The question of how diverging populations become separate species by restraining gene flow is a central issue in evolutionary biology. Assortative mating might emerge early during adaptive divergence, but the role of other types of reproductive barriers such as migration modification have recently received increased attention. We demonstrate that two recently diverged ecotypes of a freshwater isopod (Asellus aquaticus) have rapidly developed premating isolation, and this isolation barrier has emerged independently and in parallel in two south Swedish lakes. This is consistent with ecological speciation theory, which predicts that reproductive isolation arises as a byproduct of ecological divergence. We also find that in one of these lakes, habitat choice acts as the main barrier to gene flow. These observations and experimental results suggest that migration modification might be as important as assortative mating in the early stages of ecological speciation. Simulations suggest that the joint action of these two isolating barriers is likely to greatly facilitate adaptive divergence, compared to if each barrier was acting alone.
O R I G I NA L A RT I C L E doi:10.1111/j.1558-5646.2011.01327.x THE ROLE OF DIFFERENT REPRODUCTIVE BARRIERS DURING PHENOTYPIC DIVERGENCE OF ISOPOD ECOTYPES Fabrice Eroukhmanoff,1,2,3 Anders Hargeby,4,5 and Erik I Svensson1,6 Section for Animal Ecology, Ecology Building, Lund University, SE-223 62 Lund, Sweden Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, University of Oslo, P O Box 1066 Blindern, N-0316 Oslo, Norway E-mail: fabrice.eroukhmanoff@bio.uio.no ¨ ¨ Division of Biology, Linkoping University, 581 83 Linkoping, Sweden E-mail: anhar@ifm.liu.se E-mail: erik.svensson@zooekol.lu.se Received September 24, 2010 Accepted April 03, 2011 The question of how diverging populations become separate species by restraining gene flow is a central issue in evolutionary biology Assortative mating might emerge early during adaptive divergence, but the role of other types of reproductive barriers such as migration modification have recently received increased attention We demonstrate that two recently diverged ecotypes of a freshwater isopod (Asellus aquaticus) have rapidly developed premating isolation, and this isolation barrier has emerged independently and in parallel in two south Swedish lakes This is consistent with ecological speciation theory, which predicts that reproductive isolation arises as a byproduct of ecological divergence We also find that in one of these lakes, habitat choice acts as the main barrier to gene flow These observations and experimental results suggest that migration modification might be as important as assortative mating in the early stages of ecological speciation Simulations suggest that the joint action of these two isolating barriers is likely to greatly facilitate adaptive divergence, compared to if each barrier was acting alone KEY WORDS: Adaptive divergence, assortative mating, contemporary evolution, ecological speciation, migration modification Empirical evidence has accumulated over the last decade pointing to an important role of ecology and natural selection in speciation (Schluter 2000; Coyne and Orr 2004; Nosil et al 2005; Nosil and Crespi 2006a) Several different studies have demonstrated the parallel build-up of reproductive isolation alongside phenotypic divergence between different ecological environments (Nosil et al 2002; Rundle et al 2003; Boughman et al 2005; Nosil and Crespi 2006b) The early emergence of assortative mating is crucial in the speciation process, because it will counteract the constraining effects of gene flow, which in turn will enhance the degree of phenotypic divergence (Nosil et al 2005; Coyne and Orr 2004; Rundell and Price 2009) 2631 C 2011 The Author(s) Evolution Evolution 65-9: 2631–2640 C However, other mechanisms than assortative mating can also restrain gene flow One such mechanism that has recently been discussed is the evolution of migration modification, that is behavioral shifts promoting philopatry and sedentariness (Yukilevich and True 2006; Edelaar et al 2008) Rather than limiting gene flow in situ, migration modification will reduce gene flow at the source, thereby decreasing migration load between habitats (Gavrilets et al 2000) If habitat choice is strong, migration modification might ultimately lead to allopatric or parapatric speciation (Yukilevich and True 2006; Gavrilets et al 2007; Bolnick and Nosil 2007; Edelaar et al 2008) Later, assortative mating might emerge secondarily through reinforcement of mate 2011 The Society for the Study of Evolution FA B R I C E E RO U K H M A N O F F E T A L preferences upon secondary contact (Yukilevich and True 2006; Gavrilets et al 2007; Edelaar et al 2008) Rapid emergence of reproductive isolation has been predicted by theoretical models, which suggest that assortative mating can evolve rapidly and under a broad range of selective conditions (Yukilevich and True 2006) In contrast, when divergent selection is strong, migration modification might be more efficient in restraining gene flow and causing speciation than assortative mating (Yukilevich and True 2006) With the exception of one previous empirical study on salmonids (Hendry et al 2000) which found that reproductive isolation could emerge as early as after only 13 generations, little is known about the temporal order and the rate of emergence of different isolation mechanisms during speciation (Nosil and Crespi 2006b; Rundell and Price 2009) Moreover, total reproductive isolation might also become weakened due to antagonistic interactions