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postmating, prezygotic reproductive isolation between populations. Ecology and Evolution.
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1 Received May|
Revised June|
Accepted Ju yDOI ece
O R I G I N A L R E S E A R C H
Persistent postmating, prezygotic reproductive isolation
between populations
Martin D. Garlovsky
1|
Rhonda R Snook
1,2This is an open access artic e under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided the origina work is proper y cited
The Authors Ecology and Evolution pub ished by John Wi ey Sons Ltd
1Department of Anima and P ant Sciences University of Sheffie d Sheffie d UK 2Department of Zoo ogy Stockho m University Stockho m Sweden
Correspondence
Rhonda R Snook Department of Zoo ogy Stockho m University Stockho m Sweden Emai rhonda snook zoo ogi su se
Funding information
Adapting to the Cha enges of a Changing Environment ACCE Natura Environment Research Counci Grant Award Number NE L Roya Society Leverhu me Trust Senior Research Fe owship
ABSTRACT
Studying reproductive barriers between popu ations of the same species is critica to understand how speciation may proceed Growing evidence suggests postmating prezygotic PMPZ reproductive barriers p ay an important ro e in the evo ution of ear y taxonomic divergence However the contribution of PMPZ iso ation to specia -tion is typica y studied between species in which barriers that maintain iso a-tion may not be those that contributed to reduced gene f ow between popu ations Moreover in interna y ferti izing anima s PMPZ iso ation is re ated to ma e ejacu ate fema e reproductive tract incompatibi ities but few studies have examined how mating his -tory of the sexes can affect the strength of PMPZ iso ation and the extent to which PMPZ iso ation is repeatab e or restricted to particu ar interacting genotypes We addressed these outstanding questions using mu tip e popu ations of Drosophila
montana We show a recurrent pattern of PMPZ iso ation with f ies from one popu a
-tion exhibiting reproductive incompatibi ity in crosses with a three other popu a -tions whi e those three popu a-tions were fu y ferti e with each other Reproductive incompatibi ity is due to ack of ferti ization and is asymmetrica affecting fema e fitness more than ma es There was no effect of ma e or fema e mating history on reproductive incompatibi ity indicating that PMPZ iso ation persists between popu
-ations We found no evidence of variabi ity in ferti ization outcomes attributab e to different fema e ma e genotype interactions and in combination with our other resu ts suggests that PMPZ iso ation is not driven by idiosyncratic genotype geno -type interactions Our resu ts show PMPZ iso ation as a strong consistent barrier to gene f ow ear y during speciation and suggest severa targets of se ection known to affect ejacu ate fema e reproductive tract interactions within species that may cause this PMPZ iso ation
K E Y W O R D S
Drosophila montana gametic iso ation postmating prezygotic iso ation sexua conf ict sexua
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INTRODUCTION
Speciation requires the accumu ation of barriers to gene f ow be -tween popu ations and subsequent taxa Identifying the barriers that act ear y during the evo ution of reproductive iso ation is critica to determine how speciation proceeds But in et a Coyne Orr Ture i Barton Coyne Reproductive barriers to gene f ow can broad y be c assified into three categories Premating reproductive barriers reduce the incidence of hybridization events between taxa Dopman Robbins Seaman Hoskin Higgie McDona d Moritz Lackey Boughman Murray C arke whi e postzygotic reproductive barriers are those that resu t in reduced fitness of hybrid offspring either due to in -trinsic genetic defects i e steri ity or inviabi ity or ow fitness in either of the parenta habitats Cooper Sedghifar Nash Comeau t Matute Presgraves Wu Ting The third c ass of reproductive barriers is postmating prezygotic PMPZ reproduc -tive barriers incompatibi ities re ating to interactions between the sexes that act after copu ation but before karyogamy preventing successfu ferti ization between popu ations or taxa Both premating and postzygotic reproductive barriers to gene f ow have been stud -ied extensive y however on y re ative y recent y have PMPZ repro -ductive barriers begun to be considered in more detai as potentia y important reproductive barriers
The fast paced mo ecu ar evo ution of reproductive tract tissues within popu ations acce erated by sexua se ection and sexua con -f ict is predicted to resu t in rapid divergence between popu ations in a opatry and the emergence of PMPZ reproductive incompatibi -ities between popu ations ear y during reproductive iso ation Eady
Panhuis But in Zuk Tregenza In po yandrous mat -ing systems with interna ferti ization where fema es mate mu tip y within a sing e reproductive cyc e the ejacu ates of mu tip e ma es may over ap within the fema e reproductive tract Different ma es ejacu ates must then compete to ferti ize ova sperm competition and fema es retaining sperm from mu tip e ma es may bias paternity cryptic fema e choice Such postcopu atory sexua se ection and its attendant sexua conf ict within popu ations Andersson Andersson Simmons Arnqvist Rowe Gavri ets
