REPRODUCTIVE ISOLATION AMONG ALLOPATRIC

DROSOPHILA MONTANA POPULATIONS

Jackson H. Jennings1,3,4, Rhonda R. Snook2,4,5 and Anneli Hoikkala1

Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of
Jyvskyl, Finland, +358-440-771-123; anneli.hoikkala@jyu.fi

Department of Animal and Plant Sciences, S10 2TN, University of Sheffield, UK, +44-114222-0126; r.snook@sheffield.ac.uk

Current Address: Department of Biological Sciences, University of Arkansas, USA 72701,

479-575-7437; jjenning@uark.edu
4. Joint first authors
5. Corresponding author

Running title: Reproductive isolation within a species

Data will be deposited in Dryad upon acceptance
Key words: speciation, sexual isolation, gametic isolation, speciation phenotypes,
postmating-prezygotic isolation
Word count of main document: 6398
Table count: 3
Figure count: 5

This article has been accepted for publication and undergone full peer review but has not been through the copyediting,
typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of
Record. Please cite this article as doi: 10.1111/evo.12535.
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Abstract
An outstanding goal in speciation research is to trace the mode and tempo of the evolution of
barriers to gene flow. Such research benefits from studying incipient speciation, in which
speciation between populations has not yet occurred but where multiple potential
mechanisms of reproductive isolation (RI: i.e. premating, postmating-prezygotic (PMPZ),
and postzygotic barriers) may act. We used such a system to investigate these barriers among
allopatric populations of Drosophila montana. In all heteropopulation crosses we found
premating (sexual) isolation which was either symmetric or asymmetric depending on the
population pair compared. Postmating isolation was particularly strong in crosses involving
males from one of the study populations, and while sperm were successfully transferred,
stored and motile, we experimentally demonstrated that the majority of eggs produced were
unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no
evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of preand post-mating barriers showed that populations differed in the mode and magnitude of RI
barriers. Our results indicate that incipient RI among populations can be driven by different
contributions of both premating and PMPZ barriers occurring between different population
pairs and without the evolution of postzygotic barriers.

Introduction
Reproductive isolation (RI) is the crux of speciation (Dobzhansky 1935, 1937; Mayr 1963;
The Marie Curie SPECIATION Network 2011). Without it, the homogenizing effects of gene
flow can act to prevent biological diversification. Accordingly, characterizing the strength
and nature of barriers to gene flow can offer insight into both patterns and processes involved
in species formation, bolstering our understanding of biodiversity and its maintenance (Coyne
and Orr 2004). While speciation is often a continuous process, many of the existing studies of
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this type are on already diverged species in which it is difficult to distinguish between RI
barriers that contribute to divergence and those that accumulate after divergence. Such a
distinction is important because the reproductive barriers associated with early stages of
divergence may not be the same as those that maintain genetic integrity after the species are
already established (The Marie Curie SPECIATION Network 2011). On the other hand,
studies of reproductive isolation on separate species have the benefit of at least knowing that
speciation has occurred. The continuous nature of speciation argues that studying across this
spectrum is critical in understanding the process of divergence.

Reproductive isolating barriers can occur prior to mating (premating), after mating but before
zygote formation (postmating-prezygotic; PMPZ), and/or after zygote formation
(postzygotic). Within each of these categories, different and multiple mechanisms could act
as barriers and contribute to total RI with different absolute and relative strengths and at
different stages of divergence (e.g., Ramsey et al. 2003; Coyne and Orr 2004). It is thought
that most speciation occurs as a consequence of the action of multiple reproductive isolating
barriers (Coyne and Orr 2004). Thus, the identification of the order of appearance and the
magnitude of each isolating barrier contributing to speciation has been described as the holy
grail of speciation research by Sobel et al. (2010). This endeavor is made difficult by
disagreement over which barriers to include and how to measure total RI (Sobel et al. 2010)
and because the tempo of the evolution of these barriers are not independent (e.g., Ramsey et
al. 2003; Martin and Willis 2007; Lowry et al. 2008). Studies that quantify these barriers are
typically focused on hybrid zones, where barriers are filtered by events after contact, or
ecotypes where ecological speciation via natural selection is acting and are biased to extrinsic
pre- and postmating barriers. Moreover, many are on species pairs rather than conspecific,
but divergent populations (e.g., Nosil et al. 2005; Lowry et al. 2008; Johannesson et al. 2010;

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Snchez-Guilln et al. 2012; Corl et al. 2012; see both Mullen and Shaw 2014 and Abbott et
al. 2013 for review). Studies of multiple reproductive barriers occurring in recently diverged
allopatric populations of the same species are relatively rare despite the insight they could
provide into the factors that influence the origin of isolating barriers (but see Etges and
Ahrens 2001, Etges et al. 2009, 2007 and Nosil 2007)

There is also an historical bias in the accumulation of knowledge on different categories of

reproductive isolating barriers; while both premating and postzygotic barriers have been
studied extensively in a number of taxa (Coyne and Orr 2004), PMPZ barriers have received
less attention. These barriers have been referred to as cryptic because they cannot be
revealed by studies on mating probabilities (i.e., premating isolation experiments) or hybrid
incompatibilities (i.e., tests of postzygotic isolation) (Nosil and Crespi 2006). PMPZ barriers
operate at the level of gametic and/or reproductive tissue or reproductive protein interactions.
Perhaps the best understood of PMPZ barriers have been found in externally fertilizing
species, such as abalones and sea urchins, where protein incompatibilities between gamete
surfaces result in RI (Shaw et al. 1994; Metz et al. 1994; Metz and Palumbi 1996; Palumbi
and Lessios 2005; Palumbi 2008). While such processes provide ample opportunity for
selection to drive extensive evolutionary change in internally fertilized species given the
more complex nature of male-female interactions, they are generally difficult to detect and
measure in such species (Howard et al. 1998, 2009; Knowles and Markow 2001; Snook et al.
2009).

