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\begin{document}

\title{Reproductive isolation caused by azoospermia in sterile male hybrids of
\emph{Drosophila}}



\author[1]{Hunter Davis}%
\author[2]{Nicholas Sosulski}%
\author[1]{Alberto Civetta}%
\affil[1]{University of Winnipeg}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{June 04, 2024}


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\selectlanguage{english}
\begin{abstract}
Recently diverged population in the early stages of speciation offer an
opportunity to understand mechanisms of isolation and their relative
contribution. Drosophila willistoni is a tropical species with broad
distribution from Argentina to the southern United States, including the
Caribbean islands. We have recently documented a postzygotic barrier
between Central America, North America, and the northern Caribbean
islands (D. w. willistoni) from South American and the southern
Caribbean islands (D. w. winge). Here we identify premating isolation
between strains regardless of their subspecies status, with the effect
being dependent on environment. We find no evidence of postmating
prezygotic isolation and proceed to characterize hybrid male sterility
among the subspecies. Sterile male hybrids transfer an ejaculate that is
devoid of sperm but causes elongation and expansion of the female
uterus. In sterile male hybrids, bulging of the seminal vesicle appears
to impede the movement of the sperm towards the sperm pump, where sperm
normally mixes with accessory glands products. Our results highlight a
unique form of hybrid male sterility in Drosophila that is driven by a
mechanical impediment to transfer sperm rather than by an abnormality of
the sperm itself. Interestingly, this form of sterility is reminiscent
of a form of infertility (azoospermia) that is caused by lack of sperm
in the semen due to blockages that impede the sperm from reaching the
ejaculate.%
\end{abstract}%



\sloppy


\textbf{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 \emph{et
al} ., 2011). Biological barriers that prevent hybridization can
manifest themselves at premating stages (Svensson \emph{et al} ., 2007;
Kozak\emph{et al} ., 2009; Nickel \& Civetta, 2009; Jennings \emph{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 \emph{et al} ., 1998; Jennings \emph{et al} .,
2014), or after fertilization due to reduced fitness of hybrid offspring
(postzygotic) (Haldane, 1922; Aalto \emph{et al} ., 2013;
Ishishita\emph{et al} ., 2016; Liang \& Sharakhov, 2019).

Different types of barriers can be critical to speciation.
In\emph{Drosophila} , 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 \emph{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 \emph{Drosophila} , the strength of premating \emph{vs} . postmating
barriers can be dependant on sympatry \emph{vs} . allopatry status of
the species (Kaneshiro, 1976; Carson \emph{et al} ., 1989; Kang \emph{et
al} ., 2017). Among populations of \emph{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.

\emph{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 (\emph{D. w. willistoni} ), with a different subspecies
(\emph{D. w. quechua} ) restricted to the west of the Andes in a narrow
geographic area near Lima, Peru. It has been recently found that
\emph{D. w. willistoni} is subdivided into two partially isolated
population (subspecies) that are reproductively isolated from each
other; \emph{D. w. willistoni} in North America, Central America and
northern Caribbean islands, and \emph{D. w. winge} in South America and
southern Caribbean islands (Mardiros \emph{et al} ., 2016). When a
female of \emph{D. w. willistoni} mates with a male of \emph{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. \emph{D. w. willistoni} : Guadeloupe, Puerto Rico,
and\emph{D. 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 (\emph{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 \emph{Drosophila} .

\textbf{Materials and methods}

\emph{Drosophila stocks and maintenance}

Four strains obtained from the National \emph{Drosophila} Species Stock
Center were used in this study. The stocks were \emph{Drosophila
willistoni willistoni} 14030--0814.10 (G: Guadeloupe) and 14030--0811.12
(P: Toro Negro, Puerto Rico), and \emph{D. w. winge} 14030--0811.13 (S:
Saint Vincent and the Grenadines), and 14030--0811.16 (U: Rocha,
Uruguay). A previously established postzygotic isolation of Central
America, North America, and northern Caribbean islands from South
American and southern Caribbean island strains and suggested a
subdivision of \emph{D. w. willistoni} into two subspecies named\emph{D.
w. willistoni} and \emph{D. w. winge} (Mardiros \emph{et al} ., 2016).

Flies were kept in bottles containing cornmeal-yeast-agar-molasses
(CYAM) medium and kept in a 12:12 light--dark cycle at 22-24\selectlanguage{ngerman}°C. Adult
flies used in mating experiments, in interrupted mating assays or to
produce hybrids were collected as newly emerged every 4 hours to ensure
virginity. Virgin females and naïve males were separated, maintained at
a density of 20 flies per vial, and aged post-eclosion before being
used.

