Hemipenial morphology does not provide insight on mating barriers between the two main lineages of Hierophis viridiflavus (Lacépède, 1789)

From Firenze University Press Journal: Acta Herpetologica

Federico Storniolo, Dipartimento di Scienze della Terra e dell’Ambiente, Università di Pavia

Thomas Dadda, Dipartimento di Scienze della Terra e dell’Ambiente, Università di Pavia

Stefano Scali, Museo di Storia Naturale di Milano

Marco A.L. Zuffi, Museo di Storia Naturale dell’Università di Pisa

Marco Mangiacotti, Dipartimento di Scienze della Terra e dell’Ambiente, Università di Pavia

Roberto Sacchi, Dipartimento di Scienze della Terra e dell’Ambiente, Università di Pavia

The anatomy and morphology of copulatory organs have been of great interest for herpetologists in the last century especially concerning snakes (Cadle, 2011; Fol-well et al., 2022). As a matter of fact, hemipenes are postulated to play a major role in mating success, being supposedly species-specific (Cope, 1895; Keogh, 1999), and thus with marked implications in terms of repro-ductive biology and behaviour (Tokarz, 1988; King et al., 2009; Klaczko et al., 2017). Reptilian hemipenes show highly variable morphological traits, in terms of the gross shape of the organ itself (unilobed, bilobed) as well as of its external ornamentations, which can con-sist of rigid spines (spread across the organ or aggre-gated in a specific region, i.e., basal region or the apex; Fig. 1) or soft tissue folds, or otherwise can be com-pletely absent (Zaher et al., 1999; Andonov et al., 2017).

The extent of hemipenial morphological variability can remarkably vary among different families (Cadle, 2011; Andonov et al., 2017), but also at lower taxonomic levels (Inger and Marx, 1962; Branch, 1986; Zaher, 1999; Zuffi, 2002; Bernardo et al., 2012; Klaczko et al., 2014; Myers and McDowell, 2014). From this perspective, investigat-ing the mechanisms that drive the evolution of specific features in copulatory organs can be of great interest to address phylogenetic relationships and species splitting over time as copulatory organs are some of the most rap-idly evolving traits in squamates (Brennan and Prum, 2015; Klaczko et al., 2015, 2017; Folwell et al., 2022). Hypotheses have been proposed for the development of male genitalia, first of which the “lock-and-key”, former-ly postulated by Dufour (1844), states that male genita-lia evolve to be complementary to those of females with noticeable species-specificity. Alternatively, the pleiotropy hypothesis for male genitalia differentiation has been partly supported, hypothesizing that they evolve through selective pleiotropic effects on other traits (Mayr, 1963; Edwards, 1993; Arnqvist and Thornhill, 1998; Hosken and Stockley, 2004). This hypothesis appears still unsuit-able to be applied broadly as a common rule, because it assumes that the set of genes coding for general morphol-ogy codes also for genital morphological variation, which should not be selected against, implying tight genetic correlation between genital and general morphology (Arnqvist and Thornhill, 1998). These hypotheses have been revised broadly (Shapiro and Porter, 1989; Sota and Kubota Soto et al., 2013; Brennan and Prum, 2015) in an evolutionary perspective highlighting the role of genital morphology as a barrier against hybridisation, favouring coevolution between male and female genitalia (House et al., 2020; Greenwood et al., 2022).Ophidian hemipenes have been studied vastly in the last decades (Zaher, 1999; Myers and McDowell, 2014) under the functional perspective as they are related to copulation duration as well: indeed, in New World natricines more complex and ornate hemipenes (as in Thamnophis radix) are associated to more prolonged copulations compared to the congeneric T. sirtalis, char-acterised by simple subcylindrical hemipenes with little ornamentation (King et al., 2009). From this perspective, the occurrence of abundant ornamentations on hemipe-nial surfaces of both apical and body part of the organ appears to be relevant in terms of how efficiently males remain attached to females (Friesen et al., 2014), signifi-cantly affecting the duration of copulation and thus male fitness. Additionally, Rivas et al. (2007) showed that male coiling during copulation in species that undergo mating balls (Eunectes murinus in this case) can impede other males from mating with the female and, in these cases, more conspicuously ornamented hemipenes (i.e., more abundant or large ornamentations) should favour copu-lation and operate synergically with behaviour. On the other hand, snakes that exhibit male-male combat behav-iours are subject to sexual selection prior to copulation. Therefore, hemipenis morphology should not be selected to evolve more complex structures such as calyces, spines, and hooks. However, as shown by Andonov et al. (2017), hemipenial morphology does not always correlate with behavioural strategies, so the scenario of the evolution of male genitalia is a complex task to untangle.The green whip snake Hierophis viridiflavus(Lacépède, 1789) is one of the most widespread species in Mediterranean Europe as it occurs from Northern Spain across France and throughout Italy to Northern Balkans (Sillero et al., 2014). From the phylogenetic point of view, this species has been object of debate and Mezzasalma et al. (2015), according to both molecular and morphologi-cal differences, have split the two subspecies H. v. viridi-flavus and H. v. carbonarius (Western and Eastern clade respectively) and elevated them to the rank of species. Recently, Speybroeck et al. (2020) have proposed to pool them together as a unique species; however, the debate is still open. As a matter of fact, the phylogenetic relation-ships between the two lineages are still unclear and recent research on the genetic basis of its colour polymorphism (mtDNA and nDNA; Senczuk et al., 2021) has suggest-ed that these two lineages might undergo asymmetrical gene flow from the Western into the Eastern clade, even though no decisive evidence has been gathered. Morpho-logical variability in dentition, pholidosis, and hemipenes has also been used to characterise the phylogeny of the Hierophis genusby Schätti (1987, 1988), discriminating the members of this genus with respect to sister groups (i.e., Spalerosophis, Eirenis, Platyceps genera); however, interspecific variability patterns within genus are still unexplored (Schätti and Monsch, 2004; Utiger and Schät-ti, 2004). With this respect, nevertheless, Schätti and Vanni (1986) have investigated morphological traits of the target species, among which hemipenes too, but no difference among populations was found by the authors; however, hemipenial morphology was not the key subject for investigation and no in-depth study of morphology and ornamentations was performed.In this scenario, the role of copulatory organs can be useful to cast light on the reproductive aspects of species/subspecies delimitations as marked differences in hemi-penial morphology and ornamentation might impede interbreeding driving divergence between lineages; on the other hand, similar hemipenes would not act as a barrier to hybridization, favouring gene flow and inter-lineage mating (King, 1989; Sota and Kubota, 1998; Greenwood et al., 2022). For such reasons we investigated hemipe-nial morphology to address potential morphofunctional advantages of hemipenial structures, such as increased copulation efficiency and duration according to differenc-es in size, shape, and ornamentation, in the scenario of intraspecific lineage diversification. Additionally, we com-pared the gross morphology of H. viridiflavus as a whole, to that of the sister species H. gemonensis to check the extent, if any, of morphological variability of hemipenes at the genus level.

DOI: https://doi.org/10.36253/a_h-14145

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