Recent studies have sought to identify PMPZ barriers in internally fertilized species at the
level of egg production, sperm viability, motility, transfer, storage or usage and sperm-egg
interactions (either extracellular or intracellular; Snook et al. 2009). For example, between

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some members of the D. melanogaster and D. simulans species groups, reduction in sperm
transfer, depletion of transferred sperm and/or inefficient sperm storage in heterospecific
matings have been found to contribute to PMPZ isolation (Fuyama 1983; Price et al. 2001;
Matute and Coyne 2010; Manier et al. 2013). In the D. virilis group, cases of PMPZ isolation
between species have been to found to involve incompatibility between the male ejaculate or
sperm and the female reproductive tract, resulting in the incapacitation, death or loss of sperm
after heterospecific matings (Sweigart et al. 2010; Sagga and Civetta 2011; Ahmed-Braimah
and McAllister 2012). While these studies are concentrated on species pairs, a few recent
studies have examined PMPZ in recently diverged, conspecific populations. For example, in
the walking stick, Timema cristinae, ecological divergence in host plant use results in
reduced oviposition and fecundity in crosses between ecotypes that use different host plants
(Nosil and Crespi 2006), thus PMPZ isolation was ecologically dependent. PMPZ barriers
have also been demonstrated between populations that seemingly lack ecological divergence,
and in some cases, this is the only barrier operating. For example, in the bruchid beetle,
Callosobruchus maculatus, and the ground cricket, Allonemobius socius, allopatric
populations are exclusively isolated by PMPZ mechanisms (Brown and Eady 2001; Fricke
and Arnqvist 2004; Wilkinson and Birge 2010). In one within-species case, multiple
reproductive barriers have evolved; crosses between cosmopolitan (M) and Zimbabwe (Z) D.
melanogaster show strong behavioral isolation and a lack of postzygotic isolation (Wu et al.
1995), but have an asymmetric barrier to gene flow at the sperm-egg level due to the inability
of M-type sperm to penetrate and successfully fertilize Z-type eggs (Alipaz et al. 2001).

Knowledge on the number of reproductive barriers maintaining species integrity, the stage of
speciation at which they occur, the magnitude of their contribution to species isolation, and
the order in which they have evolved is integral to understanding the speciation process. For

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example, RI between species pairs isolated by a few strong barriers is suggested to have a
less complicated genetic architecture than between species pairs in which more, but weaker,
barriers are involved (Dopman et al. 2010). Likewise, understanding the relative contribution
of each barrier to total isolation provides information on the importance of early- vs. lateacting isolation barriers (e.g., premating vs. postzygotic) in species formation. In Drosophila,
comparative work has suggested that pre- and post-zygotic isolation evolve in allopatry at
approximately the same rate across evolutionary time (Coyne and Orr 1989, 1997, 2004). A
more controversial hypothesis is that early acting barriers may be more important in limiting
gene flow, regardless of whether late acting barriers evolved first, largely due to the
sequential nature of these barriers; that is, earlier acting barriers disproportionately restrict
gene flow because they operate at an earlier life stage and thus affect the probability of the
action of barriers acting at later stages (Coyne and Orr 2004; Martin and Willis 2007; Lowry
et al. 2008).

Here we address incipient reproductive isolation barriers among populations of the same
species, quantifying absolute and relative contributions of premating, PMPZ, and postzygotic
RI barriers. We use diverging allopatric populations of Drosophila montana. We test for the
contributions of sexual premating isolation, a variety of different PMPZ mechanisms
including sperm transfer, storage and usage, and postzygotic hybrid inviabilty and sterility in
all pairwise comparisons using two allopatric populations of D. montana from North America
(Colorado USA and Vancouver Canada) and one population from Europe (Oulanka Finland).
Previous work using microsatellites and mtDNA has estimated that European and North
American clades diverged between 450,000 and 900,000 years ago and show no evidence of
recent gene exchange (Mirol et. al. 2007). Our work sheds light on potential traits whose
divergence contributes either directly or indirectly to reduction of gene flow during the

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speciation process (speciation phenotypes, Shaw and Mullen 2011), and offers insights into
the action of different RI barriers and how these can contribute differentially and
asymmetrically across populations exhibiting nascent reproductive isolation.

