Journal of Heredity Advance Access originally published online on September 27, 2007
Journal of Heredity 2007 98(7):712-715; doi:10.1093/jhered/esm077
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Brief Communications |
Multiple Paternity Analysis in the Thornback Ray Raja clavata L.
From the Department of Marine Benthic Ecology and Evolution, Center for Ecological and Evolutionary Sciences, Biological Center, University of Groningen, Postbus 14, 9750 AA Haren, The Netherlands (Chevolot, Stam, and Olsen); Centre for Environment, Fisheries and Aquaculture Science, Lowestoft Laboratory, Pakefield Road, Lowestoft, Suffolk, NR33 0HT, UK (Ellis); and Wageningen Institute for Marine Resources and Ecological Studies, PO Box 68, 1970 AB IJmuiden, The Netherlands (Rijnsdorp). Malia Chevolot is now at UMR 547-PIAF, INRA/Université Blaise Pascal, 24 avenue des landais, 63177 Aubière Cedex, France
Address correspondence to M. Chevolot at the address above, or e-mail: malia.chevolot{at}univ-bpclermont.fr.
Skates (Rajidae) are characterized by slow growth rate, low fecundity, and late maturity and are thus considered to be vulnerable to exploitation. Although understanding mating systems and behavior are important for long-term conservation and fisheries management, this aspect of life history is poorly understood in skates. Using 5 highly polymorphic microsatellite loci, we analyzed egg clutches collected from 4 female Raja clavata captured in the wild to test for multiple paternity. Using the reconstructed multilocus genotypes method to explain the progeny genotype array, we showed that all 4 clutches were sired by a minimum of 4–6 fathers and, thus, female thornback rays are polyandrous. Whether polyandry in R. clavata is natural or a consequence of overexploitation remains uncertain. This is the first report of multiple paternity in a rajiform species and any oviparous elasmobranch.
Knowledge about life histories and mating systems is important for developing long-term fisheries management and conservation (Rowe and Hutchings 2003). Depending on the mating systems, exploitation may actually increase the rate of decline and the time of recovery of a species (Rowe and Hutchings 2003). For example, species in which the number of males contributing to the gene pool is small, have a smaller effective population size (Sugg and Chesser 1994), and thus are more vulnerable to exploitation. Elasmobranchs (sharks, rays, and skates) are characterized by slow growth rate, low fecundity, late maturity, and long generation time and thus are considered to be vulnerable to overexploitation (Walker and Heessen 1996). This is well illustrated by the reported disappearance of the common skate Dipturus batis (Brander 1981), the white skate Rostroraja alba (Dulvy et al. 2000), and the spinytail skate Bathyraja spinicauda (Devine et al. 2006). Interestingly, elasmobranchs show a high variability in their reproductive modes, from oviparous to viviparous (Hamlett 2005), and in their mating systems from monoandry to polyandry (Chapman et al. 2004). Despite their vulnerability, many aspects of the reproductive biology of elasmobranchs, such as mating systems, are poorly known.
Thornback ray, Raja clavata L., has declined by nearly 80% in the North Sea over the past 40 years and is reported as locally extinct in some areas (Walker and Heessen 1996) and has declined elsewhere (Dulvy et al. 2000). Its decrease in abundance and distribution in the North Sea has led to concerns about the sustainability of skate populations in depleted areas (Walker and Heessen 1996), especially given their life history. Raja clavata has internal fertilization, is oviparous, and has a low fecundity in comparison to most teleosts and invertebrates (48–150 eggs/female/year) (Holden 1975; Ryland and Ajayi 1984; Ellis and Shackley 1995). The egg-laying season extends from February/March to September for the population as a whole, with a peak in the late spring/early summer, with young hatching at a length of about 10 cm after 4–5 months (Ryland and Ajayi 1984; Ellis and Shackley 1995).
Direct observations of the mating behavior have not been made for R. clavata. In the few observations that exist for other rajiformes, a complex courtship has been documented in which the male follows and bites the pectoral fins of the female. When the male finally grasps the pectoral fins of the female, he inserts one of his claspers into her cloaca (Chapman et al. 2003). As females suffer from biting wounds, the cost of copulation for females is high, especially in the absence of later paternal care. Thus, monoandry may be less costly to females (Feldheim et al. 2004). However, the nidamental gland of rajiformes is known to store sperm and some observations have described females copulating with a second male, though mating success could not be established (Chapman et al. 2003). These observations suggest that female rajiformes may be polyandrous (mating with multiple males) and suggest the possibility of multiple paternity. In the present study, we had the opportunity to assess multiple paternity in 4 females that had been captured in the wild and thus test whether female thornback rays are monoandrous or polyandrous.
