THE ROLE OF DIFFERENT REPRODUCTIVE BARRIERS DURING PHENOTYPIC DIVERGENCE OF ISOPOD ECOTYPES

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.

THE ROLE OF DIFFERENT REPRODUCTIVE

BARRIERS DURING PHENOTYPIC DIVERGENCE

OF ISOPOD ECOTYPES

Fabrice Eroukhmanoff, 1 ,2,3 Anders Hargeby, 4 ,5 and Erik I Svensson 1 ,6

1 Section for Animal Ecology, Ecology Building, Lund University, SE-223 62 Lund, Sweden

2 Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, University of Oslo, P O Box 1066 Blindern, N-0316 Oslo, Norway

3 E-mail: fabrice.eroukhmanoff@bio.uio.no

4 Division of Biology, Link ¨oping University, 581 83 Link ¨oping, Sweden

5 E-mail: anhar@ifm.liu.se

6 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.

K E Y W O R D S : 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)

However, other mechanisms than assortative mating can also restrain gene flow One such mechanism that has re-cently 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 lim-iting 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, mi-gration modification might ultimately lead to allopatric or parap-atric 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

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

re-straining 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

an-tagonisms 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

gener-ations, 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

(F ST –Q ST 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

rel-atively recent and took place over a few decades, this system

provides a unique opportunity to study the emergence and

tem-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 es-timated 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 con-clusions 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 habi-tats in lakes, and mainly occur in reed stands (P australis) (Smock

and Harlowe 1983) Two shallow Swedish lakes have (start-ing 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,

form-ing a massive area of submerged vegetation in the limnetic zone (Hargeby et al 2007) Following the establishment of these exten-sive 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 re-sulted 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

con-trast, in the original source habitat (reed), invertebrate predators

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

diversi-fication 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 previ-ously published study (Eroukhmanoff et al 2009a) We measured pigmentation brightness (V) over the entire body (with values ranging from 0 [completely dark] to 1 [extremely lightly pig-mented individuals]) For the frequency distribution of pigmen-tation brightness (V), a total of 805 individuals were measured (Fig 1) We calculated the phenotypic variance from all individ-uals 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 quan-tify the degree of sexual isolation between lakes or ecotypes, we

Lake Tåkern

Pigmentation

0 0 0

0 0 0 0 0 0 0

0 0.

0

2

4

6

8

10

12

14

16

18

Reed

Stonewort

Lake Krankesjön

Pigmentation

0 0.

0 0 0 0 0 0 0 0 0 0 0.

0

2

4

6

8

10

12

14

16

18

Reed

Stonewort

Figure 1.Variation in coloration in the reed and stonewort isopod populations Shown are the frequency distribution for pigmentation brightness for both ecotypes in the two study lakes (Lake Krankesj ¨on and Lake T ˚akern) 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 Krankesj ¨on and in 2000 in Lake T ˚akern (equivalent to 40 and 14 isopod generations, respectively).

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 0 (did not mate) or

1 (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

indi-viduals 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)

(Abbrevia-tions 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

sub-strate 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

sub-strate 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

“experimen-tal 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

un-known substrate through repeated samplings and successive

dis-persal 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,

Table 1. Generalized linear model (GLZ) of how mating probabil-ity is affected by female and male ecotype and lake, as well as all their possible interactions.

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 <0.001

Male lake× female lake × male ecotype 0.68 0.41 Male lake× female lake × female ecotype 9.09 <0.001

Male lake× male ecotype × female ecotype 11.52 <0.001

Female lake× male ecotype × female ecotype

0.17 0.68 Male lake× female lake × male ecotype ×

female ecotype

1.40 0.24

short-lived and slow-moving animals are likely to suffer from high energetic expenses and high predation risk in the open water

We used three different replicates of each possible combination, for a total of 12 replicates Because of logistical difficulties, we were not able to perform this migration modification experiment also in Lake T˚akern, and hence only results from Lake Krankesj¨on are reported here

STATISTICS

We used a fully factorial generalized linear model (GLZ) (Type III) to investigate which factors influenced mating prob-ability In this model, we assumed that mating probability (the dependent variable) followed a binomial distribution, and we in-cluded female’s and male’s ecotype and lake as fixed factors and all their possible interactions (both two- and three-way, as in-dependent variables (Table 1) To correct for overdispersion, we rescaled the deviance parameter when it was needed To assess

to what extent lake-specific factors influenced the emergence of assortative mating, we categorized the trials as being either within lake/between lakes or within ecotype/between ecotypes, follow-ing the procedure of previous studies on ecological speciation (Rundle et al 2000; Boughman et al 2005) We estimated the av-erage mating probability for each type of combination Each of the four categories involved four different average mating probabili-ties from four different mating combinations We then compared

these four categories using a two-tailed t-test.