can shape the evo ution of intersexua interactions during copu ation and ferti ization Bernasconi et a Birkhead Pizzari Firman Gasparini Manier Pizzari Rapid evo ution of such phenotypes is supported by evidence that genes encoding reproductive tract proteins are among the fastest evo v -ing show-ing rapid protein sequence and gene expression evo u -tion Ho is Hou e Yan Kawecki Ke er Perry et a Swanson Vacquier Ve tsos Fang Cossins Snook Ritchie
Postmating prezygotic iso ation in externa ferti izers is most y imited to incompatibi ities re ating to chemo attraction between gametes Weber et a and or gamete interactions at the ce surface Vacquier Swanson For interna ferti izers an additiona array of potentia PMPZ reproductive barriers can act as a resu t of the comp ex series of events that take p ace within
the fema e reproductive tract after mating B och Qazi Heifetz Wo fner Orr Brennan Schnakenberg Siega B och Qazi In sing e heterospecific matings successfu fer -ti iza-tion can be decreased or prevented by reduced sperm trans -fer by ma es and or reduced transport storage and viabi ity of hetero specific sperm in fema es Ahmed Braimah Ke eher
Markow Kohyama Matsubayashi Katakura Larson Hume Andr s Harrison Manier et a Reinhardt Rose Brand Wi kinson PMPZ iso ation has a so been suggested to occur when hetero specific matings resu t in reduced egg production compared to con specific mat -ings even though ferti ization is successfu e g Matute Coyne
Turissini McGirr Pate David Matute PMPZ iso -ation in interna y ferti izing anima s may a so be manifested on y when con and hetero specific ejacu ates are in competition For instance con specific sperm precedence occurs when paternity is biased to sperm from the con specific ma e even though hetero specific ma e sperm may ferti ize ova in sing e matings Casti o Moy e Cramer und McFar ane Johnsen Qvarnstr m
Price Yeates et a
A growing body of iterature now shows PMPZ iso ation is the primary or on y barrier to gene f ow in some c ose y re ated taxa suggesting an important ro e in the ear y evo ution of reproductive iso ation Ahmed Braimah Unck ess C ark Bono Matzkin Hoang Brandsmeier Cramer et a Dean Nachman
Soudi Reinho d Engqvist Turissini et a However the majority of research has focused on incompatibi ities arising between species even though barriers that maintain repro -ductive iso ation after divergence may not be the same barriers that were important in reducing gene f ow during the initia stages of the speciation process But in et a Coyne Orr Ture i et a Therefore to understand the factors important during the initia stages of divergence more focus is needed on the repro -ductive barriers acting between recent y diverged popu ations of the same species
Drosophila montana provides an opportunity to study the ro e of
PMPZ iso ation and the ear y stages of the speciation process This species is distributed across the northern hemisphere at high a ti -tudes and ati-tudes with a we documented eco ogy and phy ogeo -graphic history Aspi Lumme Hoikka a Heikkinen Miro et a Investigating the contribution of both pre and post -mating reproductive barriers between three D. montana popu ations
iso ation may be especia y important ear y during the evo ution of reproductive iso ation
Yet there remain severa open questions about the evo ution of PMPZ iso ation both specifica y for this system and genera y Are patterns of PMPZ iso ation unique to these popu ations or more widespread Are PMPZ iso ation patterns repeatab e with individu -a s tested from the s-ame oc-ation but co ected -at different times Do a individua s show simi ar strengths of PMPZ iso ation or is PMPZ iso ation idiosyncratic between some individua s Additiona y ma e and fema e mating history may a so inf uence the expression and strength of PMPZ iso ation with consequences for the importance of PMPZ iso ation in imiting gene f ow between popu ations Mating history is known to have both ame iorating and exacerbating effects on other types of reproductive incompatibi ity such as cytop as -mic incompatibi ity between Wolbachia infected D. simulans ma es
and uninfected fema es which is ame iorated if ma es have remated frequent y Awrahman Champion de Crespigny Wede Karr Yang Feder In contrast receipt of mu tip e foreign ejacu ates by fema es may amp ify inferti ity due to receipt of toxic foreign ejacu ates Ke eher Markow Know es Markow Do ma e and fema e mating history inf uence the strength of PMPZ iso ation
We addressed these outstanding questions about the evo ution of PMPZ iso ation by testing both recent co ections from the same North American ocations as previous y described and additiona new popu ations We a so assessed whether PMPZ iso ation is acting at the popu ation eve or on y between specific genotype geno -type interactions from different popu ations Furthermore we de -termined whether the presence and strength PMPZ iso ation are affected by intrinsic inferti ity or ma e and fema e mating history
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METHODS
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F y stocks
Adu t D. montana were co ected from riparian habitats using ma t
bait buckets and mouth aspirators from Ashford Washington USA in referred to as Ashford and abbreviated as A Crested Butte Co orado USA in and referred to as Co orado abbreviated as C Jackson Wyoming USA in referred to as Jackson abbreviated as J and Vancouver British Co umbia Canada