\emph{Premating isolation and fecundity}

We measure premating isolation among strains of the same subspecies
(i.e., G×P; S×U) as well as different subspecies (i.e., G×U; P×S) using
multiple\selectlanguage{english}-choice mating experiments (Jennings et al. 2011). Virgin
females and naive males from different strains were allowed to feed in
either red or blue colored food overnight and then placed together in
bottles containing CYMA food supplemented with yeast in groups of 30
males and females per strain. Mating pairs were observed until half of
all possible matings had occurred but for no longer than an hour, mating
pairs were removed and identified based on the color of their abdomen
(Gilbert \& Starmer, 1985; Casares \emph{et al} ., 1998). The
experiments were run over 3-4 replicates (different days) of each cross.
Data was pooled over replicates and the index of sexual isolation,
I\textsubscript{PSI}, was calculated using the program JMating
(Rolan\selectlanguage{english}-Alvarez \& Caballero, 2000). Positive Ipsi values are indicative
of positive assortative mating and suggest premating isolation. We also
estimated the asymmetry index (I\textsubscript{APSI(AB/BA)}), which
measures possible asymmetric differences in female preference for
heterotypic males. Statistically significant deviations from random
mating (i.e. I\textsubscript{PSI}= 0) were determined by bootstrapping
10,000 in JMating.

We tested fecundity of crosses between individuals of the same
population as well as between individuals of different subspecies. We
followed a protocol described in Gomes and Civetta (2014). Briefly, five
5 to 6 days-old naive males and virgin females were placed together for
48 h in a vial containing CYMA food. Males were removed after 48 h and
females transferred to a fresh vial five days after the initial set-up.
Females were discarded after 5 days and progeny counted from both vials
23 days after the initial set-up. Each cross was replicated at least 5
times.

\emph{Interrupted matings}

Virgin male and female pairs of the same strain/population were
aspirated into vials without anesthetization. Similarly, sterile hybrid
males were paired with females of each population used to generate them.
Vials containing a single male and female pair were observed
continuously for a period of 3-4 hours. Mating pairs were stopped by
freezing at either 2 or 6 minutes into copulation and stored frozen for
later dissection. The frozen couples were retrieved from the freezer and
allowed to briefly thaw over the course of a few minutes. All
dissections were done in a drop of 1x phosphate buffered saline (PBS).
The frozen copulating pair was gently separated using forceps and the
male was checked for an ejaculate mass on the tip of its aedeagus, in
case the separation had pulled it from the female, before being
discarded. The removal of the female reproductive tract was done
following a protocol described by Adams and Wolfner (2007). Briefly,
forceps and pins were used to separate cuticular tissue and open the
abdomen. Once the uterus was visible, we gently removed it from the
abdomen without disturbing the ejaculate when present. Pins were used to
clean excess tissue; no coverslips were placed over samples and images
were captured using an inverted Olympus CKX41 microscope. The samples
were checked for morphological shifts in the female's uterus and the
presence of a darker mass that denoted an ejaculate. When present,
ejaculates were removed from the female's uterus using dissecting pins
and placed on a drop of NucBlue Fixed Cell DAPI stain (Thermo Fisher
Scientific). A coverslip was placed over the drop containing the
ejaculate and incubated in the dark for 30 minutes. The samples were
observed for presence of sperm under both phase contrast and UV light
using a Zeiss AX10 microscope.

\emph{Testes measures of sterile and fertile males}

Males from each strain as well as sterile hybrid males were collected
and kept in vials containing CYAM medium with no more than 20 males per
vial to avoid crowding. The males were aged to either 5 or 10 days old
before being frozen to preserve them. Frozen males were allowed to
briefly thaw over the course of a few minutes and dissected in a drop of
1x PBS. Forceps were used to grip the thorax while the other pinched the
male's abdomen just above the aedeagus. Then, by simply pulling on the
gripped abdomen the entire male's reproductive tract was pulled out into
a drop of 1xPBS. The testes were isolated using dissecting pins and
moved onto a fresh drop of 1xPBS. Testes images were captured using an
inverted Olympus CKX41 microscope and the image processing and analysis
software Image J (https://imagej.nih.gov/ij/) was used to measure an
area towards the basal end of the testes where mature sperm is found and
which we refer to as the seminal vesicle. The area can be approximately
identified by an apical pinch followed by a basal pinch or torsion. One
testis from each male was measured.

\emph{Statistical Analysis}

Data from the interrupted matings was analyzed using Fisher exact test
on nominal variables (i.e. presence vs. absence). One-way analysis of
variance (ANOVA) was used to compare differences in fecundity and on
area measures of testes. If significant differences were detected by
ANOVA, Scheffe's post-hoc test was used to determine if the variance was
statistically significant between specific samples
(e.g.\emph{Drosophila} strains). All analysis was done using SPSS
software.