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Four mature females were caught off East Anglia (United Kingdom) by a commercial fisherman in 2004 and 2 specimens were collected in "Sand Pit" (53.67°N; 1.54°E) during the IBTS (International Bottom Trawl Survey) survey in 2006 in the North Sea. Females were maintained in isolated tanks until they stopped laying eggs. As egg cases were released, they were transferred to separate aquaria and each clutch was maintained separately. In total, only 4 females laid fertilized eggs, and clutch sizes were 38, 39, 44, and 52. Small tissue samples were collected from embryos after 3–4 months of development and from the pectoral fins of the mothers. Tissue was stored in 70% ethanol.
DNA was extracted using a silica-based protocol (Elphinstone et al. 2003). Offspring and mothers were genotyped using 5 microsatellite loci as described in Chevolot et al (2005). These loci showed no null alleles, stuttering, large allele dropout, or significant multilocus f (Weir and Cockerham 1984; see Chevolot et al. 2005, 2006). Polymerase chain reaction products were separated on a 6% polyacrylamide gel and visualized with an ABI Prism-377 automatic sequencer (Applied Biosystems, Foster City, CA). Allele size was determined using an internal lane standard (Genescan 350 RoxTM) and GenescanTM software.
Power to detect multiple paternity depends on the level of polymorphism of microsatellite loci, clutch size, number of putative fathers, and their respective reproductive success (Neff and Pitcher 2002). We ran a number of simulations using the program PrDM (Neff and Pitcher 2002) to test the power to detect multiple paternity with the 5 microsatellite loci. Simulation scenarios were guided by reported clutch size in R. clavata and degree of paternity observed in sharks (Saville et al. 2002; Chapman et al; 2004; Feldheim et al. 2004). In total, 7 different scenarios were simulated with different combinations of number of fathers with their relative reproductive success: 2 males with equal (50:50), skewed (66.7:33.3), and strongly skewed reproductive success (90:10); 3 males with equal (33.3:33.3:33.3), skewed (57:28.5:14.5), and strongly skewed success (80:15:5); and 4 males with equal reproductive success. Each of these scenarios was run with a range of clutch sizes, 20, 40, 60, 80, and 100, and for each of them the probability of detecting multiple mating with our microsatellite loci was estimated.
The possibility of multiple paternity was only considered if more than 2 nonmaternal alleles were found in at least 2 microsatellite loci to allow for microsatellite mutation at a single locus, for each offspring. The minimum number of fathers for each clutch was estimated using the software GERUD 1.0 (Jones 2001). The software uses a reconstructed genotype method based on the multilocus genotype of known parent (the mother in this case) and the multilocus genotype of all offspring. By subtracting maternal alleles from the offspring genotype for each locus, it is possible to derive the paternal alleles. The software combines all pairwise paternal alleles for each locus, and then all single-locus genotypes are combined to create all the possible multilocus genotypes. Through an exhaustive search, the minimum number of fathers necessary to explain the progeny array is estimated.
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Under all scenarios, the power to detect multiple paternity was very high and ranged between 0.859 and 1 (Table 1). As expected, probabilities increased with clutch size and the number of fathers. This high power to detect multiple mating is likely due to loci Rc-B4 and Rc-B6, which are highly polymorphic with an average of 17.6 alleles for Rc-B4 and 19.6 alleles for Rc-B6 (Chevolot et al. 2006). Maternal alleles were detected in all offspring, in all clutches, and thus, null alleles or mutations are unlikely to have biased our results. Two to six paternal alleles/locus were found in all progeny arrays, and a minimum of 4–6 fathers were needed to explain the progeny array (Table 2). Thus, female R. clavata are polyandrous, and we have provided the first evidence of multiple paternity in both skates and oviparous elasmobranch.
