Male courtship preferences demonstrate discrimination against allopatric colour morphs in a cichlid fish

Whether premating isolation is achieved by male‐specific, female‐specific or sex‐independent assortative preferences often depends on the underlying evolutionary processes. Here we test mate preferences of males presented with females of different allopatric colour variants of the cichlid fish Tropheus sp., a Lake Tanganyika endemic with rich geographical colour pattern variation, in which the strength of sexual isolation varies between populations. We conducted two‐way mate choice experiments to compare behaviour of males of a red‐bodied morph (population Moliro) towards females from their own population with behaviour towards females from four allopatric populations at different stages of phylogenetic and phenotypic divergence. Males courted same‐population females significantly more intensely than females of other populations, and reduced their heteromorphic courtship efforts both with increasing genetic and increasing phenotypic distinctness of the females. In particular, females of a closely related red‐bodied population received significantly more courtship than either genetically distinct, similarly coloured females (‘Kirschfleck’ morph) or genetically related, differently coloured females (‘yellow‐blotch’ morph), both of which were courted similarly. Genetically and phenotypically distinct females (Tropheus polli) were not courted at all. Consistent with previous female‐choice experiments, female courtship activity also decreased with increasing genetic distance from the males’ population. Given successful experimental and natural introgression between colour morphs and the pervasive allopatry of related variants, we consider it unlikely that assortative preferences of both sexes were driven by direct selection during periods of secondary contact or, in turn, drove colour pattern differentiation in allopatry. Rather, we suggest that sexual isolation evolved as by‐product of allopatric divergence.     

Assortative mating preferences between colour morphs of the endemic Lake Tanganyika cichlid genus Tropheus

Female mate preferences effectuate reproductive isolation among and sexual selection within species. Both mechanisms have been associated with the diversification and speciation of cichlid species flocks of the East African Great Lakes. In Lake Tanganyika, the endemic genus Tropheus has diversified into >100 geographic colour morphs. Although distributed allopatrically at present, water level fluctuations have repeatedly displaced and merged the benthic, rock-dwelling populations. Tests for assortative mating were performed to explore the potential for reproductive isolation between morphs in secondary contact, and to assess the importance of sexual selection for the diversification of this group. In contrast to other haplochromine cichlids, Tropheus is a sexually monochromatic, territorial and maternally mouthbrooding fish, which establishes temporary pair bonds prior to spawning. Female mate preference trials involved two-way choices between a homotypic and a heterotypic male and were conducted on allopatric populations of red and blue morphs from the southern part of Lake Tanganyika. Female affiliation time near each male’s compartment did not predict the mate preferences subsequently expressed in unrestrained interactions after removal of the compartment separators (spawning, pseudospawning and courtship). Consequently, mate preferences were inferred from unrestrained interactions with one test male at a time in replicate observation sessions. Of the 23 females tested, 13 courted, pseudospawned or spawned with the homotypic male, one blue female courted a red male, and nine females expressed no sexual motivation. The assortative mate preferences in the experiments (P < 0.01) suggest that colour differentiation between Tropheus populations can effectuate reproductive isolation, and is consistent with the notion that sexual selection contributed to the diversification of the genus. Guest editors: T. Wilke, R. V?in?l? & F. Riedel Patterns and Processes of Speciation in Ancient Lakes: Proceedings of the Fourth Symposium on Speciation in Ancient Lakes, Berlin, Germany, September 4–8, 2006     

A single mitochondrial haplotype and nuclear genetic differentiation in sympatric colour morphs of a riverine cichlid fish

Some of the diversity of lacustrine cichlid fishes has been ascribed to sympatric divergence, whereas diversification in rivers is generally driven by vicariance and geographic isolation. In the riverine Pseudocrenilabrus philander species complex, several morphologically highly distinct populations are restricted to particular river systems, sinkholes and springs in southern Africa. One of these populations consists of a prevalent yellow morph in sympatry with a less frequent blue morph, and no individuals bear intermediate phenotypes. Genetic variation in microsatellites and AFLP markers was very low in both morphs and one single mtDNA haplotype was fixed in all samples, indicating a very young evolutionary age and small effective population size. Nevertheless, the nuclear markers detected low but significant differentiation between the two morphs. The data suggest recent and perhaps sympatric divergence in the riverine habitat.     