referred to as Vancouver abbreviated as V in and Figure Tab e S Both iso fema e ines and popu ation cages were tested for PMPZ iso ation Popu ation cages for Co orado and Vancouver were estab ished by combining F prog -eny of each sex from each of iso fema e ines The popu ation cage for Vancouver was estab ished in the same way except F progeny from iso fema e ines were merged A popu ations and iso fema e ines were cu tured in the aboratory on Lakovaara ma t medium Lakovaara in over apping generations at C in constant ight Jennings et a F ies used for experimenta -tion were co ected within days of ec osion as ma e reproductive
maturity does not occur unti at east days postec osion Pitnick Markow Spicer A experiments were carried out using f ies aged between and days from ec osion In each experiment we carried out a four possib e crosses between the two foca popu a -tions being tested where the fema e popu ation is a ways indicated first e g AA is a cross between Ashford fema es and Ashford ma es and AC is a cross between Ashford fema es and Co orado ma es
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Statistica ana ysis
We out ine specific statistica tests for each experiment and trait we ana yze at the end of each section described be ow A statistica ana yses were performed in R version R Core Team Genera ized inear mixed effects mode s GLMMs and paramet -ric bootstrap simu ations to obtain mode predicted va ues confidence interva s were fitted using the me package Bates M ch er Bo ker Wa ker We tested for significance of fixed effects and interactions via ike ihood ratio tests LRT or parametric bootstrapped simu ations using the PBmodcomp function from the
pbkrtest package Ha ekoh Højsgaard When necessary we performed post hoc Tukey s honest significant difference HSD tests using the glht function from the mu tcomp package Hothorn
Bretz Westfa
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Postmating prezygotic iso ation between
North American popu ations of
D. montana
To test the pattern of PMPZ iso ation previous y reported Jennings et a with a new Co orado popu ation and to identify other popu ations showing evidence of PMPZ iso ation we performed a
F I G U R E Co ection ocations of Drosophila montana
popu ations Maps created using the ggmap package in R Kah e Wickham
Vancouver
Ashford
Jackson
Colorado
series of crosses between Co orado and Vancouver and two previ -ous y untested popu ations Ashford and Jackson PMPZ iso ation is measured by egg hatch success number of eggs oviposited that hatched and or number of progeny produced For each pair of foca popu ations we performed fu y factoria experiments generating data from both parenta crosses and the two reciproca between popu ation crosses We refer to the four crosses within each pair wise comparison as the cross type Fina samp e sizes and detai s of the specific strain x strain cross types performed in each of the pair wise combinations between the four popu ations and a summary of PMPZ outcomes are presented in Figures S and S
For each cross we assessed PMPZ iso ation by mating sing e virgin ma es and fema es n per cross type per b ock Note
a crosses were observed for mating over a hr period to exc ude confounding sources of reproductive iso ation If mating did not occur within this timeframe then we discarded that pair If mat -ing occurred then the pair was mouth aspirated into a chamber of an oviposition manifo d after mating Jennings et a Manifo ds were connected to oviposition p ates containing a mo asses agar egg aying medium with a drop of dried yeast paste added and incubated at C Fema es were eft to oviposit for days before changing the oviposition p ate and a owing a fur -ther days of oviposition Fo owing the second day oviposi -tion period f ies were discarded the numbers of eggs aid were counted fecundity and the oviposition p ate returned to the in -cubator Two days ater the numbers of unhatched eggs on the second oviposition p ate were counted again Fema es that did not oviposit were exc uded from ana yses
To assess differences between cross types in fecundity we fitted GLMMs with Poisson errors and a og ink using the tota number of eggs aid as the response variab e To assess differences between cross types in hatching success rates we fitted GLMMs with binomia errors and a ogit ink using the numbers of hatched eggs successes and unhatched eggs fai ures as the response variab e A mode s inc uded cross type as the on y fixed effect and we performed ana yses on each of the six crosses separate y We inc uded random effects for the specific strains tested from within each popu ation and for experimenta b ock to account for variation between strains tested in each cross type and variation between b ocks testing each cross between popu ations respective y A mode s a so inc uded an observation eve random effect OLRE to account for overdispersion Harrison To test whether there was a significant effect of cross type on each measure of PMPZ iso ation we compared each mode to a nu mode inc uding the g oba intercept as the on y fixed effect but with the same random effects structure with parametric bootstrapped simu ations
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Postmating prezygotic iso ation mechanism
Previous work showed that PMPZ iso ation was manifested by the ack of ferti ization despite ma es transferring and fema es storing
