Assessment of multiple paternity in spinner shark (Carcharhinus brevipinna) litters from eastern Australian waters

Study locations and sampling

Tissue samples (fin clips and liver tissue) were obtained from two mother–litter groups of C. brevipinna to be assessed for multiple paternity. The two gravid females were caught in the Queensland Shark Control Program (QSCP) between 2020 and 2021. The pregnant females were captured in shark nets (gillnets) off the Gold Coast and the Sunshine Coast in south-eastern Queensland, Australia (Fig. 1). Full litters were made available, except for a single female pup retained by the Gold Coast Shark Control contractors (Table 1). Thus, genotypes were obtained for a total of 18 embryos (12 from the Gold Coast and 6 from the Sunshine Coast), as well as from the 2 mothers. All samples were stored in 80% ethanol and frozen at −80°C until laboratory processing. Specimens were retained under Queensland General Fisheries permit number 208761, and the University of the Sunshine Coast’s Animal Ethics Committee approval number ANE2161.

Fig. 1.

Capture locations of spinner shark (Carcharhinus brevipinna) mother–litter groups (n = 2) caught in the Queensland Shark Control Program (QSCP) in south-eastern Queensland, Australia, between February 2020 and February 2021.

DNA extraction and SNP genotyping

Genomic DNA extraction was conducted using QIAGEN DNeasy Blood and Tissue extraction kits following standard protocols (QIAGEN 2020). DNA was successfully extracted from the 20 individuals (18 embryos and 2 mothers) and sent to Diversity Arrays Technology (DArT, Canberra, ACT, Australia) for genotyping. DArT processed the DNA isolates by using their trademarked DArTseq protocol to generate SNP data. This is a genotyping-by-sequencing approach that involves reducing complexity of the genome through digestion using refined restriction enzymes (PstI and SphI), and next generation sequencing of selected size fragments (Sansaloni et al. 2011; Kilian et al. 2012).

SNP filtering

A preliminary SNP dataset received from DArT was imported into R (ver. 4.2.1, R Foundation for Statistical Computing, Vienna, Austria, see https://www.r-project.org/) as a genlight object using the dartR package (ver. 2.0.4, see https://cran.r-project.org/web/packages/dartR; Gruber et al. 2018). To mitigate errors and maintain a dataset with minimal missing data, stringent filtering criteria were applied. The filtering pipeline proceeded with the removal of monomorphic loci, retaining loci with a repeatability of >0.9, retaining loci with a call rate threshold of 0.9, removal of secondaries, and retaining loci with a read depth between 5 and 50. The dataset was then filtered at a moderate minor allele frequency (MAF) threshold of 0.3 (e.g. Premachandra et al. 2019). Alternative filtering stringencies were also tested showing the same results and, thus, the aforementioned filtering pipeline was considered to be most appropriate to obtain a final dataset with a robust number of SNPs and minimal missing data. Finally, loci showing a significant departure from Hardy–Weinberg equilibrium (HWE) proportions were filtered at two significance levels (0.01 and 0.001) to assess variation in stringency and ensure the loci segregated according to Mendelian expectations.

Sibship analysis

To determine the most likely number of paternal genotypes for each litter, a sibship analysis was performed using likelihood assignment methods, implemented in COLONY (ver. 2.0.6.8, see https://www.zsl.org/about-zsl/resources/software/colony; Jones and Wang 2010; Wang 2018). To address the potential genotyping errors in the SNP data, two allelic dropout rates were tested (0.25 and 0), which approximately correspond to sequencing coverages of 4 and 10 (Wang 2019). Additionally, two false allele error rates were tested (0.02 and 0.1). Therefore, the combination of significance thresholds for HWE, allelic dropout rates and false allele error rates resulted in eight COLONY input files that were analysed for each litter (16 total). The two litters were analysed separately, and the genotypes of the pups and their known mother were included in the sibship analysis, with this known maternal relationship being specified in the input file. Within all input files, the following parameters remained consistent. The full-likelihood method was selected, with medium precision, and medium run length. The dioecious and diploid options were selected, and a polygamous mating system was selected for both males and females. Allele frequencies were not updated, no sibship prior was selected, sibship scaling was set to the default, the seed for the random number generator was set to 1234 and markers were noted as co-dominant. COLONY reports two likelihood probabilities to determine whether full-sibling groups have not been over- or underestimated in the analysis. The inclusion probability (Prob(Inc.)) describes the probability that individuals assigned to an inferred sire are actual full-siblings, and the group is not under-split. The exclusion probability (Prob(Exc.)) describes the probability that all individuals assigned to an inferred sire are full-siblings and the group has not been over-split. Reporting higher values for both metrics indicates that the number of full-sibling groupings (i.e. the number of inferred sires) is not over- or under-split.