Materials and Methods

FLIES
Drosophila montana is a cold-adapted member of the D. virilis group found at extreme
latitudes (up to 70N) and altitudes (up to 3200m) around the northern hemisphere
(Throckmorton 1982). Throughout its distribution, it carries out its life cycle near water, in
the moist, decaying phloem tissues of various aspen and alder tree species. Adult D. montana
were collected from riparian habitats in Oulanka (Finland) and Vancouver (Canada) in the
summer of 2008 and in Colorado (USA) in summer 2009 using malt bait buckets and
aspirators. Isofemale lines were established from the progenies of wild-caught females and
maintained in half-pint bottles on malt medium until a large number of F3 flies were
available. For the Oulanka and Vancouver populations, 20 F3 males and 20 F3 females from
each isofemale line (800 flies total) were then combined in a population cage and bred in
overlapping generations. For the Colorado population, the population cage was prepared with
13 lines (520 total F3 flies) using the same procedure. Each representative population was
maintained in a 252560 cm wooden cage with a Plexiglas top and eight available food
bottles attached in holes in the floor of the cage for feeding, oviposition and larval rearing.
All flies used in premating, PMPZ and postzygotic isolation experiments were collected from
these bottles as virgins within 3 days of eclosion at least two generations after the
establishment of the cages. Flies to be used in experiments were separated by sex and kept in
groups of 10-20 flies until sexually mature at 21-28 days old. To prevent diapause, all flies
were maintained in constant light conditions at 19 1C and 70% relative humidity.

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SEXUAL ISOLATION
Sexual (premating) isolation was measured for each pairwise population comparison (i.e.,
Colorado x Oulanka, Colorado x Vancouver and Oulanka x Vancouver) using pairwise
multiple-choice mating experiments (see Jennings et al. 2011 for full description of general
methodology). To identify what populations mating flies were from, we marked flies from
each population with either red or blue colored food and, to control for any dye effects,
altered colors between trials (as in Wu et al. 1995 and Jennings and Etges 2010). If the color
of a fly was unidentifiable after dissection of the gut, then the mating pair was removed from
the analysis (n = 4 out of 720). After marking, virgin mature males and females, 30 each,
from each of two populations being tested were introduced into a 666 cm Plexiglas mating
chamber without anesthesia and allowed to mate. Mating pairs were removed with an
aspirator and subsequently identified. Experiments were terminated after half of the possible
matings had occurred, so that 30 mating pairs were identified for each replicate mating trial
(see Gilbert and Starmer 1985 for statistical justification). Eight replicate mating trials were
carried out for each population cross resulting in the identification of 716 total mating pairs.
To analyze these data we used the program JMating (Rolan-Alvarez and Caballero 2000). For
each population cross, the total numbers of each type of pair mating (AA, AB, BA, BB) were
pooled across all 8 replicate mating trials and these total numbers were then entered into
JMating accordingly, as per programme guidelines. The index of sexual isolation, IPSI, was
calculated based on the total numbers of each type of pair mating in each population. IPSI
ranges from 1 to 1, where 1 is complete sexual isolation, 0 represents random mating, and
1 is complete disassortative mating (i.e., all matings are heteropopulation). An asymmetry
index, IAPSI(ab/ba), was also calculated in JMating which assesses potential asymmetrical
differences in female preference for heteropopulation males. Significance of both parameters
was determined by bootstrapping 10,000 times in JMating.
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POSTMATING ISOLATION
Fecundity and Productivity
We first assessed general postmating isolation in all possible crosses among the three
populations (n = 9 crosses) by counting the number of eggs (fecundity) and progeny produced
(productivity) by singly mated females. This allowed us to determine whether postmating
isolation occurred, but not to discriminate between PMPZ and postzygotic isolation, which
we investigated subsequently (see below). For each mating, a single virgin mature male and
female were combined in a small plastic dish covered with netting and allowed them to mate
(see Fig. 2 for sample sizes). All copulations were observed to ensure that they did not
terminate prematurely. After mating, males were discarded and females were transferred
singly to fresh food vials and allowed 7 days of oviposition (changed to a new vial after the
first 3 days), as once-mated D. montana are known to produce offspring for approximately 6
days (Aspi and Lankinen 1992). Oviposited eggs were counted under a dissecting microscope
and the resulting progeny (males and females, see Postzygotic isolation below) were
counted after they eclosed. We used one-way ANOVA to test the effect of cross on fecundity.
We used one-way ANCOVA to analyze the effect of cross on productivity; number of eggs
laid was used as a covariate to account for differences in the total number of progeny that
could be produced arising from variation in the total number of eggs oviposited for each
female. For analysis of productivity, data were square root transformed for normality given
that certain crosses showed impaired progeny production (as expected if RI was occurring).
Tukeys HSD tests were utilized to detect differences between levels of each factor. Analyses
were performed in JMP 11.0. (SAS Inc).

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Postmating, prezygotic isolation

To determine whether the postmating isolation observed in the above tests occurred either
before or after fertilization, we measured traits that contribute to PMPZ isolation, including
sperm transfer, storage, and usage. To qualitatively determine whether differences in progeny
production might be due to inefficient sperm transfer or storage in heteropopulation crosses,
we carried out a separate set of matings for all nine possible cross combinations. The
reproductive tract of mated females was dissected either one or three days after mating in
order to assess the transfer and storage of sperm (Snook et al. 1994). For each mated female,
we separated the uterus, spermathecae and ventral (seminal) receptacle and scored each for
the presence or absence of motile sperm (n = 7-10 females/cross/time).

To evaluate whether progeny reduction in some crosses was due to failure of egg hatch,
which could be due to either fertilization failure or early embryonic developmental
abnormalities, we carried out single pair matings for all nine possible cross combinations in
empty plastic shell vials. We then transferred mated females to oviposition manifolds (as in
Crudgington et al. 2005, Snook et al. 2000) with 20 replicate chambers connected to a plate
with corresponding oviposition dishes containing molasses-yeast-agar food, sprinkled with
dried yeast. Individual females were left in a chamber for 2d and then transferred to new food
plates for another 2d of oviposition. The number of oviposited eggs was counted immediately
after the plates were removed from the manifold and the number of unhatched eggs on each
plate was counted 2d later (see Fig. 3 for sample sizes). Data were square root transformed
for normality such that we could use one-way ANCOVA to analyze the effect of cross on the
number of eggs that hatched, again using number of eggs laid as the covariate. Tukeys HSD
tests were utilized to detect differences among crosses using JMP 8 (SAS Inc).