In the migration experiments, we analyzed variation in the probability of migration using a fully factorial general linear

Table 2. Generalized linear model (GLZ) of how migration

prob-ability is affected by ecotype and experimental ecotype as well as

their interaction.

Experimental habitat 3.81 0.51

Ecotype× experimental ecotype 73.50 <0.001

model (GLM) including experimental habitat and original

eco-type and their interaction (Table 2) In this analysis, we took into

account the average migration probability of the 50 individuals

per replicate for each treatment category Thus, we used the

av-erage value per replicate, pooled across all individuals, to ensure

statistical independence and avoid pseudoreplication

We partitioned the relative contribution of assortative mating

and migration modification, where habitat isolation was

quan-tified as 1 – (% of individuals which chose the foreign habitat

in all trials) and which equals 0.5 when habitat choice is

ran-dom and sexual isolation as 1 – (heterotypic mating

frequen-cies/homotypic mating frequencies) which equals 0 when mate

choice is random following a procedure described by Ramsey

et al (2003) We used a previously developed spreadsheet to

cal-culate total isolation and the absolute contributions to the total

isolation by any number of isolating barriers (Ramsey et al 2003;

available at http://www.plantbiology.msu.edu/schemske.shtml)

Finally, we used the software JMATING ( Antonio

Carvajal-Rodr´ıguez and Rol´an-Alvarez 2006) to estimate Ipsi, the index

of sexual isolation between ecotypes

THE EFFECT OF MIGRATION MODIFICATION AND

ASSORTATIVE MATING ON ADAPTIVE DIVERGENCE

As we have shown in a previous study (Eroukhmanoff et al 2009b)

phenotypic divergence between ecotypes is likely to be adaptive

and the phenotypic changes showed evidence of high evolutionary

rates, especially the pigmentation traits The two different barriers

to gene flow that we studied might have substantially enhanced

divergence, but their relative contribution to total isolation needs

to be quantified and the total extent to which they amplify

pheno-typic divergence when working in isolation, as well as jointly To

quantify their relative importance of these two barriers to

pheno-typic barriers, we used a previously developed theoretical

frame-work for how migration–selection balance influences population

divergence in quantitative traits (Hendry et al 2001; Bolnick et al

2009)

Hendry et al (2001) showed that, when migration precedes

selection within a generation, the equilibrium adaptive difference

in a quantitative trait between populations in the process of

spe-ciation, or in our case ecotypes, can be quantified as a function of

D, the optimal trait difference, m, the sum of the migration rates in

each direction, G the genetic variance, P the phenotypic variance,

andω2 the variance of the fitness function (inversely related to the strength of stabilizing selection).The following equation de-scribes the ratio of adaptive divergence in a phenotypic trait (as

it is the case for pigmentation brightness (Eroukhmanoff et al 2009b) with versus without a given isolating barrier

D I

D = V G + m(V P − V G+ ω2)

V G + m(1 − I )(V P − V G+ ω2), (1)

where D and D Iare the optimal trait differences with and without

one or several isolating barriers, V G and V P are the genetic and phenotypic variance for one quantitative trait (here, pigmentation brightness, a trait that is know to be under divergent selection

[Eroukhmanoff et al 2009b]), m is the cumulated random

mi-gration rate from one habitat to another in both directions and as defined in Hendry et al (2001),ω2is the variance of stabilizing

selection experienced in each habitat and I is the strength of the isolating barrier Thus, calculating this ratio between D I and D

enables us to investigate the impact of isolation on adaptive di-vergence without knowing the optimal trait differences between populations and the exact nature of the fitness function for this trait From this equation, we can derive a second one taking in account the two types of isolating barriers jointly:

D I

D = V G + m(V P − V G+ ω2)