Differences in sounds made by courting males of three closely related Lake Malawi cichlid species

Courtship sounds made by three sympatric cichlid species, Pseudotropheus zebra , P. callainos and an undescribed species known as P . 'zebra gold' were recorded and compared to investigate the potential role of acoustic signals in mate choice. Sounds were emitted during 'quiver' and 'circle' components of the male courtship display and consisted of rapidly repeated pulse units. Some sound variables differed significantly among species with P. callainos generally being separated from the other two species. This species produced sounds with higher peak frequency (for a given length) and lower number of pulses than P . 'zebra gold' and higher pulse durations than P. zebra . In addition, standard length was inversely related to peak frequency in both P . 'zebra gold' and P. callainos (this relation was not tested in P. zebra due to the small sample size). These differences might indicate different regimes of intraspecific sexual selection among the three species.     

AFLP genome scans suggest divergent selection on colour patterning in allopatric colour morphs of a cichlid fish

Genome scan-based tests for selection are directly applicable to natural populations to study the genetic and evolutionary mechanisms behind phenotypic differentiation. We conducted AFLP genome scans in three distinct geographic colour morphs of the cichlid fish Tropheus moorii to assess whether the extant, allopatric colour pattern differentiation can be explained by drift and to identify markers mapping to genomic regions possibly involved in colour patterning. The tested morphs occupy adjacent shore sections in southern Lake Tanganyika and are separated from each other by major habitat barriers. The genome scans revealed significant genetic structure between morphs, but a very low proportion of loci fixed for alternative AFLP alleles in different morphs. This high level of polymorphism within morphs suggested that colour pattern differentiation did not result exclusively from neutral processes. Outlier detection methods identified six loci with excess differentiation in the comparison between a bluish and a yellow-blotch morph and five different outlier loci in comparisons of each of these morphs with a red morph. As population expansions and the genetic structure of Tropheus make the outlier approach prone to false-positive signals of selection, we examined the correlation between outlier locus alleles and colour phenotypes in a genetic and phenotypic cline between two morphs. Distributions of allele frequencies at one outlier locus were indeed consistent with linkage to a colour locus. Despite the challenges posed by population structure and demography, our results encourage the cautious application of genome scans to studies of divergent selection in subdivided and recently expanded populations.     

Asymmetrical reproductive character displacement in the house mouse

Our study addressed reproductive character displacement between two subspecies of the house mouse, Mus musculus musculus and Mus musculus domesticus that hybridize in Europe along a zone where selection against hybridization is known to occur. Based on a multi-population approach, we investigated spatial patterns of divergence of mate preference in the two taxa. Mate preference was significantly higher in the contact zone than in allopatry in both subspecies, suggesting that reproductive character displacement occurs. Moreover, patterns of preference were stronger in M. m. musculus than in M. m. domesticus, indicating an asymmetrical divergence between the two. In the context of selection against hybridization, our results may provide empirical support for the hypothesis of reinforcement in a parapatric hybrid zone. We discuss factors that could explain the asymmetrical pattern of divergence and the possible impact of a unimodal structure on the maintenance of premating divergence between the two subspecies.     