SNP filtering

In total, 30,569 SNP loci were derived from the DArTseq analysis. Stepwise filtering, with the additional filtering for departure from HWE at significance thresholds of 0.01 and 0.001 resulted in final datasets of 377 and 567 loci respectively. These datasets consisted of only 1.62 and 3.26% missing data. Most loci were excluded owing to departure from HWE (Supplementary Table S1).

Sibship analysis

The sibship analyses conducted in COLONY confirmed the presence of multiple paternity in both litters of C. brevipinna assessed in this study. Of the 16 input files analysed, the results remained consistent, yielding the same five full-sibling groupings for the Gold Coast litter from all corresponding input files (Tables 2, S2) with no evidence of over- or under-splitting. Of the eight input files corresponding to the Sunshine Coast litter, five produced the same three full-sibling groupings, with the remaining analyses producing two (Table S3). Lower exclusion probabilities were reported in the outputs indicative of three full-sibling groupings and, thus, the overall consensus is that there are two clear full-sibling groups in the Sunshine Coast litter (Table 2). Hence, reported here are the results obtained with the application of lower error rates for allelic dropout (0), and false alleles (0.1) to the less conservative dataset of 567 loci (HWE = 0.001).

Convenience polyandry

As male reproductive success theoretically increases with the number of successful mating events, polyandrous mating may be male-mediated (Byrne and Avise 2012). However, matings imposed on females often result in physical damage to flanks, dorsal and pectoral fins as males attempt copulation (Klimley 2013). Coercive strategies such as mobbing and herding have been documented in nurse sharks (Ginglymostoma cirratum), where multiple males will cooperatively harass a female into submission (Pratt and Carrier 2001). The effects of this conflict may be compounded in species that may be sexually segregated, owing to coercive behaviours exhibited by male-biased aggregations. Owing to the increased physical costs of polyandry to females, the theory of convenience polyandry suggests that females may submit to the advances of multiple males to avoid the adverse outcomes of sexual conflict, such as injury, infection or decreased fitness (Daly-Engel et al. 2006, 2007, 2010). This idea has been corroborated in several studies that also documented female sharks’ induction into ‘tonic immobility’ during courtship, a reversible catatonic state characterised by a motionless posture and unresponsiveness to the external noxious stimuli or injury (Williamson et al. 2018). As wild observations of copulation and potential breeding aggregation behaviours are absent for C. brevipinna, we were unable to infer whether convenience polyandry may contribute to the occurrence of multiple paternity in this species at this time. As this is a consistent issue in attempting to understand the reproductive strategy of elasmobranchs, and especially the vagile carcharhinids, many studies instead have given increased thought to the post-copulatory mechanisms, which may potentially influence the manifestation of multiple paternity in these species (Daly-Engel et al. 2010; Fitzpatrick et al. 2012; Green et al. 2017; Lamarca et al. 2020; Lyons et al. 2021).