V G + m(1 − I MM)(1− I AM )(V P − V G+ ω2), (2)

where I MM and I AM are the proportion of individuals that stay in their native habitats (a measure of habitat fidelity implying migra-tion modificamigra-tion, MM) or only mate with individuals from their own habitats (assortative mating, AM) In all of our simulations, the model parameters (additive genetic and phenotypic variances for the traits) were taken from our previously published studies on both ecotypes of Lake Krankesj¨on (Eroukhmanoff et al 2009a,

b) We successively used I MM and I AM alone in equation (1) to estimate their relative strengths when operating alone, as well

as jointly (eq 2) The goal of these simulations was to estimate total effect of reproductive isolation on adaptive phenotypic di-vergence These two parameters were taken directly from the raw mating and migration data, as can be seen in Figs 2A and 3B Because these values tended to slightly differ between the Reed and Stonewort habitats, as did phenotypic and genetic variances (Eroukhmanoff et al 2009a, b), we chose to present the results

of our simulations for the reed and stonewort quantitative genetic parameters independently, instead of averaging all parameters be-tween ecotypes We chose a moderate variance (ω2= 5 times the phenotypic variance for pigmentation brightness) for the fitness function in each habitat As a caveat, we note that this system is quite young and adaptive divergence might not yet have reached its equilibrium Thus these simulations will therefore provide a

Within Ecotypes Between Ecotypes

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

B

Stonewort Reed

Female Ecotype

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

A

Figure 2. Sexual isolation between ecotypes and lakes.

(A) Isopods belonging to the same ecotype form copulation pairs

more frequently, even when including all individuals from the two

different lakes in all possible population crosses (totalN = 1178).

A generalized linear model that included both female and male

ecotypes and lakes revealed a significant interaction between

fe-male ecotype and fe-male ecotype The I PSI index of total sexual

isolation between ecotypes amounted to 0.173 (SD= 0.067, P =

0.0092), showing moderate but significant assortative mating

be-tween ecotypes (B) Premating isolation bebe-tween ecotypes from

the same or different lakes Shown are the comparisons of the

average copulation probability for each experimental mating

cat-egory Comparison I: isopods belonging to the same ecotype have

the same probability of copulation, regardless of whether they are

from the same or different lakes Comparison II: isopods belonging

to the same ecotypes but from different lakes have a higher

prob-ability of copulation than isopods belonging to different ecotypes

from different lakes These two comparisons suggest a strong role

of natural selection in the emergence of reproductive isolation.

Comparison III: there was no difference in the degree of

premat-ing isolation between ecotypes among or within lakes.

conservative estimate of the extent to which adaptive divergence

might be enhanced (Hendry et al 2001; Bolnick et al 2009) We

chose to vary m, the random migration rate, to assess the role

of the isolating barriers on divergence at low, moderate, or high

dispersal rates However, in our case, dispersal is likely to be

Experimental Habitat 0.0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Stonewort Reed

B A

Figure 3. Migration modification and habitat isolation in the two different ecotypes in Lake Krankesj ¨on (A) We sampled isopods in the field for all the experimental procedures from two geograph-ically close sites that differ in habitat (less than 50 m between the sample sites,N = 600 individuals distributed in 12 experimental

replicates) (B) A fully factorial GLM that included “experimental habitat” (X-axis) and habitat of origin (ecotype) revealed a signif-icant interaction between these two factors The signifsignif-icant inter-action in this analysis reveals that isopods that were put in their native habitat dispersed to the other habitat to a lesser extent than the isopods that were put in the native habitat of the other ecotype.

quite low given both the physical and biological barriers between the two ecotypes (several meters of open water with no shelter, low food availability and predators, Eroukhmanoff and Svensson 2009)

Results

ASSORTATIVE MATING

We first conducted no-choice mating experiments that involved all possible mating combinations between the two ecotypes from the two lakes We investigated how probability of mating was affected by a male and female ecotype and lake of origin, as well as all possible interactions between these factors (Table 1 and Fig 2A) We found strong evidence for a significant female

ecotype× male ecotype interaction (GLZ: χ2= 9.02, P < 0.01).

