/12409). We obtained total DNA following the protocol described by FitzSimmons (1997), and with the AquaPure Genomic DNA Isolation Kit (Bio–Rad Laboratories, CA, USA). We amplified two oligonucleotides, OR1 (Aggarwal et al., 2004) with the 6–FAM, and Ei8 (FitzSimmons, 1997) VIC modified oligonucleotide forward. Both oligonucleotides were amplified in 25ul reactions: Buffer 1X, 0.2mM DNTPs; 2.4 mM MgCl2; 0.5 u,M of each primer; 20 µg/ml BSA; 1U Taq polimerase; 20– 50 ng of DNA, with the following program: 94°C 5 minutes, 30 cycles of 94°C 10 seconds, 45°C 10 seconds, 72°C 30 seconds and a final step of 72°C 7 minutes. We carried out electrophoresis with an AB3100 Avant unit in 0.2 ul of each PCR product and 0.25 ml of LIZ–500. We obtained the genotypes with Genotyper 4.0 from Applied Biosystems (Applied Biosystems Inc. CA, USA). Finally we identified the paternal genotypes with GERUD 2.0 (Jones, 2005).

The Ei8 oligonucleotide was monomorphic, and was eliminated from both analyses. We found 8 alleles with the OR1 oligonucleotide. In nest one, 40 hatchlings were released before we were able to collect samples, and DNA samples from 11 hatchlings were not large enough to obtain material to purify or amplify. We obtained genotypes from 50 hatchlings, which represents 49.5% of the total progeny, following the order of oviposition (sampling 50% of each nest is preferable according to FitzSimmons (1996)). We found at least two paternal genotypes. The most frequent paternal genotype fertilized 49 of the analyzed hatchlings (98% of analyzed hatchlings) and a second genotype fertilized one hatchling (2% of analyzed hatchlings).

For female two, we obtained the genotype of 65 of 69 eggs, or 94.2% of clutch size. We detected the participation of three paternal genotypes. The first fertilized 59 hatchlings (90.8% of analyzed hatchlings), the second fertilized four hatchlings (6.1% of analyzed hatchlings) and a third fertilized two hatchlings (3.1% of analyzed hatchlings).

The genotypes of males involved in the fertilization process and fertilization order for both nests are shown in Table 1 and 2. Upon analysis of the paternal genotypes we infer that there is no paternal genotype shared between the two analyzed nests. In both nests a male fertilized more than 95% of the progeny (male 158/146 and 158/150 for nests one and two, respectively), with a minor contribution from a second male for the first nest and from two additional males for the second nest.

Despite the importance of participation of one male in both nests, we did not suppose that the genital tract of the female was capable of separating semen from each copulation (through peristaltic movements). The order of fertilization was less pronounced in the second nest, where three males participated. In this nest, the second male fertilized eggs 5, 19, 20 and 68, while the third male fertilized eggs 9 and 61. Overall, the fertilization pattern could be explained through a cryptic choice mechanism. We cannot eliminate the possibility that a cryptic choice mechanism in the female is driving the proportion of the progeny fertilized by each male, favoring a genotype in particular. However, a few scattered genotypes within a dominant background suggest, an imperfect cryptic choice mechanism. Alternatively spermatic competition processes could also affect the frequency of multiple paternity, but our observations do not suggest that they determine the order of fertilization.

Even though this study has a small simple size, we present an innovative strategy that allows to study the order of fertilization in an indirect form. This strategy can be used for oviparous organisms for which there are reports of polyandrous mating systems where oviposition order can be maintained and associated to fertilization processes.



To the Colola community in Michoacán for their cooperation and assistance during the study. We thank Ericka Urbiola, Armando Hernandez, Xochiquetzal Rodriguez, Alejandra Galvez and Leticia Hernandez for valuable assistance in sample collection.



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