Discussion
We found evidence of premating isolation among populations of D. willistoni that is not determined by the subspecies status. Moreover, our detection of premating isolation is dependant on replicate (environmental) effects. While it is possible that premating reproductive barriers to gene flow are important among populations ofD. willistoni , a caveat is the use of laboratory strains that do not allow us to determine to what extent the partial but significant levels of isolation have been a consequence of laboratory conditions. While we consider it unlikely, if the levels of premating isolation we detected among populations arose or became stronger in laboratory stocks then clearly this form of isolation is not an important contributor to reproductive isolation between subspecies. If positive assortative mating among male and females of the same populations truly reflects a condition found in natural populations, then premating isolation is clearly important, but it is not a fixed condition. Contrary to premating isolation, postmating postzygotic isolation (i.e. unidirectional male hybrid sterility) is a fixed condition between subspecies (i.e. D. w. willinstoni and D. w. winge ) that is unlikely to have been created in laboratory settings for two reasons: 1) The isolation mechanism is fixed in a pattern that is geographical rather than random (north vs . south; Mardiros et al ., 2016) and 2) the same observation of hybrid male sterility among populations of north vs . south origin was made over 40 years ago by H. Winge before the laboratory stocks were established (Dobzhansky, 1975; Cordeiro & Winge, 1995).
An interesting observation regarding the levels of premating isolation detected between populations is the significant isolation between geographically distant populations (i.e. Saint Vincent and Uruguay) but not between geographically closer populations of the same subspecies (i.e. Guadeloupe and Puerto Rico). Isolation by distance rather than between geographically closer populations suggests that allopatry might have facilitated the evolution of positive assortative mating among populations, rather than premating barriers being reinforced upon possible secondary contacts among more geographically closer populations. This preliminary pattern of increased isolation by distance is reminiscent of observations made in other populations of Drosophila (Jennings et al ., 2014; Garvlosky & Snook, 2018) and, in our case, suggests that postmating isolation might be particularly important as a barrier during early divergence of these two subspecies of D. willistoni .
We have shown that sterile males manage to trigger changes in the morphology of the female’s uterus that is not different from the changes induced by fertile males. While the transfer of the seminal fluids seems to be slower in sterile males, their ability to cause the same morphological changes as fertile males suggests no major differences among subspecies in seminal fluid composition. We know from some studies in D. melanogaster that interactions between components of the male seminal fluid and the female reproductive tract trigger female responses to mating and are needed for efficient fertilization (Rezávalet al ., 2012; Avila & Wolfner, 2017; Chen et al ., 2019). Often, male seminal fluid proteins are rapidly evolving among species (reviewed in Swanson & Vacquier, 2002) and can potentially contribute to postmating barriers between species (Castillo & Moyle, 2014). Our findings suggest that components of the seminal fluids responsible for morphological changes after mating are conserved, and our results from fecundity assays show no evidence for non-competitive postmating prezygotic isolation between populations of different subspecies. However, it is possible that proteins involved in competitive processes might have substantially diverged between these subspecies. Therefore, we can not rule out the possibility that competitive postmating prezygotic isolation (e.g. conspecific sperm precedence) might exist between these two subspecies. Deciphering the existence of any form of competitive postmating isolation will have to wait for the development of tools for use in competitive paternity or different males’ sperm tracking assays.
Problems in sperm transfer has been reported before, for exampleDrosophila simulans females mate for a shorter period of time with Drosophila sechellia males than with conspecific males and very few sperm are transferred (Price et al ., 2001). However, complete failure to transfer sperm as a form of hybrid male sterility imposing postzygotic isolation between recently diverged subspecies is unique and novel among species of Drosophila . It has been previously shown that hybrid sterile males between the D. w. willistoni and D. w. winge produce normal and fully motile sperm (Gomes & Civetta, 2014) and we show here that hybrid male sterility results from a blockage impeding movement of sperm from the seminal vesicle into the vas deferens and mixing with other components of the ejaculate. This type of failure to transfer sperm due to a blockage (azoospermia) is reminiscent of cases of sterility in humans (Jarviet al ., 2010) and might be a consequence of abnormalities during the male reproductive tract developmental process. The development of the male reproductive tract during pupation is split into two parts, the genital disc which leads to the formation of most of the internal organs and the external genitalia, and the gonads which develops into the testes. Both the vas deferens and the seminal vesicles arise from the genital disc and the proper development of the testes depends on fusion with the seminal vesicles (Rothenbusch-Fender et al ., 2017). Given the overall normal male reproductive tract morphology of the sterile hybrids, we can conclude that the fusion occurs successfully. Based upon reports on the reproductive tract formation ofDrosophila melanogaster , the seminal vesicles form prior to thevas deferens (Kuckwa et al ., 2016; Rothenbusch-Fenderet al ., 2017). It is feasible that the origin of the enlargement in sterile hybrids be a consequence of subtle abnormalities at or around the time when the seminal vesicles are formed. Given that smooth musculature grows over the testes, it is possible that the musculature layer of the vas deferens grows but the interior lumen is hindered by a defect in the seminal vesicle.
Here, we have shown that non-competitive postmating prezygotic isolation is not a barrier to hybridization between D. w. willistoni andD. w. winge , but incomplete premating isolation is detectable among populations regardless of subspecies status. We have characterized a unique form of hybrid male sterility that involves an impediment of the male’s ability to transfer sperm. Detail characterization of the “speciation phenotype” is crucial in guiding future attempts to understand its genetic basis.