Even though directional natural selection and genetic drift would preclude the persistence of genetic diversity (Lewontin 1974; Nielsen 2005), colour polymorphisms exist in many invertebrate and vertebrate species, and they have even been shown to persist along species radiations (Jamie and Meier 2020). Even the same colour polymorphism can be found in closely related species, suggesting the persistence of a shared evolutionary mechanism maintaining the polymorphism across species radiation. We have studied a group of marine gastropod species within the subgenus Neritrema (genus Littorina) that share a similar shell colour polymorphism (Fig. 2; Table S1). The results of the pooling analyses of assortative mating (light vs dark) using mating pairs from natural populations of the three studied species (Littorina fabalis, L. obtusata and L. saxatilis) show a consistent negative assortative mating across all populations and species (Table 1). L. obtusata showed the greatest negative assortative mating (IPSI = -0.59) and L. fabalis from Cangas, the lowest (-0.24). A similar result is obtained from the IPSI estimates for the homogeneous groups of colour frequency (Supplementary Table S2), which have been used to correct for the scale-of-choice effect (Rolán-Alvarez et al. 2015b). These estimates support previous results obtained for L. fabalis in Abelleira (Rolán-Alvarez and Ekendahl 1996; Rolán-Alvarez et al. 2012, 2015b; Estévez et al. 2020) and are also similar to those found in other species (Takashi and Hori 2008; Field and Barrett 2012; Holman et al. 2013; Hedrick et al. 2016 and 2018). Even though our results cannot unravel the evolutionary mechanisms responsible for the observed patterns, they strongly suggest that negative assortative mating for shell colour in Neritrema species is caused by mate choice and therefore it would be at least partially responsible for the maintenance of the colour polymorphism within population via negative frequency-dependent sexual selection. There are at least two different pieces of evidence that support this claim, as we have already suggested: first, theoretical analysis have shown that any mate-choice based negative assortative mating will render a negative frequency-dependent sexual selection that could be able to maintain the polymorphism (Pusey and Wolf 1996; Hedrick et al. 2016 and 2018). Second, recently Estévez et al. (2020) have shown that this relationship between negative assortative mating, mate choice and negative frequency-dependent selection holds for L. fabalis (see similar evidences for other model organisms: Takashi and Hori 2008; Field and Barrett 2012; Holman et al. 2013; Hedrick et al. 2016 and 2018). These two strains of evidence, in addition to the results presented in this study, strongly suggest that an ancestral plesiomorphic behavioural mechanism could exist for mate choice within the Neritrema subgenus. We find the ancestral behavioural mechanism hypothesis plausible, since, from an evolutionary point of view, it is reasonable to expect similar behavioural mechanisms operating under similar ecological conditions in closely related species. This is akin to what in evolutionary studies of cognition has been dubbed the “evolutionary parsimony” principle (de Waal 1999). Thus, if L. fabalis, L. obtusata and L. saxatilis show a similar behavioural pattern, live in the same ecosystem, and they all share a (relatively) recent common ancestor, it is reasonable to expect a common behavioural mechanism behind this behavioural pattern. Nevertheless, this hypothesis, although plausible, needs to be experimentally tested in the future before concluding its validity.
In any case, there are a few potential caveats in the present study that need to be addressed in order to dispel doubts about our results. First, the IPSI and similar indexes allow us to estimate assortative mating, and are frequently used as a proxy of mate choice, at least in laboratory conditions (reviewed in Gilbert and Starmer 1985; Rolán-Alvarez and Caballero 2000; Pérez-Figueroa et al. 2008). However, their capability to estimate mate choice has been discussed and several sources of bias have been identified (Rolán-Alvarez and Caballero 2000; Pérez-Figueroa et al. 2008). In any case, when they have been exclusively used to estimate assortative mating in the laboratory, these indices performed appropriately (Conde-Padín et al. 2008; Rolán-Alvarez et al. 2015b). On the other hand, it has been shown that correlation indexes could be potentially inflated towards positive values when estimated directly in the wild, especially in species with low mobility, as well as when the study species shows great variability (e. g. colour) at a scale smaller than the scale at which the samples were collected (Rolán-Alvarez et al. 2015b). This bias could be corrected by estimating the IPSI in homogeneous groups (e. g. by similar colour), as it has been shown by simulations (Rolán-Alvarez et al. 2015b) and as it has been carried out here. In fact, this correction has been used previously in cases of negative and positive assortative mating (Rolán-Alvarez et al. 2015b; Ng et al. 2016; Estévez et al. 2018, 2020). Thus, the correction used here does not correct, a priori, towards either negative or positive values of assortative mating. For example, the same correction was used to estimate the real scale at which mate choice occurs in two species of marine gastropods (Estévez et al. 2018), one characterised by positive assortative mating (Echinolittorina malaccana) and the other by negative assortative mating (L. fabalis), highlighting that this method is suitable to estimate both types of assortative mating. Therefore, the correction that we applied in this study assures a more reliable estimate of assortative mating in natural populations.