Evolution of colour patterns in East African cichlid fish

African cichlid fishes have undergone outbursts of explosive speciation in several lakes, accompanied by rapid radiations in coloration and ecology. Little is known about the evolutionary forces that triggered these events but a hypothesis, published by Wallace Dominey in 1984, has figured prominently. It states that the evolution of colour patterns is driven by sexual selection and that these colour patterns are important in interspecific mate choice, a combination which holds the potential for rapid speciation. Here we present phylogenetic analyses that describe major events in colour evolution and test predictions yielded by Dominey's hypothesis. We assembled information on stripe patterns and the presence or absence of nuptial coloration from more than 700 cichlid species representing more than 90 taxa for which molecular phylogenetic hypotheses were available. We show that sexual selection is most likely the selection force that made male nuptial coloration arise and evolve quickly. In contrast, stripe patterns, though phylogenetically not conserved either, are constrained ecologically. The evolution of vertical bar patterns is associated with structurally complex habitats, such as rocky substrates or vegetation. The evolution of a horizontal stripe is associated with a piscivorous feeding mode. Horizontal stripes are also associated with shoaling behaviour. Strength of sexual selection, measured in terms of the mating system (weak in monogamous, strong in promiscuous species), has no detectable effects on stripe pattern evolution. In promiscuous species the frequency of difference between sister species in nuptial hue is higher than in pair bonding and harem forming species, but the frequency of difference in stripe pattern is lower. We argue that differences between the two components of coloration in their exposure to natural selection explain their very different evolutionary behaviour. Finally, we suggest that habitat-mediated selection upon chromomotor flexibility, a special form of phenotypic plasticity found in the river-dwelling outgroups of the lake-dwelling cichlids, explains the rapid and recurrent ecology-associated radiation of stripe patterns in lake environments, a new hypothesis that yields experimentally testable predictions.     

Asymmetric reproductive barriers and mosaic reproductive isolation: insights from Misty lake–stream stickleback

Ecological speciation seems to occur readily but is clearly not ubiquitous – and the relative contributions of different reproductive barriers remain unclear in most systems. We here investigate the potential importance of selection against migrants in lake/stream stickleback (Gasterosteus aculeatus) from the Misty Lake system, Canada. This system is of particular interest because one population contrast (Lake vs. Outlet stream) shows very low genetic and morphological divergence, whereas another population contrast (Lake vs. Inlet stream) shows dramatic genetic and morphological divergence apparently without strong and symmetric reproductive barriers. To test whether selection against migrants might solve this “conundrum of missing reproductive isolation”, we performed a fully factorial reciprocal transplant experiment using 225 individually marked stickleback collected from the wild. Relative fitness of the different ecotypes (Lake, Inlet, and Outlet) was assessed based on survival and mass change in experimental enclosures. We found that Inlet fish performed poorly in the lake (selection against migrants in that direction), whereas Lake fish outperformed Inlet fish in all environments (no selection against migrants in the opposite direction). As predicted from their phenotypic and genetic similarity, Outlet and Lake fish performed similarly in all environments. These results suggest that selection against migrants is asymmetric and, together with previous work, indicates that multiple reproductive barriers contribute to reproductive isolation. Similar mosaic patterns of reproductive isolation are likely in other natural systems.     

Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species

Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, we have little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. In the present study, we estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and show that male mate discrimination accounts for the majority of the current isolation between these species. We further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. Our findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process.     

Lineages of Silene nutans developed rapid,strong, asymmetric postzygotic reproductive isolation in allopatry

Reproductive isolation can rise either as a consequence of genomic divergence in allopatry or as a byproduct of divergent selection in parapatry. To determine whether reproductive isolation in gynodioecious Silene nutans results from allopatric divergence or from ecological adaptation following secondary contact, we investigated the pattern of postzygotic reproductive isolation and hybridization in natural populations using two phylogeographic lineages, western (W1) and eastern (E1). Experimental crosses between the lineages identified strong, asymmetric postzygotic isolation between the W1 and the E1 lineages, independent of geographic overlap. The proportion of ovules fertilized, seeds aborted, and seeds germinated revealed relatively little effect on the fitness of hybrids. In contrast, hybrid mortality was high and asymmetric: while half of the hybrid seedlings with western lineage mothers died, nearly all hybrid seedlings with E1 mothers died. This asymmetric mortality mirrored the proportion of chlorotic seedlings, and is congruent with cytonuclear incompatibility. We found no evidence of hybridization between the lineages in regions of co‐occurrence using nuclear and plastid markers. Together, our results are consistent with the hypothesis that strong postzygotic reproductive isolation involving cytonuclear incompatibilities arose in allopatry. We argue that the dynamics of cytonuclear gynodioecy could facilitate the evolution of reproductive isolation.     

