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 .