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Genetic monoandry and polyandry/multiple paternity have been documented in shark species (Saville et al. 2002; Chapman et al. 2004; Feldheim et al. 2004; Daly-Engel et al. 2006, 2007; Portnoy et al. 2007). Polyandry and multiple paternity are predicted to be more common in species with low dispersal rates and high levels of philopatry (Chapman et al. 2004) because polyandry may increase genetic diversity of clutches and decrease sibling competition for resources (Simmons 2005; Daly-Engel et al. 2007). This is suggested by the predominance of multiple paternity in Negaprion brevirostris (Feldheim et al. 2004) and Ginglymostoma cirratum (Saville et al. 2002), species with a high degree of philopatry and a low dispersal rate. Likewise, the predominance of genetic monoandry is found in Sphyrna tiburo, which exhibits a higher dispersal rate and lower degree of philopatry (Chapman et al. 2004). For R. clavata, tagging studies have suggested small migration distances (maximum traveling distance 130 km) and some level of philopatry (Hunter et al. 2005), which would be consistent with polyandry. However, our recent genetic survey of R. clavata showed a weak genetic differentiation among British waters populations and a high migration rate (Chevolot et al. 2006). Thus, the hypothesis of polyandry being more common in low dispersal species had to be tested in more rajiformes.
Polyandry may also provide a way to ensure a sufficient amount of viable sperm to fertilize the eggs. Some female elasmobranchs can store sperm in their nidamental gland before ovulation/fertilization (Pratt and Carrier 2001). For oviparous elasmobranchs (such as R. clavata), the ovulation/fertilization period can last for several months in which a single female will have several cycles of ovulation/fertilization followed by egg release (Ellis and Shackley 1995). Thus, sperm needs to remain viable for the whole period. Because some males may have sperm that remain viable over a longer time, polyandry may maximize the probability of having viable sperm during the whole spawning period. From the fisheries perspective, overexploitation reduces population densities and/or may bias the sex ratio toward females (Rowe and Hutchings 2003). So, if polyandry is a natural behavior in R. clavata, overexploitation may cause a sperm limitation, and this can become a major problem for the long-term conservation of the species. However, polyandry may also be the consequence of a change in female behavior due to overexploitation. Although speculative (given the limited sample size), it is a very important question in conservation and management context of this species (Rowe and Hutchings 2003). Change in mating behavior due to human exploitation has been showed for the American lobster (Homarus americanus) (Gosselin et al. 2005). Females sampled in exploited sites are mostly polyandrous, whereas in less exploited areas, they are mostly monoandrous (Gosselin et al. 2005). Bigger males (or good males) may have higher amounts of sperm and be able to fertilize the whole clutch of one female, but these males are also the main target of fisheries. As a result, females may encounter fewer and fewer "good males" (males with sufficient amount of sperm), and thus, they will seek and mate with several males to ensure all their eggs to be fertilized (Gosselin et al. 2005). Thus, polyandry can be a compensatory response to coping with an analogous Allee effect, a situation in which scarcity of mates leads to a decrease in mean fitness of the population in relation to density and decrease of one sex. Although the sex ratio of newly hatched individuals and juveniles R. clavata has been estimated to be close to 1:1 (Ellis and Shackley 1995), fisherman often report aggregations by sex in catches (Ryland and Ajayi 1984). Thus, it is not actually known whether the adult sex ratio is skewed or not. In any case, given that R. clavata is one of the most important commercially fished skates in the NE Atlantic and Mediterranean (ICES 2005), the possibility that polyandry is a compensatory behavior cannot be ruled out.
In conclusion, multiple paternity has been clearly demonstrated in R. clavata. Whether this strategy is natural or the consequence of fishing pressure needs to be further investigated by expansion of the spatial sampling and, if at all possible, including sampling from areas that harbor larger, less fished populations, thus approximating a more natural set of conditions.
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NWO-PRIORITEIT programma SUSUSE, Project Nr 885-10-311.
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We thank Dirk den Uijl and Jan van der Heul from Wageningen Institute for Marine Resources and Ecological Studies; Stuart Hetherington and Mark Smith from the Center for Environmental, Aquaculture and Fisheries Sciences for taking care of the females and clutches; and Galice Hoarau for comments.
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Corresponding Editor: Martin Tracey
Received November 1, 2006
Accepted August 23, 2007
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