Female affiliative preference depends on reproductive state in the African cichlid fish, Astatotilapia burtoni

Hormones play a pivotal role in reproductive behavior and havebeen implicated in mediating mate choice decisions. Here weasked whether the differences in female reproductive state dependenton changes in hormone levels correspond to changes in femaleaffiliation with males. In the African cichlid fish, Astatotilapiaburtoni, males shift between reproductive (territorial; T) andnon-reproductive (non-territorial; NT) states depending on socialcontext while females alternate between gravid (egg bearing;G) and non-gravid (NG) reproductive states independent of socialconditions. Moreover, the brain-pituitary-gonadal axis controllingreproduction and reproductive hormones is substantially remodeledin both males and females depending on reproductive state. Tomeasure affiliative preference, gravid and non-gravid femaleswere given the choice of associating with T or NT males. Gravidfemales preferentially associated with T males, whereas non-gravidfemales showed no preference. To discover whether gravid femalesuse male size independent of dominance status as a cue for theirchoice, gravid females were given a choice between territorialmales of different sizes. Gravid females preferred the smallerof two T males, but the smaller T males were significantly moreactive. Our results show that associative change could be animportant behavioral mediator between hormonal cues and reproductivesuccess, and that females use a hierarchy of cues in decision-making,preferring to affiliate with T over NT males and, among T males,preferring more active animals.     

The colour of paternity: extra‐pair paternity in the wild Gouldian finch does not appear to be driven by genetic incompatibility between morphs

In socially monogamous species, individuals can use extra‐pair paternity and offspring sex allocation as adaptive strategies to ameliorate costs of genetic incompatibility with their partner. Previous studies on domesticated Gouldian finches (Erythrura gouldiae) demonstrated a genetic incompatibility between head colour morphs, the effects of which are more severe in female offspring. Domesticated females use differential sex allocation, and extra‐pair paternity with males of compatible head colour, to reduce fitness costs associated with incompatibility in mixed‐morph pairings. However, laboratory studies are an oversimplification of the complex ecological factors experienced in the wild and may only reflect the biology of a domesticated species. This study aimed to examine the patterns of parentage and sex ratio bias with respect to colour pairing combinations in a wild population of the Gouldian finch. We utilized a novel PCR assay that allowed us to genotype the morph of offspring before the morph phenotype develops and to explore bias in morph paternity and selection at the nest. Contrary to previous findings in the laboratory, we found no effect of pairing combinations on patterns of extra‐pair paternity, offspring sex ratio or selection on morphs in nestlings. In the wild, the effect of morph incompatibility is likely much smaller, or absent, than was observed in the domesticated birds. Furthermore, the previously studied domesticated population is genetically differentiated from the wild population, consistent with the effects of domestication. It is possible that the domestication process fostered the emergence (or enhancement) of incompatibility between colour morphs previously demonstrated in the laboratory.     

Deep sympatric mitochondrial divergence without reproductive isolation in the common redstart Phoenicurus phoenicurus

Mitochondrial DNA usually shows low sequence variation within and high sequence divergence among species, which makes it a useful marker for phylogenetic inference and DNA barcoding. A previous study on the common redstart (Phoenicurus phoenicurus) revealed two very different mtDNA haplogroups (5% K2P distance). This divergence is comparable to that among many sister species; however, both haplogroups coexist and interbreed in Europe today. Herein, we describe the phylogeographic pattern of these lineages and test hypotheses for how such high diversity in mtDNA has evolved. We found no evidence for mitochondrial pseudogenes confirming that both haplotypes are of mitochondrial origin. When testing for possible reproductive barriers, we found no evidence for lineage‐specific assortative mating and no difference in sperm morphology, indicating that they are not examples of cryptic species, nor likely to reflect the early stages of speciation. A gene tree based on a short fragment of cytochrome c oxidase subunit 1 from the common redstart and 10 other Phoenicurus species, showed no introgression from any of the extant congenerics. However, introgression from an extinct congeneric cannot be excluded. Sequences from two nuclear introns did not show a similar differentiation into two distinct groups. Mismatch distributions indicated that the lineages have undergone similar demographic changes. Taken together, these results confirm that deeply divergent mitochondrial lineages can coexist in biological species. Sympatric mtDNA divergences are relatively rare in birds, but the fact that they occur argues against the use of threshold mtDNA divergences in species delineation.     