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10

To determine whether the decreased number of eggs that hatched in particular crosses (Fig. 3)
was due to either fertilization failure (a PMPZ mechanism) or abnormal zygote development
(a postzygotic mechanism), we scored eggs laid by mated females in all nine cross types for
development and/or sperm presence using fluorescent and differential interference (DIC)
microscopy. To obtain the large number of eggs required for these surveys, we transferred
flies (n = 30-40 flies per sex) in half-pint food bottles (two replicates per cross type), each
covered by an oviposition plate filled with molasses-yeast-agar food sprinkled with dry yeast.
Oviposition plates were removed 24 hours later and eggs were collected by washing with
dH2O, dechorionated with a 50% bleach solution, and fixed and stained with the nuclear stain
4',6-diamidino-2-phenylindole (DAPI) for microscopy (as in Snook and Karr 1998). For each
cross, we categorized these fixed eggs as either developing or non-developing (total n eggs =
381-667 per cross). Developing eggs showed either clear mitotic division or cellular
differentiation whereas non-developing eggs had fewer than four nuclei visible within the egg
(see Fig. 1A). One caveat is that non-developing eggs may appear non-developing for three
mutually exclusive reasons: 1) they may be unfertilized, 2) they may have been fertilized but
fixed for analysis prior to nuclear fusion and subsequent mitosis, or 3) they may be fertilized
but failed to develop due to incompatibilities arising after fertilization. To distinguish
between these alternatives, we scored non-developing eggs for fertilization using DIC light
microscopy. Since sperm length of D. montana is 3.34 0.02 mm (Pitnick et al. 1995), the
tail can easily be seen as a coiled structure near the anterior end of the egg under 20-40X
magnification (see Fig. 1B).

Due to the extensive protocol in processing eggs for fixation and the subsequent staining for
examination of development and presence of sperm (Snook et al. 1994), some egg loss
occurred. Because of this, eggs from multiple females were processed together; that is,

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11

multiple females oviposit on the same oviposition plate which we then process for egg
collection and staining. Females were allowed to oviposit for just 24h per plate, and we
collected eggs from multiple plates for each 24h oviposition bout, across multiple 24h blocks.
Thus, there was some pseudoreplication and eggs are collated all together for staining.
Therefore, data on fertilization are represented by only one value per cross. Here we do not
provide any statistical tests, but instead simply show the proportion of eggs examined that
were not developing in each cross and, as a subset of those, the proportion that were
unfertilized.

Postzygotic isolation
We have previously shown that F1s between two of these populations (Oulanka and
Vancouver) are viable, fertile and show no evidence of bias in offspring sex ratio (Jennings et
al. 2011). Total numbers of F1 males and females from each pair mating used in the above
tests of productivity were counted to assess potential bias in F1 offspring sex ratio. To test
for evidence of intrinsic postzygotic isolation in the form of hybrid sterility or inviability,
we carried out mass matings within each reciprocal F1 hybrid from each interpopulation
cross. Twenty to forty virgin males and females were combined in half-pint food bottles and
allowed to mate. The hybrids were then cleared from the bottle and were deemed fertile and
their offspring viable if any adult F2 offspring were seen emerging from the bottles. When F2
offspring emerged (which occurred in all crosses made), they were then kept together in the
bottles until the F3 generation to generally assess the fertility of the F2s given that some types
of incompatibilities (e.g. Dobzhansky-Muller incompatibilities due to recessive gene gene
interactions; Coyne and Orr 2004) would not be apparent until the F2 generation.

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12

ABSOLUTE AND RELATIVE CONTRIBUTIONS OF PRE- AND POST- MATING

BARRIERS
We assessed the absolute and relative contributions of pre- and post-mating isolation between
each pair of populations using the criteria of Ramsey et al. (2003). The strength of premating
isolation was estimated by calculating Coyne and Orrs (1989) proposed index of prezygotic
isolation:

RI1 = 1

Frequency of heteropopulation matings

Frequency of homopopulation matings

Postmating isolation was determined by calculating the degree of total progeny reduction in
hetero- vs. homopopulation matings (RI2) for each population cross with the equation:

RI2 =

P AA P BB P AB P (BA)
P AA P( BB )

where P is the overall mean number of progeny produced by females in homopopulation (AA,
BB) and heteropopulation (BA, AB) mating combinations.

Both of these indices range from 0 to 1, where 0 = no isolation and 1 = complete isolation.