between assortative mating and other types of barriers to gene flow such as habitat choice, and these antagonisms might slow down the process of speciation (Yukilevich and True 2006; Hendry et al 2007) Here, we have estimated the strength and importance of assortative mating and migration modification during adaptive divergence between two ecotypes of the aquatic isopod Asellus aquaticus This aquatic isopod is common in many lakes and ponds in southern Sweden In two lakes (Lake Krankesj¨on and Lake T˚akern) independent oligotrophication events have taken place during the last two decades (Hargeby et al 2004, 2007) These ecological shifts resulted in the emergence of submerged vegetation (mainly a stonewort, Chara tomentosa) which formed a new habitat in the limnetic zone of both lakes The new stonewort habitat was rapidly colonized by isopods from neighboring reed belts (Phragmites australis) along the shores of both lakes (Hargeby et al 2004) In less than 50 generations, isopods diverged phenotypically between these different habitats, resulting in the emergence of two distinct ecotypes (Eroukhmanoff et al 2009a, b) Molecular analyses (mtDNA and AFLP-markers) indicate that the novel stonewort ecotype has evolved independently in the two lakes (Eroukhmanoff et al 2009a) Pigmentation and body size have an additive genetic basis, both within (Harbeby et al 2004) and between populations (Eroukhmanoff et al 2009b) We also have indirect evidence (FST –QST analyses, Eroukhmanoff et al 2009b) for a strong role for divergent selection, and at least pigmentation traits are under divergent selective pressures in the different ecotypes Adaptive divergence in this system is likely to be a result of predator-mediated natural selection, caused by qualitative and quantitative differences in predator faunas between the reed and the stonewort habitats (Hargeby et al 2004; Eroukhmanoff and Svensson 2009) Because this diversification process is relatively recent and took place over a few decades, this system provides a unique opportunity to study the emergence and tem- 2632 EVOLUTION SEPTEMBER 2011 poral order of different reproductive isolation mechanisms that might reduce ongoing gene flow in the early stages of population divergence and speciation We have investigated the strength of assortative mating and habitat isolation between ecotypes and estimated their relative contribution to total reproductive isolation We have also performed numerical simulations using previously estimated quantitative genetic parameters from these populations to estimate the relative importance of these two isolating barriers when operating either in isolation, or jointly Our results and conclusions in this study should hopefully stimulate future research on other reproductive barriers in addition to assortative mating, such as migration modification, a factor that might have been overlooked in speciation research (Yukilevich and True 2006) Methods STUDY ORGANISM AND STUDY POPULATIONS Asellus aquaticus is a freshwater isopod that is widespread in lakes, ponds, and slow-flowing rivers in Eurasia (Smock and Harlowe 1983) Populations of A aquaticus occupy various habitats in lakes, and mainly occur in reed stands (P australis) (Smock and Harlowe 1983) Two shallow Swedish lakes have (starting in 1987 in Lake Krankesj¨on and in 2000 for Lake T˚akern) experienced dramatic ecological shifts from a phytoplankton dominant state toward a macrophyte-dominant state (Hargeby et al 2007) Following these large-scale environmental shifts, stonewort (C tomentosa) colonized the old sediment areas, forming a massive area of submerged vegetation in the limnetic zone (Hargeby et al 2007) Following the establishment of these extensive stonewort stands, isopods subsequently colonized this novel habitat in both Lake T˚akern and Lake Krankesj¨on, where they can be found at very high densities (Karlsson et al 2010) In the new stonewort habitat isopods became brighter and smaller in size, compared to darker and larger isopods in the source populations in the reed habitat (Eroukhmanoff et al 2009a, b) Variation in body size and pigmentation brightness is largely heritable, with significant additive genetic variation both within and between populations (Hargeby et al 2004; Eroukhmanoff et al 2009b) Local adaptation in isopod pigmentation is likely to have resulted from the action of divergent selection pressures, caused by different visual backgrounds and different predator faunas in the two different habitats (Hargeby et al 2005) Several fish species are efficient predators on aquatic invertebrates (Wellborn et al 1996) and A aquaticus represents a common food source (Rask and Hiisivuori 1985) Predation from fish is likely to be more intense in the stonewort than in the reed habitat (Eroukhmanoff and Svensson 2009), due to much higher densities of perch (Perca fluviatilis) in the stonewort (Wagner and Hansson 1998) In contrast, in the original source habitat (reed), invertebrate predators E C O L O G I C A L D I V E R G E N C E A N D I S O L AT I N G BA R R I E R S relying on tactile cues (i.e., dragonfly and damselfly larvae) are the main threat toward the isopods (Eroukhmanoff and Svensson 2009) Recent molecular data suggest that this ecological diversification has occurred independently in these two lakes, suggesting that this system is a case of rapid contemporary parallel evolution (Eroukhmanoff et al 2009a) SAMPLING AND PHENOTYPIC ANALYSIS Isopods were captured with a net on their original substrate and at multiple locations within their source habitats, in both Lake T˚akern and Lake Krankesj¨on during two