Second, another potential bias of our results could be caused by the actual observation of the mating pairs during the collection of the samples. For example, mating pairs in flat periwinkles (L. fabalis, L. obtusata) are commonly found among a dense canopy of Fucus vesiculosus, typically dark brown, allowing for a possible sampling bias towards the most visible mating pairs (light-light, dark-light). Although, in order to avoid this potential bias, we performed the search of mating pairs very close to the algae (~ 30 cm) and for each sampled microarea we checked all the individual algae, so it was unlikely to miss dark-dark pairs more often than the rest. Moreover, this bias in the sampling procedure has been rejected in previous studies as light-light mating pairs, which are highly noticeable on the Fucus canopy, appear at significantly lower frequencies than expected under random mating (Rolán-Alvarez et al. 2015b; Estévez et al. 2020). This potential sampling bias is even more unlikely in L. saxatilis, given that in this case all mating pairs are collected on the rock surface, as this species grazes on microalgae that grows on the bare rock. Therefore, in the light of the current results it seems plausible a hypothesis where a mating strategy of negative assortative mating for shell colour could represent an ancestral character within the subgenus Neritrema.
Recently, Estévez et al. (2020) have investigated the behavioural mechanism responsible for the negative assortative mating observed in L. fabalis from Abelleira (NW Spain) and the White Sea (NE Russia) populations. They found indirect evidence that mate choice is involved in assortative mating, with males showing preference for females with different shell colours. This, in turn, points to negative frequency-dependent selection as the evolutionary mechanism maintaining colour polymorphism in this species. However, their results didn’t provide any evidence regarding the potential contribution of colour to fitness, so that it would be favoured by mate choice. Their results neither clarify if colour, and not any other genetically linked trait, is the real target of selection. Here, we present a complementary result that could help to understand the relationship between shell colours and mating in these species. In the analysis of each colour separately (Table 1 and Supplementary Table S2), we found an interesting pattern given that, although in general all colours showed the exact same trend as in those cases in which they are grouped in dark and light colours, there was an exception. The olive shell colour in L. fabalis changes from a pattern of random (positive but not significant estimates) mating in Spain to a pattern of negative assortative mating in the Russian populations (Table 1 and Supplementary Table S2; Fig. 3). This result is unexpected if the shell colour itself is the target of the putative behavioural mechanism causing the assortative mating, but it could be expected if the colour is linked to another adaptive trait, physically or through pleiotropy. A relevant question is, therefore, what circumstances make a particular mating behaviour to be present in one location but not in others. One hypothesis is that the colour genes are associated with several other genes in a chromosomal inversion. In such scenario, different alleles of the colour gene could be associated to different chromosome variants (inversed or not). In support of this view is the fact that colour vision has not been detected and olfactory cues seems more relevant than visual cues in determining movement in aquatic gastropods (Seyer, 1992; Wyeth 2019). Actually, Cephalopods, the animals with the most complex visual system within the phylum Mollusca, have only one visual pigment and therefore cannot see in colour (Nilsson 2013). If cephalopods do not have colour vision despite having a complex visual system, it seems implausible that gastropods could have it. Because of this, previous hypotheses about mate choice for shell colour in gastropods were based on the possibility that they can sniff different colours when males follow mucus trails of females in the context of mate searching (see Estévez et al. 2020). Actually, a relationship between assortative mating for colour forms and sex chromosomes have been claimed in one finch species (Pryke 2010). Chromosomal inversions have been recently detected and associated to presumably adaptative traits in several organisms (Wellenreuther and Bernatchez 2018), including littorinids (Westram et al. 2018; Faria et al. 2019), and in at least one case has been related to negative assortative mating too (Hedrick et al. 2018). This could mean that the trait responsible for the pattern of negative assortative mating would be linked to the colour gene within the inversion, but could not be the colour itself. Although speculative, this hypothesis can be tested in the future and it opens a potentially fruitful new line of research.