After calculating the above indices, we followed Ramsey et al.s (2003) protocol to calculate
the absolute and relative contributions of each barrier to total isolation for each of the three
population crosses. Since an isolating barrier occurring earlier on in the process of
hybridization (e.g., premating isolation) may lower the probability of proceeding to a later

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13

barrier (e.g. postmating isolation), the absolute and relative contribution of each barrier to
total isolation are not independent. Hence, we calculated the absolute contributions (AC) of
each barrier and total reproductive isolation (T) with the following equations:

AC1 = RI1
AC2 = RI2(1 - AC1)
T = AC1 + AC2

where AC1 and AC2 represent absolute contributions of pre- and postmating isolation,
respectively. Finally, the relative influence of pre- and postmating isolation to total
reproductive isolation (relative contributions, or RC1 and RC2, respectively) in each
population cross was calculated by dividing their absolute contributions by total isolation:

RC1 =

AC1
AC 2
, RC2 =
T
T

Results
SEXUAL ISOLATION
All three pairwise population crosses showed overall significant premating isolation in
multiple-choice situations (IPSI; Table 1). In the cross between the Colorado and Oulanka
populations, sexual isolation was symmetrical; both Colorado and Oulanka females prefer
males from their own population and discriminate against the heteropopulation males (IAPSI;
Table 1). In contrast, this isolation was asymmetric in both crosses involving the Vancouver
population. Vancouver males were accepted by heteropopulation females as frequently as

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14

homopopulation males whereas Vancouver females showed clear preference for their own
males versus males from allopatric populations (Table 1).
POSTMATING BARRIERS
We found no effect of cross on fecundity (Fig. 2; F8, 135 = 1.71, P = 0.10). Despite this, the
number of progeny significantly differed among crosses (Table 2a), largely due to the low
progeny production in both heteropopulation crosses involving Vancouver males (particularly
with Colorado females; Fig. 2). Additionally, Vancouver females mated to Colorado males
had reduced progeny production relative to other crosses involving either Vancouver females
or Colorado males (Fig. 2). Thus, there is evidence of reproductive isolation between
populations, particularly in interactions between Vancouver and Colorado males and females.
We found no predicted bias in offspring sex ratio in any experimental cross in this study (see
Supporting Information Table 1).

Although F1 progeny production was most severely compromised in crosses between

Colorado females and Vancouver males (Fig. 2), we assessed sperm transfer and storage in
all possible crosses. Female reproductive tract dissections revealed that, in all crosses, sperm
was successfully transferred and stored in both storage organs (spermathecae and seminal
receptacle) and sperm were motile at both 1 and 3 days after mating in 100% of the females.
We could not quantify the amount of sperm in either of the sperm storage organ types due to
the length of sperm. However, the fact that sperm were transferred, stored and motile - even
in the crosses that produced statistically fewer offspring - suggests that the mechanism
responsible for the low progeny production occurred either at the sperm-egg level (PMPZ) or
after fertilization (postzygotic).

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15

We found that, even though the mated females successfully received and stored motile sperm
for at least 3 days after mating, the mean number of eggs that hatched significantly differed
among the crosses (Table 2b). A significant reduction was found in heteropopulation crosses
involving Vancouver males and a slightly weaker reduction in the cross between Vancouver
females and Colorado males (Fig. 3).

To determine the cause of such low egg hatching, we assessed the fertilization and
development status of the oviposited eggs. While the total number of eggs examined differed
among crosses, the proportion of developing eggs in heteropopulation crosses involving
Vancouver males was lower than that of all other crosses (Fig. 4), mirroring the significant
trends seen in overall progeny production and egg hatchability. Heteropopulation crosses
involving Vancouver males had the lowest percentage of eggs that were developing; only 30%
of eggs from Vancouver males crossed with Colorado females, and only 65% of eggs from
Vancouver males crossed with Oulanka females, were developing (Fig 4). In contrast, all
other crosses had a higher percentage of eggs developing. To determine whether lack of
development was due to lack of fertilization, we examined whether non-developing eggs had
a sperm inside them. In heteropopulation crosses involving Vancouver males, lack of
development was associated with fertilization failure rather than failure to develop after
fertilization (Fig. 5). Thus, the low number of eggs hatching from heteropopulation crosses
with Vancouver males was a consequence of prezygotic barriers. Some crosses did have a
relatively high percentage of eggs that were classed as non-developing but that did have
sperm; these were cases where eggs were fertilized, but were fixed shortly after fertilization,
before syngamy could occur.

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16

We also examined crosses for postzygotic sterility. 100% of all crosses produced F2 progeny,
indicating that if eggs were fertilized, then F1 individuals arising from that cross were fertile
and their offspring viable. Supporting this was the fact that if eggs were fertilized in crosses
for which PMPZ occurred, these embryos appeared normal (Jennings, pers. obs). Further, 100%
of crosses between F2 individuals resulted in high numbers of progeny. Overall, postzygotic
isolation appears to be absent in all heteropopulation crosses, suggesting no evidence of
downstream postzygotic incompatibilities. However, as matings were done en masse, it is
possible that some offspring were only partially fertile.

ABSOLUTE AND RELATIVE CONTRIBUTIONS OF PRE- AND POSTMATING

BARRIERS
We calculated the absolute and relative contributions of pre- and postmating isolation for
each pairwise population comparison. These comparisons clearly show that pre- and
postmating isolation evolve effectively independently. The strength of the premating, but not
PMPZ, barrier may be associated with divergence time (Table 3). In the cross where
premating isolation was strongest (CO), postmating isolation played little to no role whereas
the low level of premating isolation in the CV cross was accompanied by substantial
postmating isolation in the form of a PMPZ barrier.

