Introduction
Among sexually reproducing organisms, barriers that can impede interbreeding among individuals can contribute to reproductive isolation and speciation (Mayr, 1942; Coyne & Orr, 2004). While reproductive isolation mechanisms interact, barriers to gene flow can be broadly divided into those imposed by environmental conditions and considered extrinsic, or due to changes in the biology of individuals, independent of the external environment, and considered intrinsic (Bierne et al ., 2011). Biological barriers that prevent hybridization can manifest themselves at premating stages (Svensson et al ., 2007; Kozaket al ., 2009; Nickel & Civetta, 2009; Jennings et al ., 2011) or after mating has taken place. Postmating reproductive isolation can take place before fertilization through competitive or non-competitive mechanisms (postmating prezygotic) (Gregory & Howard, 1994; Price, 1997; Howard et al ., 1998; Jennings et al ., 2014), or after fertilization due to reduced fitness of hybrid offspring (postzygotic) (Haldane, 1922; Aalto et al ., 2013; Ishishitaet al ., 2016; Liang & Sharakhov, 2019).
Different types of barriers can be critical to speciation. InDrosophila , studies on the rate at which different barriers evolve have shown that, on average, prezygotic isolation evolves faster than postzygotic isolation (Coyne & Orr, 1989; 1997), with premating barriers evolving faster than postmating prezygotic and postzygotic isolation being even slower (Turissini et al ., 2018). However, the average rate of evolution of such barriers among species is not necessarily indicative that premating mechanisms are always more relevant in establishing isolation. For example, among Hawaiian species of Drosophila , the strength of premating vs . postmating barriers can be dependant on sympatry vs . allopatry status of the species (Kaneshiro, 1976; Carson et al ., 1989; Kang et al ., 2017). Among populations of Drosophila montana , there is evidence that premating mechanisms contribute to isolation, but premating isolation increases with distance between populations while postmating isolation is independent of distance, suggesting its important role in the early stages of speciation (Garlovski & Snook, 2018). While mechanisms of isolation have been studied extensively, it has been commonly done using species in which isolation is already fully established, thus making it difficult to differentiate between barriers that might evolve post-speciation from those that might have contributed to reduce gene flow in early stages of speciation. The identification of isolating barriers among diverging populations or partially isolated subspecies that have not yet reached a full-species status can help addressing questions on the role of different isolating mechanisms in speciation. Moreover, it has become increasingly evident that proper identification of the speciation phenotype aids in understanding not only the speciation process but its genetic basis (Mullen & Shaw, 2014). In turn, fine phenotypic characterization is crucial to functionally annotate genes.
Drosophila willistoni is a non-human commensal that uses flowers and fruits as substrates (Markow & O’Grady, 2008). The species was once believed to continuously spread from the southern United States to South America (D. w. willistoni ), with a different subspecies (D. w. quechua ) restricted to the west of the Andes in a narrow geographic area near Lima, Peru. It has been recently found that D. w. willistoni is subdivided into two partially isolated population (subspecies) that are reproductively isolated from each other; D. w. willistoni in North America, Central America and northern Caribbean islands, and D. w. winge in South America and southern Caribbean islands (Mardiros et al ., 2016). When a female of D. w. willistoni mates with a male of D. w. winge , the resulting males are sterile, but the females are fertile. In the reciprocal cross, all offspring are fertile. It has been also previously determined that copulation duration is similar for sterile hybrid males and parental species and that the external male genitalia shows no differences between the subspecies. Further, examination of the internal genitalia found no evidence of major atrophy in the hybrids relative to parental species, and the sterile males produced motile sperm but failed to place sperm within the female reproductive storage organs after mating (Civetta & Gaudreau, 2015). Whether hybrid male sterility due to failure to transfer sperm is unique to the two populations previously assayed (Civetta & Gaudreau, 2015) remains unclear. Moreover, we lack clear phenotypic characterization of what causes sterile male hybrids failure to transfer sperm and whether any form of assortative mating, or postmating prezygotic incompatibility, prevents gene flow between populations of these two different subspecies.
Here, we use strains derived from different populations of the two subspecies (i.e. D. w. willistoni : Guadeloupe, Puerto Rico, andD. w. winge : Uruguay and Saint Vincent). We found mating preferences among individuals of the same populations and no evidence of non-competitive postmating prezygotic isolation. Using a series of interrupted mating assays to track the fate of sperm and ejaculate of sterile male hybrids, we find that the sterile males manage to transfer an ejaculate that triggers the expected responses of elongation and expansion of the female uterus. However, the ejaculate is devoid of sperm. We identify a large mass forming a bulge at the basal end of the testes (i.e . the seminal vesicle) in sterile males that appears to impede the movement of the sperm towards the sperm pump, where sperm normally mixes with secretions produced by the accessory glands to produce the ejaculate. This mechanical impediment to transfer sperm represents a novel form of hybrid male sterility in Drosophila .