This provides the first significant evidence for assortative

mat-ing between the different ecotypes in both lakes and reveals that

mating was preferentially within, rather than between ecotypes

The coefficient of total sexual isolation Ipsi(−1 = disassortative

mating; 0= random mating, 1 = assortative mating,

Carvajal-Rodr´ıguez and Rol´an-Alvarez 2006) was equal to 0.173 (SD=

0.067) After bootstrapping the data (n= 10,000 runs), it was

found to be significantly different from 0 (P = 0.0092) This

suggests moderate but significant assortative mating

To further investigate the role of ecological diversification in

assortative mating (Rundle et al 2000; Boughman et al 2005),

we compared the average mating probability for all

individu-als crossed in each type of category (Fig 2B) The three types

of comparisons were performed using the average probability

of copulation of each type of trial We used criteria that have

been outlined before in a previous study on ecological speciation

(Rundle et al 2000) First, we compared categories of

individu-als from similar ecotypes from the same or different lakes They

had the same probability of mating (t6= 0.352, P = 0.74) This

indicates that there is no overall isolation between the two lakes

Isopods belonging to the same ecotype from different lakes also

had a higher probability of mating than isopods belonging to

dif-ferent ecotypes from difdif-ferent lakes (t6= 2.84, P = 0.029) This

shows that assortative mating operates with respect to ecotype

across lakes, with little or no role of long-distance geographic

isolation (Fig 1) Isopods from different lakes will thus only

dis-criminate against isopods from a different ecotype, but not against

their own ecotype (Fig 2B) Finally, we compared crosses

in-volving individuals from different ecotypes, but from the same or

different lakes There was no significant difference between these

two types of crosses in the probability of mating, which suggests

that premating isolation has emerged in a similar fashion across

lakes (t6= 1.53, P = 0.17).

MIGRATION MODIFICATION

Next, we investigated if there was any evidence for migration

modification in the two ecotypes We experimentally

quanti-fied the strength of habitat choice using isopods from Lake

Krankesj¨on We found that isopods from each of the two

eco-types clearly preferred their own habitats during all trials (Fig 3)

A fully factorial GLM identified a significant interaction between

experimental habitat and source ecotype (F1,8 = 64.00, P<0.001).

There was no significant main effect of habitat (F1,8 = 2.46, P =

0.15) and no intrinsic main ecotype effect in the tendency to

dis-perse (F1,8 = 1.00, P = 0.34) This demonstrates that when placed

in their original source habitat, individuals do not migrate as

of-ten as when placed in a foreign experimental habitat (Table 2 and

Fig 3)

REPRODUCTIVE ISOLATION AND ADAPTIVE DIVERGENCE

Because both assortative mating and habitat choice seem to oper-ate in this isopod system, we quantified their relative contribution

to total isolation between ecotypes (Ramsey et al 2003) Total isolation between ecotypes amounted to 0.78 (0= no isolation,

1= complete isolation) We found that the relative contribution of assortative mating to total isolation was relatively weak (11.9%) compared to habitat choice (88.1%) Our numerical simulations revealed that even with low random dispersal and moderate se-lective pressures, the joint emergence of both these barriers is likely to have a strong positive effect on adaptive divergence (Fig 4) We explored the effects of different values ofω2(from 1 (strong stabilizing selection within each habitat) to 15 times (weak selection) the phenotypic variance) to investigate the robustness of our findings and conclusions Our results remained qualitatively the same across a wide range of parameter values, suggesting that our general conclusions are robust Again, migration modification was likely to have a stronger effect on adaptive divergence than assortative mating In combination, these two mechanisms appear

to have enhanced divergence three- to fivefold compared to if they would have operated in isolation, especially if one considers the upper limits of each curve (Fig 4)

Discussion

Colonization of novel environments might lead to ecological spe-ciation as a byproduct of adaptation to divergent selection (Rundel

et al 2000; Nosil and Crespi 2006a; Nosil et al 2000) Although the importance of ecology in speciation is acknowledged by many, relatively little is known about the evolutionary rate by which reproductive isolation might evolve (Hendry et al 2000, 2007) Some recent studies indicate that reproductive isolation can evolve over a few dozen generations (Hendry et al 2007) In contrast, the more traditional view is that speciation might take hundreds

of thousands of generations In this isopod species, rapid adap-tive divergence in pigmentation seems to have been facilitated by sorting of pre-existing variation in the ancestral ecotype (Fig 1; Eroukhmanoff et al 2009a; F Eroukhmanoff and E I Svensson, unpubl ms) The relatively rapid evolution that has taken place in this system might indicate that some degree of reproductive iso-lation might have reduced the constraining effects of gene flow between ecotypes Here, we have shown that different ecotypes of

an aquatic isopod mate assortatively, probably as an indirect con-sequence and correlated response of selection for local adaptation

to different predation regimes Assortative mating has emerged rapidly in this system, in as short time as 50 generations or less (Fig 2A)

Some additional analyses suggest that it is local adaptation rather than geographic isolation that has indirectly resulted in

Figure 4.Numerical simulations of the effect of migration

mod-ification (MM) and assortative mating (AM) on adaptive

diver-gence for pigmentation brightness in Lake Krankesj ¨on

Pigmen-tation brightness is a trait that is known to be under divergent

selection in this system and significantly heritable Even with low

random dispersal between habitats, each reproductive barrier at

the levels measured in this study will enhance adaptive

diver-gence When both migration modification and assortative mating

are present (MM + AM), divergence might even be enhanced by

between three- to fivefold, compared to when only assortative

mating operates.