moti e sperm Jennings et a To confirm that egg hatch -ing fai ure was due to ack of ferti ization in additiona crosses and popu ations eggs from a subset of crosses were scored for deve opment fo owing the same protoco as previous y used Jennings et a Brief y for each of the four cross types in the Co orado Jackson and Co orado Vancouver cross Tab e S we mouth aspirated f ies of each sex into ha f pint bott es covered with an oviposition p ate con -taining mo asses agar egg aying medium with a drop of dried yeast paste added Oviposition p ates were rep aced every hr and eggs were co ected en masse fixed and stained using DAPI
Eggs were inspected using f uorescence microscopy to score for deve opment Nondeve oping eggs were further inspected using differentia interference contrast microscopy to score eggs for presence or absence of sperm in the egg indicating whether fer -ti iza-tion had been successfu We tested for differences in the numbers of ferti ized eggs in each cross type using Pearson s chi squared test Note that ferti ization fai ure cannot be due to cyto -p asmic incom-patibi ity as a consequence of Wolbachia infection
in our stocks because we found no visua evidence of Wolbachia
Stouthamer Breeuwer Hurst and previous ana yses in -vestigating Wolbachia preva ence across the Drosophila phy og -eny found no mo ecu ar evidence of Wolbachia in the virilis group
Bourtzis Nirgianaki Markakis Savakis Mateos et a
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Testing intrinsic ma e inferti ity
To assess whether ow ferti ization success in between popu ation crosses cou d be confounded by poor ma e ferti ity irrespective of the identity of his mate we mated foca ma es to both a between and within popu ation fema e For this experi -ment we used f ies from the Co orado and Vancouver popu ation cages Tab e S Foca ma es were paired individu -a y with two virgin fem-a es on consecutive d-ays one within -and one between popu ation fema e To account for any mating order effects we random y assigned ha f of ma es n per cross
partner first or second Vancouver ma es having a between popu ation partner first or second
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Consistency of postmating prezygotic
isolation across different genotypes
Reproductive incompatibi ity cou d be the resu t of idiosyn -cratic genotype genotype interactions between ma es and fema es from different popu ations rather than a popu ation eve effect To assess whether specific fema e genotype ma e genotype interactions yie ded variab e ferti ization outcomes we used matings within and between individua s from the Co orado and Vancouver popu ation cages Tab e S Foca ma es n per cross type were paired individu -a y with -a virgin fem-a e -and monitored for m-ating M-ated fema es were mouth aspirated sing y to a manifo d chamber after mating whi e ma es were transferred to new via s con -taining ma t medium The next day foca ma es were presented with another virgin fema e from the same popu ation as on the previous day Mated fema es were mouth aspirated sin -g y to a manifo d chamber after matin-g We repeated this for five consecutive days Mated fema es were processed for egg hatch success as previous y described To assess the between individua variance in hatching success for those ma es that mated three or more times we fitted a GLMM with binomia errors and a ogit ink using egg hatch success i e counts of hatched and unhatched eggs as the response variab e and mating day as the on y fixed effect We fitted a mode for each cross type separate y as combining groups across the differ -ent cross types wou d artificia y inf ate the between group variance Mode s inc uded a random effect for ma e identify and an OLRE
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Effects of ma e mu tip e mating on postmating
prezygotic isolation
To assess the effect of mu tip e mating on ma e ferti ization suc -cess we used the data co ected from Consistency of postmating prezygotic iso ation across different genotypes above and fitted a GLMM with binomia errors and a ogit ink using egg hatch success i e counts of hatched and unhatched eggs as the response vari -ab e with cross type mating number and the cross type mating number interaction as fixed effects and ma e identity as a random effect and an OLRE
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Effects of fema e mu tip e mating on
postmating, prezygotic isolation
To test whether mu tip e insemination affected the strength of PMPZ iso ation we mated foca fema es to mu tip e ma es For each of the four cross type combinations between the Co orado and Vancouver popu ation cages Tab e S foca fema es
n per cross type were paired individua y with a virgin ma e
Ma es were discarded immediate y after mating The next day foca fema es were mouth aspirated into a new via housing a virgin ma e from the same popu ation as on the previous day We repeated this for five consecutive days On y fema es who mated on three or more consecutive days were kept for ana ysis A progeny ec osing from each oviposition via were subsequent y counted and sexed To test the effect of mu tip e insemination on the tota strength of PMPZ iso ation we fitted a GLMM with Poisson errors using the tota number of progeny enc osed as the response variab e with cross type mating number and the cross type mating number interac -tion as fixed effects and a random effect for fema e identity and an OLRE
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RESULTS