Flickers of speciation: Sympatric colour morphs of the arc‐eye hawkfish,Paracirrhites arcatus,reveal key elements of divergence with gene flow

One of the primary challenges of evolutionary research is to identify ecological factors that favour reproductive isolation. Therefore, studying partially isolated taxa has the potential to provide novel insight into the mechanisms of evolutionary divergence. Our study utilizes an adaptive colour polymorphism in the arc‐eye hawkfish (Paracirrhites arcatus) to explore the evolution of reproductive barriers in the absence of geographic isolation. Dark and light morphs are ecologically partitioned into basaltic and coral microhabitats a few metres apart. To test whether ecological barriers have reduced gene flow among dark and light phenotypes, we evaluated genetic variation at 30 microsatellite loci and a nuclear exon (Mc1r) associated with melanistic coloration. We report low, but significant microsatellite differentiation among colour morphs and stronger divergence in the coding region of Mc1r indicating signatures of selection. Critically, we observed greater genetic divergence between colour morphs on the same reefs than that between the same morphs in different geographic locations. We hypothesize that adaptation to the contrasting microhabitats is overriding gene flow and is responsible for the partial reproductive isolation observed between sympatric colour morphs. Combined with complementary studies of hawkfish ecology and behaviour, these genetic results indicate an ecological barrier to gene flow initiated by habitat selection and enhanced by assortative mating. Hence, the arc‐eye hawkfish fulfil theoretical expectations for the earliest phase of speciation with gene flow.     

Asymmetric reinforcement in Lucania killifish: assessing reproductive isolation when both sexes choose

Reinforcement can occur when maladaptive hybridization in sympatry favors the evolution of conspecific preferences and target traits that promote behavioral isolation (BI). In many systems, enhanced BI is due to increased female preference for conspecifics. In others, BI is driven by male preference, and in other systems both sexes exert preferences. Some of these patterns can be attributed to classic sex-specific costs and benefits of preference. Alternatively, sex differences in conspecific preference can emerge due to asymmetric postzygotic isolation (e.g., hybrid offspring from female A × male B have lower fitness than hybrid offspring from female B × male A), which can lead to asymmetric BI (e.g., female A and male B are less likely to mate than female B and male A). Understanding reinforcement requires understanding how conspecific preferences evolve in sympatry. Yet, estimating conspecific preferences can be difficult when both sexes are choosy. In this study, we use Lucania killifish to test the hypothesis that patterns of reinforcement are driven by asymmetric postzygotic isolation between species. If true, we predicted that sympatric female Lucania goodei and sympatric male L. parva should have lower levels of BI compared with their sympatric counterparts, as they produce hybrid offspring with the highest fitness. To address the problem of measuring BI when both sexes are choosy, we inferred the contribution to BI of each partner using assays where one sex in the mating pair comes from an allopatric population with potentially low preference, whereas the other comes from a sympatric population with high preference. For one hybrid cross direction, we found that both female L. parva and male L. goodei have high contributions to BI in sympatry. In the other hybrid cross direction, we found that only female L. goodei contribute to BI. Sympatric male L. parva readily engaged in hybrid spawnings with allopatric L. goodei females. These results indicate that both asymmetric postzygotic isolation and the traditional sex-specific costs to preference likely affect the nature of selection on conspecific preferences and target traits.     

Testing for mating isolation between ecotypes: laboratory experiments with lake, stream and hybrid stickleback

Mating isolation is a frequent contributor to ecological speciation – but how consistently does it evolve as a result of divergent selection? We tested for genetically based mating isolation between lake and stream threespine stickleback (Gasterosteus aculeatus L.) from the Misty watershed, Vancouver Island, British Columbia. We combined several design elements that are uncommon in the studies of stickleback mate choice: (i) we used second‐generation laboratory‐reared fish (to reduce environmental and maternal effects), (ii) we allowed for male–male competitive interactions (instead of the typical no‐choice trials) and (iii) we included hybrids along with pure types. Males of different types (Lake, Inlet, hybrid) were paired in aquaria, allowed to build nests and then exposed sequentially to females of all three types. We found that Lake and Inlet males differed in behaviours thought to influence stickleback mate choice (inter‐ and intra‐sexual aggression, display and nest activities), whereas hybrids were either intermediate or apparently ‘inferior’ in these behaviours. Despite these differences, Lake and Inlet fish did not mate assortatively and hybrid males did not have a mating disadvantage. Our study reinforces the noninevitability of mating isolation evolving in response to ecological differences and highlights the need to further investigate the factors promoting and constraining progress towards ecological speciation.     

