According to the analyses, sperm storage and multiple paternity represent plesiomorphic states in chondrichthyans and elasmobranchs, respectively, with possible independent losses in distinct clades. Although these behaviors appear to be related, one does not require the other to occur as each has been independently observed in Galeocerdo cuvier (Holmes et al. 2018, Pirog et al. 2020, Pratt 1993) and Isurus oxyrhincus (Corrigan et al. 2015, Liu et al. 2020, Pratt 1993). Additionally, our findings suggest that the lack or low frequency of sperm storage may be a driver of diversity loss in Chondrichthyes, with implications for female reproductive fitness.
Ancestral reconstruction
We found that SS is a plesiomorphic condition in Chondrichthyes, based on the relationship hypothesis of Stein et al. (2018), with the common ancestor probably exhibiting this behavior. The condition is lost in the Lamniformes and Rhinopristiformes, and considering the distant relationships between representatives of both orders, it is plausible to suggest an independent loss of this character regardless of the phylogenetic framework. According to Pratt (1993), female Lamniformes keep the viable sperm in the OG for only a few days, and repeated inseminations (repeated copulations) are probably the rule in these females. That author also hypothesized that this trait may be due to their reproductive mode (oophagous viviparity), where perhaps the large volume of eggs and shell material characteristic of this order also prohibits the occurrence of SS in the OG. In Rhinopristiformes, the presence of sperm in the female reproductive tract was not observed in any sample of the six species studied (Márquez-Farías, 2007, Jordan et al., 2021), indicating that SS may be absent or occur at low frequencies in those species.
The present reconstruction of MP based on the relationship hypothesis of Stein et al. (2018) also suggested it as a plesiomorphic condition at least in elasmobranchs, corroborating Lamarca et al. (2020a), as the behavior has not yet been investigated in Chimaeriformes. All investigated groups exhibited the behavior, except for Galeocerdo cuvier which demonstrated clear evidence of lacking multiple paternity, as offspring of nine litters revealed to bear parental contribution of a single male, each (Holmes et al., 2018, Pirog et al., 2020). MP may be absent or occur at low frequency in this species.
These results answer our first question, indicating that SS and MP, although plesiomorphic for cartilaginous fishes, do not share the same evolutionary history and may have suffered independent secondary losses in the Lamniformes, Rhinopristiformes, and Galeocerdonidae clades, showing the independence of both conditions, i.e. a female could produce a litter with a single sire by storing sperm from one or more males, as observed in the species Galeocerdo cuvier (Holmes et al. 2018, Pirog et al. 2020, Pratt 1993), or she could produce a litter with MP without storing sperm, as observed in some species, for example Isurus oxyrinchus (Corrigan et al. 2015, Liu et al. 2020, Pratt 1993)
In tiger sharks, the occurrence of monogamy with SS could be explained by the female not mating with several males during the reproductive period, or by the effective action of post-copulatory mechanisms of intrauterine selection (sperm competition) during ovulation to select the sperm of the best male to fertilize the entire litter (Lyons et al. 2017), the last of which could be an evolutionary advantage for the species. On the other hand, in species that perform MP and present an absence or low frequency of SS, the occurrence of such behavior without sperm storage could be explained by multiple matings with several males in sequence exactly during the ovulatory period, which would require the occurrence of mating aggregations in periods synchronized with ovulation, the occurrence of intrauterine sperm competition would depend on the number of males that the female would be able to mate with during this period (Corrigan et al. 2015), for these species, mating with more than one male may not be so costly for the female. For gregarious species, such as the spiny dogfish, this scenario is plausible, since these species performs mating aggregations even with the occurrence of sexual segregation (Silva & Ross, 1993, Dell'Apa et al. 2014). However, for solitary and highly migratory species such as the mako shark (Corrigan et al. 2018), this scenario is curious because it would imply that mating aggregations would occur in a specific geographic region. While such aggregations have not yet been observed, they may be plausible, as aggregations of the species have been observed in specific locations, although mating has not been confirmed during those events (Canani, 2020, Francis et al. 2019). These results suggest that the hypothesis elaborated by Pratt (1993) may not be applicable to species of the order Lamniformes, as like the mako shark, all others representatives of the group are migratory species with low population densities, but do not seem to perform SS (Compagno, 2001, Byrne et al. 2017, Corrigan et al., 2018, Francis et al. 2019).