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Postmating prezygotic iso ation between
North American popu ations of
D. montana
We performed a series of pair wise fu y factoria crosses between four D. montana popu ations from across North America to identify
popu ations showing evidence of PMPZ iso ation Previous studies have inc uded reduced fema e fecundity fo owing mating with a foreign ma e as a PMPZ reproductive barrier Matute Matute Coyne Turissini et a Here we found a significant effect of cross type on fema e fecundity in three Ashford Jackson Ashford Vancouver and Jackson Vancouver crosses of the six pair wise popu ation crosses Tab e However these responses were asymmetric and not in the predicted direction if PMPZ iso ation was acting Figures S and S In Ashford Jackson crosses one between popu ation cross had greater fecundity than both the reciproca cross and one of the parenta crosses Jackson ma es e evated Ashford fe -ma e fecundity above that of the reciproca cross AJ vs JA Tukey s HSD p Tab e S and the within popu ation Ashford cross
AJ vs AA Tukey s HSD p Tab e S The same pattern
was found for the Ashford Vancouver cross Ashford ma es e e -vated Vancouver fema e fecundity above the reciproca cross VA vs AV Tukey s HSD p Tab e S and the within popu ation
Vancouver cross VA vs VV Tukey s HSD p Tab e S In
the Jackson Vancouver cross between popu ation crosses differed from each other but not from either within popu ation cross JV had ower fecundity than VJ Tukey s HSD p Tab e S but these
did not differ from parenta crosses Moreover these pair wise popu ation comparisons showed no effect of cross type on hatching success a p Tab e Figures S and S Thus there is no
evidence of PMPZ iso ation between these three popu ations The three pair wise popu ation comparisons invo ving the Co orado popu ation showed no difference in fecundity between cross types Tab e However there was a significant effect of cross type on hatching success Figure Tab e Hatching success was high and simi ar for within popu ation crosses Tukey s HSD a p Tab es S S and S whereas the reciproca
Tukey s HSD a p Tab es S S and S Co orado fema es
mated to a foreign ma e had hatching success and in the re -ciproca crosses foreign fema es mated to Co orado ma es had hatching success Figure In summary crosses that invo ved f ies from Co orado exhibited asymmetrica PMPZ iso ation with a three other popu ations tested In contrast the crosses between pairs of those three popu ations showed no evidence of PMPZ iso ation
Figures S and S
To determine whether the strength of PMPZ iso ation with the Co orado popu ation depended on the non Co orado pop -u ation we tested whether there was a significant difference in hatching success by poo ing data across a experimenta b ocks for a between popu ation crosses invo ving on y Co orado fema es or Co orado ma es i e incompatib e crosses Co orado fema es showed equa y ow hatching success regard ess of the origin of the between popu ation ma e LRT df p Likewise
Co orado ma es showed equiva ent y ow hatching success regard -ess of the origin of the between popu ation fema e LRT
df p To determine whether egg hatch success varied
between compatib e crosses we poo ed data across a experimenta b ocks but exc uded a between popu ation crosses invo ving both Co orado ma es and Co orado fema es Compatib e crosses showed high hatching success that did not differ between crosses LRT
df p In summary the strength of PMPZ iso ation invo v -ing Co orado was equa across a popu ations regard ess of the pop -u ation origin of the foreign mating partner see egend in Fig-ure whereas a other between popu ation crosses had hatching success equiva ent to within popu ation success Figure S
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Postmating prezygotic iso ation mechanism
After surveying a popu ations for evidence of PMPZ iso ation we scored oviposited eggs for deve opment and ferti ization status in the Co orado Vancouver cross to confirm ow hatch -ing rates were due to the same pattern of ferti ization fai ure previ -ous y reported Jennings et a We a so scored eggs from the Co orado Jackson cross to confirm whether this was a consistent PMPZ iso ating mechanism We found a significant effect
TA B L E Measures of postmating prezygotic iso ation fecundity and hatching success between North American popu ations of
Drosophila montana
Cross
A × C (3) A × J (1) A × V (3) C × J (1) C × V (7) J × V (1)
Measure of PMPZ χ2 p χ2 p χ2 p χ2 p χ2 p χ2 p
Fecundity 11.56 0.015 10.14 0.050 0.144 10.20 0.023
Hatch success 21.74 0.005 0.802 107.08 <0.001 39.571 <0.001
Note. p Va ues obtained from parametric bootstrap simu ations comparing the mode inc uding cross type as the on y fixed effect against the
nu intercept on y mode Cross ists the two popu ations being fu y reciproca y crossed e g A C AA AC CA CC where A Ashford C Co orado J Jackson V Vancouver the popu ation of the fema e is isted first Because each cross contained a four cross types for each measure of PMPZ
df for a mode s Number in parentheses after the cross is the number of rep icate b ocks Tota samp e sizes for each cross provided in Figure
Bo d va ues indicate significance of
