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.