\textbf{Results}

\emph{Positive assortative mating among populations and no evidence of
non-competitive postmating prezygotic isolation}

Multiple mating tests show, for the most part, significant sexual
isolation among strains. Tests between strains of different subspecies
(heterotypic) showed strong positive assortative mating with no
asymmetry in female's preference. This departure from random mating
indicates strong, but not complete, premating isolation between the
subspecies. The same assay using strains of the same subspecies
(homotypic) also showed positive assortative mating between island and
geographically distant mainland populations (Saint Vincent and Uruguay)
but not between geographically closer island populations (Guadeloupe and
Puerto Rico) (I\textsubscript{PSI}= 0.20; \emph{P} = 0.145). There was
also no evidence of asymmetry in female's preference in homotypic mating
assays (I\textsubscript{APSI}) (Table 1). When the data was analyzed
within each replicate, we found that the detected premating isolation
was not consistent across replicates (Table S1). Altogether, any
indication of premating isolation is not driven by the subspecies status
of the strains assayed but rather by its population origin.

We compared overall fecundity of females crossed to males of the same
population to estimates from crosses between individuals of different
subspecies. This is a non-competitive setting, as females were not
offered an opportunity to doubly-mate with both males of the same
population and of a different population or\emph{.} subspecies. We found
significant differences among crosses in fecundity
(F\textsubscript{7,37}= 9.8; p\textless{} 0.001). Crosses among
individuals of the same populations were non-significantly different
from crosses between individuals of different subspecies (Figure 1).

\emph{Sterile hybrids and fertile males transfer seminal products that
trigger morphological changes of the uterus}

Interrupted copulations showed significant differences between males
from parental population and sterile male hybrids in proportion of
seminal fluid masses present at two minutes interruptions (parentals=
87.5\%; sterile males= 33.3\%; Fisher-Exact test \emph{P} =
1.3x10\textsuperscript{-8}) but by six minutes the differences were
non-significant (parentals= 97.7\%; sterile males= 86.5\%; Fisher-Exact
test \emph{P} = 0.068) (Table 2). This result shows that sterile males
take longer, but effectively transfer seminal fluids. Moreover, the
transfer of seminal fluids triggers similar morphological responses in
the female reproductive tract of females mated to fertile or sterile
males. The uterus is compacted in virgin females, with the seminal
receptacle located ventrally (Figure 2A). By two minutes, the uterus
experiences partial elongation acquiring a more oval shape and the
seminal receptacle is displaced opposite to the gonopod (Figure 2B). At
this point, the seminal fluid and possibly sperm (the ejaculate) can be
sometimes detected as a darker mass inside the uterus (Figure 2B). By
six minutes the uterus is fully elongated, with an oval shape, and both
the ejaculate mass and the ejaculate plug are visible (Figure 2C). In
females mated to sterile males, besides taking longer for the seminal
fluid mass to be transferred (Table 2), a major noticeable difference is
the lack of sperm within the seminal fluid mass transferred by sterile
males into the uterus (Table 2) (Figures 2 D and 2E).

\emph{Failure to transfer sperm by sterile hybrids is caused by a
testis's blockage at its basal end}

Visual inspection of the testes identified a region towards its basal
end that looked enlarged and more oval in sterile hybrid males than
fertile parentals (Figure 3). Because this structure contains
individualized sperm, we refer to it as the seminal vesicle. In 5
days-old fertile males, a pinch in the testes often defines the apical
end of what we identified as the seminal vesicle (i.e. mature sperm
storage) with the basal end almost continuously extending into a tubular
structure that we refer to as the \emph{vas deferens} (Figure 3A). The
basal end of the vesicle can be harder to identify in fertile males, in
some a subtle torsion or pinch and the presence of a darker mass of
sperm can be used as a landmark (Figure 3A) while in others the basal
end is more distinguishable (Figure 3B). In both cases, the vesicle
continues into a thicker tubular structure (Figures 3A and B) compared
to sterile males (Figure 3C). In sterile hybrids, the seminal vesicle is
more clearly distinguishable by an apical and basal pinch, and it is
more oval and enlarged (Figure 3C) compared to the more elongated form
found in the fertile testes. We used the landmarks described above to
delimit this structure and to provide approximate measurements of its
area in fertile and sterile males of different age. Analysis of variance
showed significant differences in the size of the seminal vesicle
between fertile parental males and sterile hybrids at five
(F\textsubscript{1,59}= 148.6; P\textless{} 0.001), and ten-days of age
(F\textsubscript{1,59}= 379.5; P\textless{} 0.001) (Table 3). When the
analysis is partitioned by population types (Uruguay, Guadeloupe, Saint
Vincent and Puerto Rico), Scheffe's post-hoc tests showed that parental
populations are significantly smaller than sterile male hybrids at all
ages and not significantly different from each other. In sterile hybrids
the seminal vesicle is approximately two times larger than in fertile
males and grows as males ages (Table 3; Figures 3D and E).