F I G U R E Proportion of eggs hatching mean confidence interva s in crosses invo ving Co orado Within each pane different etters indicate significant differences from post hoc Tukey s HSD Letters are recyc ed in each pane however supp ementary ana yses showed that etters shared across pane s a so represent statistica y equiva ent groups see Section Cross types are abbreviated with the fema e popu ation given first A Ashford C Co orado J Jackson V Vancouver N number of mating pairs over a experimenta b ocks
N B crosses not showing PMPZ iso ation are shown in Figure S a
b
c a
n = 56 n = 52 n = 57 n = 55
a
c
b
a
n = 21 n = 20 n = 19 n = 19
a
c
b
a
n = 176 n = 165 n = 164 n = 160
Colorado x Ashford
Colorado x Jackson
Colorado x Vancouver
CC CA AC AA CC CJ JC JJ CC CV VC VV
0.00 0.25 0.50 0.75 1.00
Pr
opor
tion hatched (mean
±
of cross type on the number of eggs ferti ized in the Co orado Vancouver χ2 df p and in the Co orado
Jackson χ2 df p crosses Whi e most eggs
were deve oping in a within popu ation crosses eggs oviposited by Co orado fema es mated to foreign ma es had of eggs fer -ti ized and foreign fema es mated to Co orado ma es had of eggs ferti ized Figure
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Testing intrinsic ma e inferti ity
To test whether reduced ferti ization success cou d be due to intrin -sic ma e inferti ity we ca cu ated Spearman s rank corre ation coef -ficient for the proportion of eggs that hatched for ma es mated to both a within and between popu ation fema e Tab e Figure S There was no corre ation in the eve of ferti ity between the first and second mating regard ess of mating order Spearman s rank corre ation a p Tab e Therefore hatching success rates
can be attributed to the cross type a one and are not confounded by ma e inferti ity
|
Consistency of postmating prezygotic
isolation across different genotypes
To test whether PMPZ iso ation was due to either specific fema e genotype ma e genotype interactions or a popu ation eve phe -nomenon in the Co orado Vancouver popu ation cage cross Tab e S we assessed the between individua variance in hatching success for ma es that mated at east three times over con -secutive days most ma es mated the maximum of times median
number of mates Ha f of ma es were mated to virgin fema es from their own popu ation and the other ha f of ma es were mated to foreign fema es and we mode ed each cross type separate y to proper y partition between group variance In a cases estimates of between individua variance ma e identity random effect variance were signifying inc usion of the ma e identity random effect was not warranted in the mode s and mode s inc uding ma e identity as a random effect had higher AICc scores than those without Tab e S
Thus between ma e variance in hatching success was neg igib e in -dicating a consistent pattern of PMPZ iso ation acting across a range of fema e ma e genotype interactions between popu ations
|
Effects of ma e and fema e mu tip e mating on
postmating, prezygotic isolation
To test whether ma e mu tip e mating affected the strength of PMPZ iso ation in the Co orado Vancouver popu ation cage cross Tab e S we assessed the effect of mu tip e mating of ma es on ferti ization success Figure There was a significant effect of cross type LRT df p incompatib e crosses
had ow egg hatch success and a margina y significant effect of mating number on egg hatch success LRT df p
suggesting that ma es improve ferti ization success as they mate more The cross type mating number interaction was not signifi -cant LRT df p Thus there was no effect of ma e
mating history on the strength of PMPZ iso ation
We a so tested the effect of fema es receiving mu tip e ejacu -ates on the strength of PMPZ iso ation by counting the tota num -ber of adu t progeny produced each day by fema es inseminated by
F I G U R E Proportion of deve oping ight gray and unferti ized dark gray eggs in each cross type Cross types are abbreviated with the fema e popu ation given first C Co orado J Jackson V Vancouver Numbers in bars indicate the tota number of eggs counted
116
31
45
115 19
99
52
16
403
85
122
327 14
374
128
19
Colorado x Jackson Colorado x Vancouver
CC CJ JC JJ CC CV VC VV
0.00 0.25 0.50 0.75 1.00
Pr
opor
tion
Developing Unfertilised
Male population
Female population
N Spearman s rho p
First mating Second mating
Co orado Co orado Vancouver 20
Vancouver Co orado 18 0.482
Vancouver Vancouver Co orado 18
Co orado Vancouver
up to five ma es As with ma es a most every fema e mated every day median number of mates Like ma es we found a signifi -cant effect of cross type LRT df p incompat -ib e crosses had ow ferti ity Un ike ma es we saw a strong effect of mating number of progeny production per day LRT
df p the rate of progeny production increased with
mating number simi ar y in a four crosses Figure However we sti found no effect of the cross type mating number interaction LRT df p thus there was no effect of fema e
mating history on the strength of PMPZ iso ation
|
DISCUSSION
Identifying ear y acting reproductive barriers is centra to understand -ing the factors that contribute to the initia stages of the speciation process Whi e recent efforts have increasing y identified PMPZ iso a -tion as critica in these ear y stages Devigi i et a Soudi et a
Turissini et a outstanding questions remain about fac -tors that cou d inf uence the extent of gene f ow between popu a -tions exhibiting PMPZ iso ation We addressed the repeatabi ity and consistency of PMPZ iso ation acting between different popu ations