Conspecific encounters and female choice

A heavily cited hypothesis for the prevalence of multiple paternity in elasmobranchs is the ‘encounter rate theory’, which proposes that multiple paternity may be dependent on the number of potential mates a receptive female encounters during a breeding season (Daly-Engel et al. 2007, 2010). This theory suggests that increased rates of multiple paternity would be likely to occur in high-density populations, such as schooling or aggregating species. Under this idea, the detection of multiple paternity in C. brevipinna could simply be attributed to the species’ active schooling behaviour (Compagno 1984; Ebert et al. 2021). Similarly, considerably high rates of multiple paternity have been attributed to aggregatory behaviours in common smoothhound sharks (Mustelus mustelus) and finetooth sharks (Carcharhinus isodon) (Rossouw et al. 2016; Nash et al. 2021). The rate of encounter between females and males will invariably fluctuate in time and space, and between species and populations (Rossouw et al. 2016). Extraneous factors such as climate-driven shifts in habitat use and fisheries-related extractions may also affect population density and sex ratios, shifting the prevalence and detection rate of multiple paternity at different times.

Lengthy gestation periods, and the baring of large, live young, are energetically expensive to viviparous species such as C. brevipinna. Further, the species typically exhibits low fecundity, and the gestation period of ≥12 months results in offspring production once every 2 years (Allen and Cliff 2000; Joung et al. 2005; Geraghty et al. 2016). As such, it may be more beneficial for C. brevipinna females to engage in polyandrous mating, allowing them to optimise their potential reproductive output and minimise the chances of fertilisation by inadequate males (Jennions and Petrie 2000). The increased opportunity to mate with a viable male (or males) may outweigh the physical implications posed by multiple copulations, because the lifetime reproductive output potential of species with these life-history traits may be more constrained otherwise (Lyons et al. 2021). This notion may explain the prevalence of multiple paternity identified in species that exhibit life-history and reproductive parameters similar to those of C. brevipinna, such as C. plumbeus (Daly-Engel et al. 2006, 2007; Portnoy et al. 2007), C. isodon (Nash et al. 2021), bull shark (Carcharhinus leucas; Pirog et al. 2019) and blue shark (Prionace glauca; Armada-Tapia et al. 2023).

The notion of cryptic female choice has been discussed in some literature because post-copulatory investment is heavily skewed towards females (Conrath and Musick 2012; Fitzpatrick et al. 2012; Lyons et al. 2017, 2021). In instances where receptive females cannot evaluate the fitness or compatibility of potential mates, multiple maternity may be facilitated by some degree of female choice (Neff and Pitcher 2005). In C. brevipinna, we identified a considerable number of putative sires per litter in both the larger litter (Gold Coast, N_pups = 12, N_sires = 5) and the smaller litter (Sunshine Coast, N_pups = 6, N_sires = 2). Notably, the paternal skew in the Sunshine Coast litter, where one male sired four pups, and the other sired two, may be an indication of female selectivity; perhaps a result of biased sperm utilisation or the biochemical promotion of sperm competition within the oviducal glands (Jennions and Petrie 2000; Byrne and Avise 2012; Lyons et al. 2021).

Sperm storage

Whereas multiple paternity is thought to be facilitated by females’ capacity to store sperm and enact potential selection biases (Conrath and Musick 2012; Fitzpatrick et al. 2012; Lyons et al. 2021), sperm storage remains understudied in most carcharhinids, including C. brevipinna. One unpublished study conducted a histological evaluation of female C. brevipinna oviducal glands, and reported no evidence of sperm storage (Palmrose 2021). However, the study was limited to using historical tissues (preserved between 2003 and 2005), and potential degradation of the samples was not discussed. Notwithstanding, it is plausible that these findings may be attributed to the species’ schooling nature and implied access to conspecifics, because sperm storage may not be required as a pre-emptive measure for fertilisation in the presence of regular, repeated fresh inseminations. Lamarca et al. (2020) determined that the ability to store sperm in elasmobranchs was not a necessary pre-cursor for multiple paternity to occur in subsequent litters. In a more recent study, Lamarca et al. (2024) further suggested that an absence of the ability to store sperm may increase extinction rates in elasmobranchs, through a reduction in genetic diversity. Because our results confirmed multiple paternity as a mating strategy for C. brevipinna, the perceived lack of sperm storage in this species does not lend concern to these implied consequences. However, further research on sperm storge, and its function in C. brevipinna, is needed to resolve these ideas.