Closer examination of the seminal vesicle and \emph{vas deferens}allowed
us to find some interesting structural abnormalities in the sterile
hybrids that might at least partially explain how the enlargement of the
seminal vesicle might affect the transfer of sperm: 1) the seminal
vesicle is more vascularized in sterile males, with sperm present but
displaced to the edges (Figure 4A), 2) sperm is not found in the
\emph{vas deferens} of sterile hybrids (passing a pinch at the base of
the seminal vesicle, which is enlarged in sterile males) suggesting that
the enlarged and vascularized structure impedes proper movement of sperm
into the \emph{vas deferens} (Figure 4B), and 3) Some level of atrophy
of the \emph{vas deferens} in sterile hybrid males is apparent by the
observation of its irregular shape and the lack of lumen suggesting
collapsing and obstruction of the duct (Figure 4C).

\textbf{Discussion}

We found evidence of premating isolation among populations of \emph{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
of\emph{D. 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. \emph{D. w. willinstoni} and \emph{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 \emph{vs} . south; Mardiros \emph{et al} .,
2016) and 2) the same observation of hybrid male sterility among
populations of north \emph{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 (\emph{i.e.} Saint Vincent and
Uruguay) but not between geographically closer populations of the same
subspecies (\emph{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 \emph{Drosophila} (Jennings \emph{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 \emph{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 \emph{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
(Rezaval\emph{et al} ., 2012; Avila \& Wolfner, 2017; Chen \emph{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
example\emph{Drosophila simulans} females mate for a shorter period of
time with \emph{Drosophila sechellia} males than with conspecific males
and very few sperm are transferred (Price \emph{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 \emph{Drosophila} . It
has been previously shown that hybrid sterile males between the \emph{D.
w. willistoni} and \emph{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 \emph{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 (Jarvi\emph{et 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 \emph{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
\emph{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 of\emph{Drosophila melanogaster} , the seminal vesicles form
prior to the\emph{vas deferens} (Kuckwa \emph{et al} ., 2016;
Rothenbusch-Fender\emph{et 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 \emph{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 \emph{D. w. willistoni}
and\emph{D. 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.

\textbf{Acknowledgements:}

\textbf{Conflict of Interest} : The authors declare no conflict of
interest

\textbf{Data Accessibility} : Data supporting this publication will be
deposit in the Dryad Digital Repository upon acceptance.