the mechanism of PMPZ iso ation and how ma e and fema e mating history inf uences the strength of PMPZ incompatibi ity We found a recurrent and robust pattern of PMPZ iso ation between D. montana
popu ations Crosses invo ving either ma es or particu ar y fema es from Co orado exhibited PMPZ iso ation with three other popu ations whi e crosses between those three popu ations remained ferti e with each other Incompatibi ity was due to ferti ization fai ure but was not a consequence of intrinsic ma e inferti ity As reproductive iso ation is not comp ete between these popu ations incompatibi ities may on y be present between specific fema e ma e genotype interactions but we found no variation in hatching success attributab e to ma e iden -tity Thus we show that PMPZ iso ation at east between Co orado and Vancouver is acting at the popu ation eve Mu tip e mating by ma es did not inf uence ferti ization competency compatib e crosses remained compatib e and incompatib e crosses remained incom -patib e Likewise whi e mu tip e insemination of fema es increased the number of progeny produced per day in a crosses incompat -ib e crosses sti produced significant y fewer progeny compared to within popu ation crosses Thus ma e or fema e mu tip e mating nei -ther exacerbated nor ame iorated incompatibi ity These patterns sug -gest that gene f ow wi be imited between Co orado individua s and the other popu ations at east under these conditions
F I G U R E Proportion of eggs hatching mean and mode predicted va ues CI per day for ma es mated to between three and five within or between popu ation fema es over consecutive days Cross types are abbreviated with the fema e popu ation given first C Co orado V Vancouver Numbers be ow points indicate samp e sizes number of mating pairs each day
●
●
● ●
●
10 10 10 10 9
CC
●
● ●
● ●
10 10 10 10 9
CV
● ●
●
● ●
6 6 6 5 5
VC
●
● ●
●
●
10 9 10 9 9
VV
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
0.00 0.25 0.50 0.75 1.00
Mating number
Pr
opor
tion hatched (mean
±
95% CI
)
F I G U R E Per day progeny production mean and mode predicted va ues CI for fema es mated to mu tip e within or between popu ation ma es over consecutive days Cross types are abbreviated with the fema e popu ation given first C Co orado V Vancouver Numbers be ow points indicate samp e sizes number of mating pairs each day
● ●
● ●
●
14 13 13 12 10
CC
● ●
●
● ●
15 14 13 8 7
CV
● ●
● ●
●
14 13 12 10 8
VC
● ●
● ●
●
15 12 10 9 7
VV
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
0 10 20 30
Mating number
Pr
ogen
y pr
oduced (mean
±
95% CI
Other studies of PMPZ iso ation between species have sug -gested that reduced fecundity is a PMPZ reproductive barrier even if ferti ization occurs norma y Matute Turissini et a Here we show that in some between popu ation crosses fecundity is the same as at east one of the parenta crosses so PMPZ iso a -tion is not due to a reduc-tion in fecundity Instead PMPZ iso a-tion is manifested as a consequence of reduced ferti ization rates For norma and efficient ferti ization a coordinated series of ejacu ate fema e reproductive tract interactions are required Avi a Sirot LaF amme Rubinstein Wo fner B och Qazi et a Mattei Riccio Avi a Wo fner Pitnick Wo fner Suarez
Wo fner The emergence of PMPZ reproductive barri -ers may be due to mismatched ejacu ate fema e reproductive tract interactions deriving from popu ation differentiation arising from se ection and or genetic drift However Jennings et a found no re ationship between genetic distance and the strength of PMPZ iso ation suggesting divergence is not simp y a resu t of iso ation by distance Instead PMPZ iso ation ike y emerges as a by product of both sexua se ection and sexua conf ict which are important in shaping the rapid co evo ution of ejacu ate fema e reproductive tract interactions Ahmed Braimah et a Bono et a Mende son Martin F axman Pitnick et a Given that D. montana ma es transfer and fema es store moti e sperm for
ferti ization but most of these sperm do not penetrate eggs incom -patibi ity is ike y because of mismatches between sperm and egg re ease These incompatibi ities may arise due to variation between popu ations in semina f uid proteins Sfps in the ma e ejacu ate that cause profound behaviora morpho ogica and physio ogica changes in the mated fema e Avi a Ravi Ram et a Perry Sirot Wigby Pitnick et a Ravi Ram Wo fner Wo fner Candidate Sfps inc ude sex peptide SP which binds to the fema e sex peptide receptor SPR in the mated fema e and is essentia for proper re ease of sperm from storage to ensure effi -cient ferti ization in D. melanogaster Avi a Mattei Wo fner
Avi a Ravi Ram Qazi Wo fner and or Acp DE and ovu in which are required for efficient sperm storage and oocyte re ease in
D. melanogaster Avi a Wo fner Mattei et a Future
work shou d examine popu ation variation in D. montana Sfp com -position to test their potentia ro e in mediating PMPZ iso ation and to identify under ying speciation genes But in et a Nosi Sch uter Presgraves
Reproductive incompatibi ity in this system is asymmetrica which may a so he p to understand the evo ution of ejacu ate fema e reproductive tract interactions and the emergence of PMPZ reproductive barriers Ferti ization was reduced more in crosses in -vo ving Co orado fema es of eggs hatched than in crosses invo ving Co orado ma es ca of eggs hatched Asymmetries in reproductive barriers cou d resu t from differences between popu ations in the strength of sexua se ection Boughman Rund e Sch uter and the action of sexua conf ict Arnqvist Edvardsson Friberg Ni sson For examp e considering the ma e ejacu ate as a po ygenic trait in popu ations where fema es have evo ved preferences for high trait va ues of ma es fema es wi