premating isolation The first two comparisons of mating

propen-sity reveal that assortative mating is mostly based on ecotype,

and not on lake of origin (Fig 2B) Moreover, comparison

III (Fig 2B) also suggests a limited role for historical

contin-gency on the emergence of sexual isolation (Rundle et al 2000;

Langerhans and De Witt 2004; Langerhans et al 2006) These

findings suggest parallel emergence of premating isolation in the

two lakes and it is consistent with our previous findings of strong

parallel divergence in morphological and behavioral traits in this system (Eroukhmanoff et al 2009a; Eroukhmanoff and Svensson 2009) Moreover, in a previous study (Eroukhmanoff et al 2009a)

a haplotype network (mtDNA) revealed that the most likely sce-nario for ecotype divergence is in situ independent emergence of the stonewort ecotype in each of the lakes, which is also likely to apply to the independent emergence of assortative mating in both lakes

Our experiments from Lake Krankesj¨on strongly suggest that migration modification contributes to maintain reproductive iso-lation of the two ecotypes (Fig 3) Habitat fidelity is strong and due to the design we used, density-dependence is unlikely to have played a strong role Indeed, in the beginning of each trial individ-uals were all placed on one substrate, thus automatically favoring migration to the other substrate present if migration was density dependent However, no such effect was detected (Table 2) The results in this study suggest that migration modification has a stronger role than assortative mating in restraining gene flow be-tween the two ecotypes (Fig 3), which is also an inherent property

of philopatry as it intervenes as an early reproductive barrier Our simulations showed that for pigmentation brightness phenotypic divergence is maximized by the joint emergence

of both assortative mating and migration modification (Fig 4) Migration modification is an alternative isolating mechanism that might counteract gene flow, before assortative mating has emerged (Yukilevich and True 2006) Ecologically divergent se-lection can of course be solely responsible for the evolution of as-sortative mating and migration modification, but has so far mainly

be studied in the context of reinforcement both theoretically (Yukilevich and True 2006) and empirically (Nosil and Yukilevich 2008) The intensity of indirect selection against mi-grants or hybrids is likely to play a role too in the adaptive di-vergence process described here (Nosil and Yukilevich 2008) Unfortunately, we do not have any data on hybrid fitness to assess the role of hybridization in this system

The findings in this study are largely consistent with a sce-nario of ongoing ecological speciation strengthened through mi-gration modification, although complete reproductive isolation has not yet been achieved The fact that this process has been relatively fast, taking only a few dozen generations (Hargeby

et al 2004), suggests that under contrasting ecological conditions and under sufficiently strong divergent selection, local adaptation might be accompanied by reproductive isolation, even in the early stages of ecological speciation The total level of premating re-productive isolation (including both sexual and habitat isolation)

is very high in this system (0.78, although the index of sexual isolation is relatively weak, 0.173), especially, when keeping in mind that the phenotypic divergence between ecotypes is rela-tively recent A survey of the pattern of isolation between species

of the genus Drosophila (Coyne and Orr 1997) revealed that most

premating isolation indices estimated between pairs of sympatric

species were close to 0.8 Hence, the system we have studied here

has comparable levels of premating isolation as several sympatric

species-pairs of Drosophila, which have been diverging for

sev-eral hundreds of thousands of years, if one takes in account the

effects of migration modification (Coyne and Orr 1997)

In conclusion, we have demonstrated that both assortative

mating and habitat choice operate in this isopod system Our

experimental results and simulations suggest that both these

iso-lating barriers are likely to efficiently and jointly restrain gene

flow between the ecologically divergent populations These

bar-riers to gene flow are likely to be especially important in the early

stages of divergence and speciation In this isopod system,

migra-tion modificamigra-tion turned out to be more important in contributing

to total isolation, and seems to play a more pronounced role

in promoting adaptive divergence, at least in Lake Krankesj¨on

Our study adds to the increasing evidence that assortative mating

can emerge extremely rapidly (Hendry et al 2000) but our

re-sults also suggest an additional and important role for migration

modification

ACKNOWLEDGMENTS

We thank S Gu´echot, N Nowshiravani-Arnberg, and K Karlsson for

their help with field-work and P Edelaar, A Hendry, A Qvarnstr¨om,

J Kotiaho, K Rengefors and “The Svensson Lab” for their comments on

earlier drafts of the manuscript This study was financially supported by

the Swedish Research Council to FE and ES.

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Associate Editor: C C Nice