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\textbf{Table 1:} Premating isolation I\textsubscript{PSI} between
intraspecific (homotypic) and interspecific (heterotypic) strains
of\emph{D. w. willistoni} and \emph{D. w. winge} as well as
interspecific mating asymmetry (I\textsubscript{APSI(AB/BA}). S= Saint
Vincent; U= Uruguay; G= Guadeloupe; P= Puerto Rico. P\selectlanguage{english}-values were
determined by bootstrapping 10,000 times. *\emph{P} \textless{} 0.05;
**\emph{P} \textless{} 0.01; ***\emph{P} \textless{} 0.001.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Strain A \selectlanguage{ngerman}× Strain B & Mating type &
A\textsubscript{}\selectlanguage{ngerman}×A\selectlanguage{english}\textsubscript{} &
A\textsubscript{}\selectlanguage{ngerman}×B\selectlanguage{english}\textsubscript{} &
B\textsubscript{}\selectlanguage{ngerman}×A\selectlanguage{english}\textsubscript{} &
B\textsubscript{}\selectlanguage{ngerman}×B\selectlanguage{english}\textsubscript{} & I\textsubscript{PSI} &
I\textsubscript{APSI(AB/BA)}\tabularnewline
\midrule
\endhead
S \selectlanguage{ngerman}× U & homotypic & 32 & 12 & 5 & 51 & 0.67*** & 0.57\tabularnewline
G \selectlanguage{ngerman}× P & homotypic & 12 & 18 & 8 & 27 & 0.20 & 0.85\tabularnewline
P \selectlanguage{ngerman}× S & heterotypic & 50 & 18 & 18 & 34 & 0.40*** & 1.01\tabularnewline
G \selectlanguage{ngerman}× U & heterotypic & 39 & 15 & 27 & 32 & 0.28** & 1.20\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2:} Interrupted matings of \emph{D. willistoni} at
different times during copulation between flies from parental
populations (P) and mattings involving sterile hybrids (SH). Proportion
of females with visible seminal fluid mass inside the bursa (Prop SF)
and with sperm within the ejaculate (Prop Sperm) are shown. P-values are
from Fisher Exact test comparisons between proportions in parental
crosses (P) and crosses with sterile male hybrids (SH).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Time & Cross & Prop SF & P-value & Prop Sperm & P-value\tabularnewline
\midrule
\endhead
2min & P & 0.875 (48) & \textless{}0.0001* & 0.976 (42) &
\textless{}0.0001*\tabularnewline
& SH & 0.333 (57) & & 0 (19) &\tabularnewline
6min & P & 0.977 (43) & 0.068 & 1 (42) &
\textless{}0.0001*\tabularnewline
& SH & 0.865 (52) & & 0 (45) &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3} : Measurement of the area of the male seminal vesicle
(in mm\textsuperscript{2} \selectlanguage{ngerman}× 1,000) in 5 and 10 days old sterile male
hybrids (SH) and fertile parental (FP) males. Sample size is shown in
parenthesis.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Male & 5 Day & 10 Day\tabularnewline
\midrule
\endhead
FP (G) & 14.1 (10) & 21.2 (10)\tabularnewline
FP (U) & 18.2 (10) & 22.8 (11)\tabularnewline
FP (P) & 18.8 (9) & 30.9 (10)\tabularnewline
FP (S) & 17.6 (10) & 23.0 (10)\tabularnewline
\textbf{Avg. FP} & \textbf{17.2 (39)} & \textbf{24.5
(41)}\tabularnewline
SH (G\selectlanguage{ngerman}×U) & 31.9 (12) & 54.5 (9)\tabularnewline
SH (P\selectlanguage{ngerman}×S) & 31.3 (10) & 61.3 (11)\tabularnewline
\textbf{Avg. SH} & \textbf{33.3 (22)} & \textbf{57.9
(20)}\tabularnewline
\textbf{Ratio SH/FP} & 1.9 & 2.4\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure 1} . Average fecundity, and 95\% confidence interval, of
females mated to males of the same subspecies or a different subspecies.
A) Results are shown for crosses using Puerto Rico (\emph{D. w.
willistoni} ) and Saint Vincent (\emph{D. w. winge} ) and B) Guadeloupe
(\emph{D. w. willistoni} ) and Uruguay (\emph{D. w. winge} ) flies.
Shared letters above bars indicate that the groups did not differ
significantly.

\textbf{Figure 2} . Expansion of the uterus (Ut) as a result of mating
and content of the male transferred ejaculate. A) In virgin females the
uterus (Ut) is compact between the gonopod long bristles (GLB) and the
sperm storage organs (SR). B) Two minutes into copulation, the Uterus is
semi-elongated, and the ejaculate (Ej) has been transferred. C) Six
minutes into copulation, the uterus is fully elongated, an ejaculate
plug (Ep) has formed in the posterior part of the uterus. D) Ejaculate
removed from the uterus of a female mated to a fertile male. Sperm tails
(black thin lines) are clearly visible. E) Mass of seminal fluid removed
from the uterus of a female mated to a sterile hybrid male is devoid of
sperm. SR= Seminal Receptacle; Spt= Spermathecae; An= Anus.

\textbf{Figure 3} . Testes of fertile and sterile hybrid males. A) and
B) Testes of a 5-days old fertile male and C) testes of a 5-days old
sterile male. Arrowheads indicate the seminal vesicle and the thin arrow
the \emph{vas deferens} . D and E) Testes of a 5 and 10 days old sterile
male showing enlargement of the seminal vesicle.

\textbf{Figure 4} . Seminal vesicle and \emph{vas deferens} of fertile
and sterile hybrid males. A) Seminal vesicle of a sterile hybrid male
showing vacuolar formations and sperm (arrow) displaced to the edge
(left) and seminal vesicle of a fertile male with arrow pointing at
sperm (right). B) \emph{Vas deferens} (VD) of a fertile male (above)
with sperm (arrow) present in its lumen \emph{vs} . sperm (arrow) within
the testes (T) of a sterile hybrid not entering the VD. C) VD of a
fertile male (above) \emph{vs} . sterile hybrid (bellow) showing an
irregular shape and lack of lumen.

\textbf{Fig. 1}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

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\end{figure}

\textbf{Fig. 2}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}



\textbf{Fig. 3}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}



\textbf{Fig. 4}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

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