DISCUSSION
Understanding how barriers to reproduction evolve may be the sine qua non of speciation
research. However, whether this is a focus on reproductive isolation per se (e.g. barriers) or
on causes of reproductive isolation (e.g. traits) has been controversial (Shaw and Mullen
2011; Mullen and Shaw 2014). Moreover, while understanding the relative contribution of all

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17

forms of reproductive isolating barriers among species pairs across different taxa has been
promoted (Ramsey et al. 2003; Sanchez-Guillen et al. 2012), there have been few studies that
document such variation among populations of the same species that exhibit incipient
speciation nor that measure the relative contributions of reproductive barriers. Likewise,
recent research from a variety of taxa has emphasized the existence of postmating-prezygotic
barriers, although this is usually in the context of the evolution of reproductive isolation
between sympatric ecotypes or along hybrid zones where current gene flow may impact
responses (e.g., Larson et al. 2012; Tyler et al. 2013; Butlin 1998). Finally, previous work on
Drosophila has suggested that pre- and post-zygotic isolation evolve in allopatry at
approximately the same rate across evolutionary time, although the mechanistic details and
information on very early isolation barriers are lacking in these surveys (Coyne and Orr, 1989,
1997, 2004). Similar studies in different taxa have not always supported this pattern (e.g.,
Mendelson 2003; Moyle et al. 2004; Stelkens et al. 2010). Here we take advantage of using
the genus Drosophila as a model system for understanding species isolation. By using
populations of the same species (i.e. do not have different species status) with limited gene
flow, we have documented both symmetric and asymmetric premating and postmating
prezygotic barriers to reproduction, which evolve independently. Studies of other taxa,
ranging from Drosophila species pairs to mice, have found support for aspects of our results,
which we discuss below; as a set of results, the combined work presented here provides
evidence addressing a number of outstanding questions on the evolution of reproductive
isolation.

In this research, we have identified intrinsic reproductive barriers that could play a role in the
early stages of speciation within a species among three allopatric populations in which
barriers to gene flow are mostly incomplete. Because other studies on these populations have

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18

identified divergence in mating traits known to be important for reproductive success in other
species (Klappert et al. 2007; Veltsos et al. 2012; Jennings et al. 2013), these traits (i.e.,
speciation phenotypes) can be putatively linked to these reproductive barriers. Additionally,
we find that premating and PMPZ barriers have both arisen among populations and that these
can be asymmetric or symmetric depending on the pairwise combinations of the three
populations. The strength of postmating, prezygotic barriers does not seem to be influenced
by time since divergence whereas there is a trend for premating barriers to increase with
increasing genetic distance (based on microsatellite measures; Table 3). Thus, we critically
show that early in the speciation continuum, pre- and post-mating, prezygotic isolation can
evolve effectively independently from one another and apparently in the complete absence of
intrinsic postzygotic isolation. These results shed light on the mode and tempo of the
evolution of early reproductive barriers occurring prior to complete speciation.

The premating isolation barriers between D. montana populations appeared to be largely

based on females discrimination against heteropopulation males. This barrier was symmetric
in one pair of crosses (Oulanka - Colorado) and asymmetric in the other two pairs
(Vancouver - Oulanka and Vancouver - Corado), where Vancouver females discriminate
against both Oulanka and Colorado males but Colorado and Oulanka females do not
discriminate against Vancouver males. In contrast, both Colorado and Oulanka females
symmetrically discriminate against each others males. A variety of candidate traits
(speciation phenotypes) could be implicated in this barrier, perhaps driven via sexual
selection. While sexual selection may be a particularly effective mechanism to reduce gene
flow between populations due to the build-up of premating male signal-female preference
functions, this idea remains controversial for a variety of reasons (Ritchie 2007; Safran et al
2013). Some evidence suggests however that sexual selection could rapidly drive speciation

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19

phenotypes, for example, in the genus Laupala, in which allopatric morphologically cryptic
species are distinguishable based on courtship song, and in which female preference is
exhibited (Mendelson and Shaw 2005).

In the D. montana system, females exercise strong mate preference on conspecific males
(Aspi and Hoikkala 1995). Both cuticular hydrocarbons (CHCs) and male courtship song are
divergent in the populations we have studied here and both are associated with courtship
success (Klappert et al. 2007; Veltsos et al. 2012; Jennings et al. 2013). Selection surfaces
(i.e., female preference functions) for male courtship song, as well as the song itself, have
been shown to vary among these populations (Klappert et al. 2007; Veltsos et al. 2012) and
CHCs, which also differ among populations, have recently been implicated as playing a role
in mate choice (Jennings et al. 2013; Veltsos et al. 2012), suggesting that sexual selection
may partially explain premating isolation between populations. Thus, differences in either
single or multimodal signals and female preferences occur in this system, potentially
contributing to sexual isolation and incipient speciation when males and females from
different populations are brought together in a mating environment.

In addition to sexual isolation, we found a PMPZ barrier that was caused by the inability of
sperm from Vancouver males to successfully penetrate and fertilize the eggs of Colorado
(and to a lesser extent, Oulanka) females. While the actual mechanism of this fertilization
inefficiency is unknown, females stored many motile sperm for at least three days, suggesting
that either the sperm cannot leave the sperm storage organs for fertilization and/or that sperm
entrance into the egg is prevented at the egg surface (i.e., the sperm cannot penetrate the
micropyle). This PMPZ barrier is not complete; 30-60% of eggs produced in
heteropopulation crosses involving Vancouver males were fertilized and properly developing.

