Multiple paternity was demonstrated for seven clutches of eggs and 40 offspring sampled from these clutches in the cuttlefish Sepiella japonica from Fujian Shacheng Harbor Cultivation Base (Fujian Province, China), using four microsatellite DNA markers. It was observed that female cuttlefish copulated with different males. In this study, genotyping data suggest that at least three paternal allele genotypes were present in all seven clutches indicating that at least two males were responsible for each brood. Combined with behavioral observations, this study provides evidence for sperm competition and multiple paternity in S. japonica.

genetic diversity, mating, polyandry, reproductive strategy, sperm competition

The cuttlefish Sepiella japonica Sasaki, 1929 (Mollusca, Cephalopoda) is a commercially important marine species in China. Production from wild stocks reached 60,000 tons in Zhejiang Province and accounted for more than 9.3% of provincial fishing catches in 1957 (Liu 2002; Wu et al. 2010). The resource of S. japonica has declined since the 1980s due to over-fishing and pollution (Jiang et al. 2014). To enhance production, artificial breeding methods are being developed in China and successful aquaculture techniques have been established in recent years (Yin et al. 2013). However, studies have revealed that the populations and individual genetic diversity in this species has declined under artificial conditions (Song and Wang 2009; Xu et al. 2011). The factors affecting the maintenance of genetic diversity have been a primary concern of conservation biologists.

An important factor that affects the genetic diversity of a population is the effective population size (Ne) which in turn is greatly influenced by the mating system of a species (Hoekert et al. 2002). The mating system influences Ne through changing the number of individuals contributing to subsequent generations (Brown et al. 2005). In a polyandrous mating system, females mate with several males within a single reproductive cycle in which the clustered offspring are descended from multiple males (Pearse and Anderson 2009). In such a mating system, Ne increases, and, as a result, maximizes the genetic diversity of the offspring within a single reproductive season (Sugg and Chesser 1994; Balloux and Lehmann 2003). Some studies have confirmed that a polyandrous mating system is frequent in marine cephalopods including Octopus vulgaris Sasaki, 1929 (Quinteiro et al. 2011), Graneledone boreopacifica Nesis, 1982 (Voight and Feldheim 2009), Sepioteuthis australis Quoy & Gaimard, 1832 (van Camp et al. 2004), Sepia apama Gray, 1849 (Naud et al. 2005), Loligo pealeii LeSueur, 1821 (Buresch et al. 2001), and Loligo bleekeri Keferstein, 1866 (Iwata et al. 2005). It is worth noting that the female of these species carries stored sperm from more than one male, and Ne will therefore be significantly higher (Pearse and Anderson 2009). Previous studies have shown that female S. japonica store sperm in the seminal receptacle found in the buccal membrane (Hanlon et al. 1999; Naud et al. 2005). All else being equal, long-term sperm storage enhances the opportunity for multiple matings of this species (Olsson et al. 1994; Ross 2001). Moreover, multiple matings of female S. japonica has actually been observed (Wada et al. 2006). Polyandry, coupled with sperm storage, is an important reproductive strategy for maximizing the genetic diversity of offspring in S. japonica.

In recent years, multiple paternity in several marine species has been documented using different genetic markers including allozymes, DNA fingerprinting, RAPDs, and microsatellites. Microsatellites are the preferred marker because they are widely distributed in the genomes of most organisms and are highly polymorphic (Jarne and Lagoda 1996). Paternity studies based on microsatellites have become increasingly common, and the number of studies using microsatellites has increased (Hoekert et al. 2002; Laloi et al. 2004; Takagi et al. 2008). Several microsatellite markers have been isolated and characterized for S. japonica and used to evaluate the genetic structure of its populations (Wu et al. 2010; Lü et al. 2017). In this study, we used the previously described microsatellite markers to investigate whether multiple paternity occurs in S. japonica. We observed multiple mating and paternity in this species and discussed the possible factors contributing to this reproductive strategy.

We observed female S. japonica mating with different males during the reproductive period, a behavior also recorded in other species of cephalopods (Hall and Hanlon 2002; Naud et al. 2005). The benefits of multiple mating not only may raise the potential for genetic diversity but also increases the possibility of offspring survival (Mann et al. 1966; Jennions and Petrie 2000). Female Euprymna tasmanica Pfeffer, 1884 that mated with different males had larger eggs than those that mated with one male, indicating that females may obtain nourishment from the seminal fluid of several males (Squires et al. 2012). Male cephalopods exhibit “flushing behavior” in which they remove fresh spermatangia from previous males (Hanlon et al. 1999). In Sepia esculenta Hoyle, 1885, the males remove sperm by using the hectocotylus instead of flushing water (Wada et al. 2005). The males in this study also exhibited such behavior, flushing the buccal area of the female with water, when mating with a previously mated female.

Microsatellite markers are particularly useful in paternity studies because of their polymorphism, codominance, and repeatability. Cephalopod biologists have determined multiple paternity in many species, including squid (van Camp et al. 2004; Shaw and Sauer 2004; Iwata et al. 2005), and Graneledone boreopacifica Nesis, 1982 (Voight and Feldheim 2009). In this study, at least three paternal allele genotypes were found in all seven clutches indicating that at least two males were responsible for each brood. This result was in accordance with that of Naud et al. (2004), where multiple paternity was also found in Sepia apama. Multiple paternity in S. japonica offspring indicates that sperm from different males must be mixed within the female’s reproductive tract. These sperm deposited around the buccal mass were used differentially to fertilize eggs (Shaw and Sauer 2004; Walker et al. 2006), after a process of sperm competition (Hanlon et al. 1999; Hall and Hanlon 2002) or mediation by female choice (Eberhard 1996).

Despite the prevalence of multiple paternity in cephalopod species, these studies show widely differing incidences of multiple paternity. In our study, multiple paternity was demonstrated in all sampled clutches (100%). In Sepia apama, one-third of the females mated with multiple males and 67% of females’ eggs had multiple sires (Naud et al. 2004). Several factors have been confirmed to be related to the variance in incidence of multiple paternity observed in cephalopod species, e.g., sperm allocation, mating systems, sperm competition, and female choice (Wada et al. 2005; Wada et al. 2010). Moreover, as suggested for the squid Loligo bleekeri by Iwata et al (2005), males who were the last to mate fertilized 85–100% eggs in four broods tested. However, in the multiple paternity study of Loligo pealeii, the mate order is not the most important factor in determining paternity (Naud et al. 2004; Buresch et al. 2009); however, no clear hypothesis has yet emerged to explain which factor is essential in the multiple paternity of S. japonica. Further work should be carried out to understand paternity patterns and to investigate different factors affecting multiple paternity in this species.

This work was financially supported by the Chinese National Natural Science Foundation (41406138), Natural Science Foundation of Zhejiang Province (LY130190001).