impose stronger se ection on ma es thus reproductive iso ation wi be stronger in crosses invo ving those fema es However fema es from a popu ation where trait va ues are ower on average may sti accept ma es from another popu ation having higher trait va ues Boughman et a generating asymmetries in reproductive iso ation Such asymmetries generate predictions to test in future research if postmating sexua se ection is stronger in the Co orado popu ation then Co orado ma es shou d have more competitive and or otherwise preferred ejacu ates than Vancouver ma es Whi e a previous study did not find PMPZ iso ation between these two popu ations under a sperm competitive scenario A a Honko a Ritchie Ve tsos the Co orado popu ation they used had very ow within popu ation ferti ization success and subsequent y went extinct in the aboratory suggesting some kind of inbreeding depression Our current research shows recurrent strong PMPZ iso ation between these popu ations that is not dependent on a particu ar co ection from a particu ar time and we conc ude that PMPZ iso ation occurs consistent y between these popu ations see a so Moorhead
Reproductive iso ation is not comp ete between the Co orado popu ation and any of the others we tested it against so it was important to estab ish whether PMPZ iso ation was an interaction between specific fema e ma e genotypes or a more widespread pattern acting across a range of genotypes We tested foca ma es against mu tip e incompatib e fema es and found itt e between ma e variance in ferti ization success which did not warrant inc uding ma e identity in the mode Low between ma e variance in ferti iza -tion success indicates PMPZ iso a-tion was acting consistent y across the range of genotype genotype interactions tested and was pres -ent at the popu ation eve It may be that genotypes were imited after being in cu ture for a period of time however we observe high ferti ization success in within popu ation crosses suggesting no in -breeding depression Moreover our resu ts between Co orado and Vancouver popu ations were simi ar regard ess of which Co orado and Vancouver popu ations iso fema e ines were being tested this study and Jennings et a Even if genetic variabi ity has been eroded during the course of aboratory cu ture then this means that a e es of arge effect are ike y fixed within popu ations making fu -ture studies identifying speciation genes oci causing PMPZ iso a -tion easier to detect
incompatibi ities between popu ations Both ma e and to a greater extent fema e reproductive success was increased by mu tip e mating but this increase was the same re ative amount for a crosses This cou d be due to severa different mechanisms such as fema es becoming more efficient ferti izers as they age increased sperm viabi ity and or sperm number and ejacu ate composition modification
In summary we focussed on recent y diverged popu ations of the same species to better understand PMPZ reproductive bar -riers that cou d act at the very ear iest stages of the speciation process But in et a Servedio Boughman Shaw Mu en Tinghite a et a the extent to which these barriers are consistent between popu ations co ected at differ -ent times and between differ-ent genotypes and how mating histories of the sexes inf uenced the strength of PMPZ iso ation Whi e there is no guarantee that these popu ations wi continue a ong the speciation process we showed consistent persistent and reproducib e iso ation between D. montana popu ations that
is manifested at the popu ation eve and not inf uenced by either ma e or fema e mating history PMPZ iso ation was asymmetrica and occurred between Co orado individua s crossed with a other tested popu ations and was a consequence of ferti ization fai ure ike y due to mismatches between ejacu ate fema e reproductive tract interactions Future work wi determine the nature of these mismatches and aim to identify the oci contributing to PMPZ iso ation
ACKNOWLEDGMENTS
Anne i Hoikka a offered the entry into this amazing system and kind y provided f ies used throughout the study Roger But in provided usefu comments that great y improved the manuscript We thank two anonymous reviewers for comments that improved the fina manuscript Joe Pick and John Jackson offered he pfu discussion about statistica ana yses and Joe Baxter he ped with data co ec -tion This work was funded by an ACCE Adapting to the Cha enges of a Changing Environment a NERC doctora training partnership ACCE DTP NE L and a Roya Society Leverhu me Trust Fe owship to RRS
CONFLIC T OF INTEREST
None dec ared
AUTHOR CONTRIBUTIONS
MDG co ected the data and performed ana yses MDG and RRS de -signed the experiments and wrote the manuscript
DATA ACCESSIBILIT Y
The fina dataset is avai ab e on Dryad https doi org dryad n g
ORCID
Martin D. Garlovsky http orcid org
Rhonda R. Snook http orcid org
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SUPPORTING INFORMATION
Additiona supporting information may be found on ine in the Supporting Information section at the end of the artic e
How to cite this artic e Gar ovsky MD Snook RR Persistent
postmating prezygotic reproductive iso ation between popu ations Ecol Evol. 2018;00:1–12. https doi