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20

Like premating sexual isolation, PMPZ is also asymmetric. Crosses involving Vancouver
females generally exhibit less evidence of such incompatibilities, although Vancouver
females mated to Colorado males may have impaired fertilization. The incomplete and
asymmetric patterns of fertilization success suggest that speciation phenotypes are associated
with the ejaculate of Vancouver males, either via sperm and/or seminal fluid, interacting
negatively with either the reproductive tract and/or eggs of heteropopulation females,
whereas Vancouver females are mostly resistant to any such effects from both coevolved and
non-coevolved males.

A number of potential reproductive traits are known to negatively affect fertilization in other
systems (e.g. conspecific sperm precedence, CSP; Gregory and Howard 1994). Given that
ejaculate proteins show rapid positive evolution (Price 1997; Gavrilets 2000; Swanson and
Vacquier 2002), are known to influence sperm storage and sperm competition (Bertram et al.
1996; Tram and Wolfner 1999) and can contribute to sexual conflict (Chapman et al. 1995),
interactions between the sexes regarding these proteins may be manifest as isolation in a
variety of ways. Most studies examining such interactions are between species but provide
good evidence for such negative interactions. For example, Marshall et al. (2011) found that
the ejaculate proteins of two species of crickets, Allonemobius fasciatus and A. socius, which
are only isolated by PMPZ barriers and are thought to have diverged only 30,000 years ago,
have rapidly and positively evolved between the species, arguing that ejaculate protein
evolution may mediate CSP. Reproductive isolation in the European hybrid zone of Mus
musculus and M. domesticus, which are thought to have diverged 500,000 years ago, is at
least partially determined by asymmetric sperm precedence in which M. musculus sperm
always outcompete M. domesticus sperm regardless of which species ova is being fertilized
(Dean and Nachman 2009). Ejaculate proteins between these species are also divergent

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21

(Ramm et al. 2008) giving weight to the idea that such differences may mediate PMPZ
between these species.

Within Drosophila, potential speciation phenotypes between the two species, D. simulans and
D. mauritiana, are related to CSP and include aspects of sperm transfer, storage, usage and
ejection and usage from female sperm storage organs (Manier et al. 2013). Likewise, in
species crosses between D. americana and D. novamexicana (Ahmed-Braimah and
McAllister 2012), and between D. virilis females and D. novamexicana males (Sagga and
Civetta 2011), problems in sperm storage and usage were implicated in reduced fertilization
and egg hatch rate. And, in heterospecific crosses between D. arizonae and D. mojavensis,
differential gene regulation was found in the female reproductive tract dependent on whether
females had conspecific or heterospecific partners (Bono et al. 2011). While these studies
show that PMPZ isolation can evolve, the above-mentioned species are those that have
already reached separate species status based on non-PMPZ barriers and thus, it is not
possible to determine whether PMPZ barriers arose first, playing a potentially vital role in the
speciation process, or have followed from other forms of isolation after speciation was
complete.

Our work on divergent populations suggests that, qualitatively, sperm transfer and storage
appear similar in all population crosses, implying more subtle interaction effects on
fertilization efficiency between the male ejaculate and female reproductive tract and/or eggs.
And while among population comparisons are rare, two previous studies have suggested traits
related to poor fertilization efficiency. In the stalk-eyed fly, Teleopsis dalmanni, four
populations with various divergence times showed reproductive incompatibilities caused by a
mismatch between male and female reproductive elements resulting in faulty sperm storage

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22

and fertilization failure (Rose et al. 2014). And in crosses between the Zimbabwe (Z) and
Cosmopolitan (M) strains of D. melanogaster, there is both asymmetric premating isolation
(Hollocher et al. 1997; Wu et al. 1995) and PMPZ due to either lack of fertilization or
incomplete fertlilization despite sperm being motile and stored in heteropopulation crosses
(Alipaz et al. 2001). Data from our study on crosses between populations of D. montana is
strikingly similar to that of D. melanogaster, suggesting that both premating and PMPZ, but
not postzygotic barriers, evolve rapidly, albeit asymmetrically, and putatively act as the
primary initiators of speciation.

Similar to sexual isolation, the PMPZ barrier we observed here could evolve as a
consequence of sexual selection (Fricke and Arnqvist 2004). Female D. montana remate
rapidly and often contain sperm from multiple males in their reproductive tracts (Aspi and
Lankinen 1992), setting the stage for sexual selection to drive the swift evolution of sperm
and/or ejaculate characteristics and corresponding female reproductive traits. Alternatively,
reinforcement, natural selection and/or genetic drift may contribute to cryptic RI (Coyne and
Orr 2004). Reinforcement-like processes may drive PMPZ barriers but this is only expected
in sympatric populations or between those with secondary contact, where maladaptive
hybrids can be produced (Servedio and Noor 2003; Coyne and Orr 1989). Here we used
allopatric populations that have no evidence of recent gene flow, are not currently in
secondary contact, and that apparently exhibit no postzygotic reproductive isolation. Thus, it
is unlikely that PMPZ in this system could have been driven by reinforcement. Whether the
observed differences between populations may be a consequence of natural selection as a
pleiotropic by-product of ecological divergence between populations is unknown. The
geographically widespread (circumboreal) distribution of D. montana populations and their
adaptation to different kinds of environmental conditions (temperature, humidity, light cycle,

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23

etc.) means that genetic changes in response to natural selection may not be consistent
between allopatric populations. Along with different temperature and humidity regimes found
in the three different locations of flies used in this study, flies have also adapted to different
photoperiods, which has been shown to influence important adaptive traits, including cold
hardening, cold tolerance and the timing of the onset of reproductive diapause (Tyukmaeva et
al. 2011; Vesala and Hoikkala 2011).