Hybridization and asymmetric reproductive isolation between the closely related bryophyte taxa Polytrichum commune and P. uliginosum

Based on genetic differentiation, the haploid dioecious bryophyte taxa Polytrichum commune and P. uliginosum have been inferred to be completely reproductively isolated. However, analysing diploid sporophytes from a sympatric population for three diagnostic microsatellite markers, we show here that reproductive isolation between these taxa is far more complex and highly asymmetric. Isolation between female-P. communex male-P. uliginosum seems to be complete and prezygotic (or early postzygotic) as no hybrid sporophytes were observed on P. commune females. In the other direction ( female-P. uliginosumx male-P. commune) isolation was clearly postzygotic as high frequencies of hybrid sporophytes were found on P. uliginosum females. However, during maturation these sporophytes showed irregular development, indicating that hybrid sporophytes are unlikely to produce ripe spores. Mechanisms possibly underlying this asymmetric reproductive isolation pattern are discussed. Notwithstanding hybrid offspring being unlikely, the high frequency of hybrid sporophytes observed suggests that viable spores may be formed occasionally through such rare processes as chromosome nondisjunction, possibly giving rise to allodiploids. Allodiploids have been reported in Polytrichum (and other bryophyte genera). Studies such as the one presented here will therefore help to elucidate the evolutionary importance of interspecific hybridization and allodiploidization in bryophyte speciation.     

Context-dependent reproductive isolation mediated by floral scent and color

Reproductive isolation due to pollinator behavior is considered a key mode of speciation in flowering plants. Although floral scent is thought to mediate pollinator behavior, little is known about its effects on pollinator attraction and floral visitation in the wild. We used field experiments with wild hawkmoths and laboratory experiments with naïve hawkmoths to investigate attraction to and probing of flowers in response to indole, a volatile emitted by Ipomopsis tenuituba but not its close relative I. aggregata, both alone and in combination with floral color differences. We demonstrated that indole attracts wild hawkmoths to flowers, but has little effect on the rate at which those attracted moths probe flowers. In contrast, white flower color did not influence hawkmoth attraction in the field, but caused more attracted moths to probe flowers. Thus, the moths require both scent and high visual contrast, in that order, to feed at flowers at dusk. Their preference for indole-scented flowers is innate, but species-specific preference is mitigated by previous experience and plant spatial patterning. This context-dependent behavior helps explain why these Ipomopsis species show geographical variation in the extent of hybridization and may potentially explain formation of hybrid bridges in other systems of hawkmoth-pollinated plants.     

Female mating preferences and male coloration covary with water transparency in a Lake Victoria cichlid fish

Rapid speciation in Lake Victoria cichlid fish of the genus Pundamilia may be facilitated by sexual selection: female mate choice exerts sexual selection on male nuptial coloration within species and maintains reproductive isolation between species. However, declining water transparency coincides with increasingly dull coloration and increasing hybridization. In the present study, we investigated the mechanism underlying this pattern in Pundamilia nyererei, a species that interbreeds with a sister species in turbid but not in clear water. We compared measures of intraspecific sexual selection between two populations from locations that differ in water transparency. First, in laboratory mate‐choice experiments, conducted in clear water and under broad‐spectrum illumination, we found that females originating from turbid water have significantly weaker preferences for male coloration than females originating from clear water. Second, both the hue and body coverage of male coloration differ between populations, which is consistent with adaptation to different photic habitats. These findings suggest that the observed relationship between male coloration and water transparency is not mediated by environmental variation alone. Rather, female mating preferences are indicated to have changed in response to this variation, constituting the first evidence for intraspecific preference‐trait co‐evolution in cichlid fish. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 398–406.