reproductive seasons (February–June) in 2005 and 2006 We only used individuals captured as pairs in precopula, where the male holds the female until molt and receptive to mating We did this to ensure that both males and females used in the experiments had reached sexual maturity All individuals were photographed live in a Petri dish with water under natural light conditions Pictures were analyzed with our own software (more information is available in a previously published study (Eroukhmanoff et al 2009a) We measured pigmentation brightness (V) over the entire body (with values ranging from [completely dark] to [extremely lightly pigmented individuals]) For the frequency distribution of pigmentation brightness (V), a total of 805 individuals were measured (Fig 1) We calculated the phenotypic variance from all individuals from both ecotypes of Lake Krankesj¨on for further use in the simulations described below MATING EXPERIMENTS To investigate if assortative mating was present and to quantify the degree of sexual isolation between lakes or ecotypes, we Lake Tåkern 18 Reed Stonewort Frequency 16 14 12 10 0.83 0.89 0.85 0.90 0.71 0.77 0.79 0.64 0.58 0.46 0.52 0.40 0.34 0.22 0.28 0.15 Pigmentation Lake Krankesjön 18 Reed Stonewort Frequency 16 14 12 10 0.74 0.68 0.63 0.57 0.52 0.46 0.41 0.35 0.30 0.24 Pigmentation Figure Variation in coloration in the reed and stonewort isopod populations Shown are the frequency distribution for pigmentation ¨ and Lake Takern) ˚ brightness for both ecotypes in the two study lakes (Lake Krankesjon in southern Sweden Isopods in the reed habitat are larger and darker, whereas the isopods in the novel stonewort habitat are smaller and lighter in pigmentation, as can also be seen in the photographs These phenotypic changes separating the ecotypes happened since the last oligotrophication of both lakes that caused ¨ rapid emergence and growth of submerged stonewort vegetation, a process that did not start earlier than in 1987 in Lake Krankesjon ˚ and in 2000 in Lake Takern (equivalent to 40 and 14 isopod generations, respectively) EVOLUTION SEPTEMBER 2011 2633 FA B R I C E E RO U K H M A N O F F E T A L performed no-choice experiments (Jennions and Petrie 1997) We randomly paired one sexually active male and one sexually active female from two given populations and observed them in a Petri dish filled with water From these trials, we were able to estimate the average propensity to form a precopula We used the same threshold time as in a previous study (520 s, Eroukhmanoff et al 2009a) to determine if individuals would have mated or not under natural conditions Couples were attributed to either the value (did not mate) or (mated) We conducted these mating experiments and tested all possible mating combinations between the two ecotypes from the two different lakes (four different crosses involving individuals of the same lake and ecotype (KR-KR, KS-KS TR-TR, TS-TS), two heterotypic crosses between lakes (TR-KS, KR-TS), two heterotypic crosses within lakes (KR-KS, TR-TS), and two homotypic crosses between lakes (TR-KR, TS-KS) (Abbreviations above: KR: Krankesj¨on Reed, KS: Krankesj¨on Stonewort, TR: T˚akern Reed, TS: T˚akern Stonewort) In total we performed a total of 589 such experimental mating trials (involving a total of 1178 individuals) These mating trials were distributed across 16 different pair combinations, and involved male and female ecotype and lake in the different categories MIGRATION MODIFICATION EXPERIMENTS To investigate whether habitat isolation was present in this system, we conducted additional experiments A total of 300 individuals from each ecotype from Lake Krankesj¨on were captured in the field and transported to our laboratory Isopods were acclimated for a period of two days They were fed on their original substrate sampled at the study sites during this period Animals were thereafter randomly divided into 50 individuals in each replicate, and placed in an aquarium (30 cm × 70 cm) containing the substrate from their original habitat (stonewort shoots or decaying reed leaves) in one end, and the substrate of the other habitat on the other end, separated by a distance of 40 cm, which formed a “neutral” zone where no substrate of any kind was present Isopods were then either placed in what we called “experimental habitat,” which could either be their own source habitat or a different habitat than from which they originated After 24 h, we counted the number of isopods within each substrate, to estimate the proportion of individuals that moved between substrates It is possible that a longer duration of the experiment might have enabled some sort of behavioral accommodation to an unknown substrate through repeated samplings and successive dispersal events by the individuals, and that habitat fidelity would decline over time However, this is unlikely to bear any strong significance in natural conditions, as both habitats are usually separated by at least 10 m of water and it is quite unlikely that isopods would migrate forth and back several times during their life under natural conditions, due to the fact that these small, 2634 EVOLUTION SEPTEMBER 2011 Table Generalized linear model (GLZ) of how mating probability is affected by female and male ecotype and lake, as well as all their possible interactions Effect χ2 P Male lake 2.97 0.08 Female lake 3.96 0.05 Male ecotype 0.05 0.83 Female ecotype 2.14 0.14 Male lake female lake 6.59 0.01 Male lake × male ecotype 6.19 0.01 Female lake × male ecotype 0.58 0.44 Male lake × female ecotype 5.79 0.02 Female lake × female ecotype 1.57 0.21 Male ecotype × female ecotype 9.02