The effect of sperm storage in females on Chondrichthyes diversity
The present analyses revealed a direct correlation between the diversification rates and sperm storage as its loss is consistent with high extinction rates and consequently with the decrease of diversity rates, clades in which SS is lacking presented. In turn, the clades that did store sperm, presented highest diversity rates with lower extinctions rates. The correlation between those variables may be considered one of the explanations for the low number of extant species in Lamniformes (16 spp) and Rhinopristiformes (70 spp), the sole lineages identified herein as lacking SS. Considering the general hypothesis adocating the advantages of SS, it loss may ultimately result in the decrease of the reproductive fitness of females influencing species diversification and extinction rates.
On the other hand, the presence of SS alone is not a drive for lineage diversification, as it is present in poorly diversified groups, as the Hexanchidae (7 spp), Hemiscylliidae (17 spp), Dalatidae (10 spp), Oxynotidae (5 spp), Somniosidae (17 spp), Callorhinchidae (3 spp), Chimaeridae (45 spp) and Rhinochimaeridae (9 spp), which did not show increased diversification rates in the MEDUSA and MiSSE results. For those groups the HiSSE recovered a correction between the observed state (SS) with a hidden states. Such combination of observed and hidden showed an increase in net diversity rate. This may indicate that diversification patterns are also driven by the hidden state(s), as taxa with SS present were not only concentrated in clades that showed increased net diversification rates.
According to Beaulieu & O'Meara (2016) diversification models can approach the effect of focal traits (e.g. BiSSE, Maddinson et al. 2007, MuSSE, Fitzjohn, 2012), adjust tree rates while completely ignoring trait information (e.g. MEDUSA, Alfaro et al. 2009). The HiSSE framework addresses all these types of models, if the true model has diversification rates varying directly dependent on traits, HiSSE can detect this, just as BiSSE would. If the true model has rates varying due to other unexamined factors, as MEDUSA does, HiSSE would also detect it. However, only HiSSE is able to provide an intermediate answer, where the focal trait explains part, but not all, of the variation in diversification rates along the phylogeny, as in the case of SS evolution in Chondrychthyans investigated here. The study by Mull et al. (2022) investigated if the diversification of the Chondrichthyes clades is associated with the evolution of reproductive modes in the group, using the partial tree of the phylogenetic hypothesis of Stein et al. (2018). Our results from the MEDUSA algorithm analysis are the same as those found in the study by Mull et al. (2022), they also identified shifts in net diversification rates in the Rajiformes (including Arhynchobatidae), Potamotrygonidae and Carcharhiniformes clades. In the results of the diversification associated with the evolution of reproductive modes, the authors identified higher diversification rates in viviparous and matrotrophic lineages. However, they pointed out that increases in diversification rates are probably associated with the colonization of new ecological spaces, rather than being systematically driven solely by the evolution of reproductive modes, here we suggest that SS may also have acted as a driver of diversity in these clades.
Both Lamniformes and Rhinopristiformes show low species diversity, low numbers of species per family, and strong morphological and ecological disparities (in the case of Lamniformes) compared to their sister groups. These may indicate the occurrence of extinctions in the past with a decrease in diversity during evolutionary history (Naylor et al. 1997, Underwood, 2006, Last et al. 2016, Marramà et al. 2021). According to Condamine et al. (2019), the diversity of extant Lamniformes is influenced by both an increase in extinction rate and a decrease in speciation rate over time, which are related to species body size, temperature, and competition between clades within the order. Here, we find evidence that the absence of sperm storage may also influence speciation and extinction rates in Lamniformes. However, due to the premises we assumed for the HiSSE analysis, we should be cautious about stating that the absence of SS or its low occurrence could be a driver of reduced diversity in Chondrichthyes. There are few records of this behavior in the group, as it has only been investigated in 72 species, while the group has approximately 1.200 species. More research is needed on species and families of the orders Heterodontiformes, Squatiniformes, Pristiophoriformes, and Torpediniformes, and on taxons from more families of the orders Orectolobiformes and Myliobatiformes.