Finally, there is a positive correlation between the strength of premating and postzygotic
reproductive isolation and genetic divergence in Drosophila (e.g., Coyne and Orr 1997) and
in other organisms (e.g., Sanchez-Guillen et al. 2014 and references therein). In our study of
allopatric populations of D. montana, we find that premating and PMPZ have evolved first
with no apparent postzygotic isolation. Likewise, allopatric M and Z populations of D.
melanogaster show mostly pre-mating and PMPZ reproductive barriers (Alipaz et al. 2001).
The lack of evidence for postzygotic RI among populations in these two Drosophila species
in the face of the strong pattern among Drosophila species between time since divergence and
the strengths of premating and postzygotic RI suggests that postzygotic processes may occur
later on the speciation continuum, although its influence on preventing gene flow must then
catch up with that of prezygotic mechanisms at some later point along that continuum.

This article is protected by copyright. All rights reserved.

24

ACKNOWLEDGEMENTS
We thank Mike Ritchie, Outi Ala-Honkola, Patrik Nosil, Roger Butlin and Bill Etges for
helpful comments on the manuscript and Antti Miettinen for help in fly rearing and
maintenance and some data collection. This work was funded by a Marie Curie Initial
Training Network, Understanding the evolutionary origin of biological diversity (ITN2008-213780 SPECIATION). We thank all participants in the network for helpful and
stimulating discussions.

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Figure 1. Micrographs of D. montana eggs. A) Example of classification of developing (D)

and non-developing (N) eggs under fluorescent microscopy. B) Non-developing eggs
were subsequently examined for the presence of sperm since they may have undergone
fertilization, but not yet proceeded to mitotic division.

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35

Figure 2. Untransformed mean (SE) numbers of eggs oviposited (open) and progeny
produced (grey) by each singly mated female (listed first) in crosses within and between D.
montana populations. Different letters indicate significant differences in means (via Tukey
HSD tests on transformed data). Fecundity did not differ among cross types but was included
as a covariate in the analysis. Crosses are organized by male population of origin. Numbers in
bars are the number of pairs for each cross.

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36

Figure 3. Untransformed mean (SE) number of eggs oviposited (white) and number of eggs
hatching (grey) by each singly mated female (listed first) in crosses within and between D.
montana populations. Different letters indicate significant differences in means (via Tukey
HSD tests on transformed data). Fecundity (open square) was included as a covariate in the
analysis. Crosses are organized by male population of origin. Numbers in bars are the number
of pairs for each cross.

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37

Figure 4. Number of developing (open square) and nondeveloping eggs (grey squares) in
crosses between females (listed first) and males within and between D. montana populations.
Crosses are organized by male population of origin.

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38

Figure 5. Number of nondeveloping eggs that either did not have a sperm in it (open square)
or had a sperm present (grey squares) in crosses between females (listed first) and males
within and between D. montana populations. Crosses are organized by male population of
origin.

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39

Table 1. Total numbers of pair matings observed in pairwise, multiple-choice mating trials between
populations (females listed first) and overall sexual isolation (IPSI), heterospecific mating asymmetry
(IAPSI(ab/ba) and associated P-values determined by bootstrapping 10,000 times in the program Jmating.
Eight mating trials were carried out for each population cross. Significant IPSI values indicate significant
assortative mating.

Cross

Numbers of pair matings

value

IAPSI(ab/ba) SD

Colorado Oulanka

CC

CO

OC

OO

59

36

38

106

0.78

Colorado Vancouver

CC

CV

VC

VV

63

61

33

80

Oulanka Vancouver

0.0054*

P-

0.366 0.06

<

P-value

0.0001* 0.985 0.09

0.0008*

IPSI SD

1.156 0.09

0.226 0.07

0.006*

OO

OV

VO

VV

78

73

30

59

1.186 0.10

0.184 0.07

0.005*

*Indicates significant deviation from random mating

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40

Table 2. ANCOVA of the number of progeny produced (A) and the number of eggs hatched
(B) among the nine crosses with the number of eggs oviposited as the covariate. Both the
covariate and progeny number was square root transformed for normality.

A.
Source

d.f.

SS

660.77

44.50

< 0.0001

74.54

40.16

<0.0001

134

248.70

Effects
Cross

Covariate
Total egg number
Error

B.
Source

d.f.

SS

347.59

19.49

<0.0001

550.76

247.10

<0.0001

222

494.80

Effects
Cross

Covariate
Total egg number
Error

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41

Table 3. Pairwise genetic distances, absolute (AC) and relative (RC) contributions of pre- and post-mating
isolation and total isolation between populations for the three population crosses carried out in this study.

Genetic distance (Fst)

Total Isolation
(Microsats / mtDNA)*
(T)

Premating Isolation

Postmating Isolation
Population cross
(AC2 / RC2)
Colorado Oulanka
0.001 / 0.002

0.371 / 0.149
0.553

0.552 / 0.998

Colorado Vancouver
0.432 / 0.558

0.215 / 0.035
0.774

0.342 / 0.442

Oulanka Vancouver
0.102 / 0.291

0.162 / 0.163
0.350

0.248 / 0.709

(AC1 / RC1)

*From Mirol et al. (2007)

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42