Parallel and nonparallel ecological,morphological and genetic divergence in lake–stream stickleback from a single catchment

Parallel phenotypic evolution in similar environments has been well studied in evolutionary biology; however, comparatively little is known about the influence of determinism and historical contingency on the nature, extent and generality of this divergence. Taking advantage of a novel system containing multiple lake–stream stickleback populations, we examined the extent of ecological, morphological and genetic divergence between three‐spined stickleback present in parapatric environments. Consistent with other lake–stream studies, we found a shift towards a deeper body and shorter gill rakers in stream fish. Morphological shifts were concurrent with changes in diet, indicated by both stable isotope and stomach contents analysis. Performing a multivariate test for shared and unique components of evolutionary response to the distance gradient from the lake, we found a strong signature of parallel adaptation. Nonparallel divergence was also present, attributable mainly to differences between river locations. We additionally found evidence of genetic substructuring across five lake–stream transitions, indicating that some level of reproductive isolation occurs between populations in these habitats. Strong correlations between pairwise measures of morphological, ecological and genetic distance between lake and stream populations supports the hypothesis that divergent natural selection between habitats drives adaptive divergence and reproductive isolation. Lake–stream stickleback divergence in Lough Neagh provides evidence for the deterministic role of selection and supports the hypothesis that parallel selection in similar environments may initiate parallel speciation.     

Spatial phenotypic and genetic structure of threespine stickleback (Gasterosteus aculeatus) in a heterogeneous natural system,Lake Mývatn,Iceland

Eco‐evolutionary responses of natural populations to spatial environmental variation strongly depend on the relative strength of environmental differences/natural selection and dispersal/gene flow. In absence of geographic barriers, as often is the case in lake ecosystems, gene flow is expected to constrain adaptive divergence between environments – favoring phenotypic plasticity or high trait variability. However, if divergent natural selection is sufficiently strong, adaptive divergence can occur in face of gene flow. The extent of divergence is most often studied between two contrasting environments, whereas potential for multimodal divergence is little explored. We investigated phenotypic (body size, defensive structures, and feeding morphology) and genetic (microsatellites) structure in threespine stickleback (Gasterosteus aculeatus) across five habitat types and two basins (North and South) within the geologically young and highly heterogeneous Lake Mývatn, North East Iceland. We found that (1) North basin stickleback were, on average, larger and had relatively longer spines than South basin stickleback, whereas (2) feeding morphology (gill raker number and gill raker gap width) differed among three of five habitat types, and (3) there was only subtle genetic differentiation across the lake. Overall, our results indicate predator and prey mediated phenotypic divergence across multiple habitats in the lake, in face of gene flow.     

Evidence for morphological and adaptive genetic divergence between lake and stream habitats in European minnows (Phoxinus phoxinus, Cyprinidae)

Natural selection drives local adaptation, potentially even at small temporal and spatial scales. As a result, adaptive genetic and phenotypic divergence can occur among populations living in different habitats. We investigated patterns of differentiation between contrasting lake and stream habitats in the cyprinid fish European minnow (Phoxinus phoxinus) at both the morphological and genomic levels using geometric morphometrics and AFLP markers, respectively. We also used a spatial correlative approach to identify AFLP loci associated with environmental variables representing potential selective forces responsible for adaptation to divergent habitats. Our results identified different morphologies between lakes and streams, with lake fish presenting a deeper body and caudal peduncle compared to stream fish. Body shape variation conformed to a priori predictions concerning biomechanics and swimming performance in lakes vs. streams. Moreover, morphological differentiation was found to be associated with several environmental variables, which could impose selection on body and caudal peduncle shape. We found adaptive genetic divergence between these contrasting habitats in the form of 'outlier' loci (2.9%) whose genetic divergence exceeded neutral expectations. We also detected additional loci (6.6%) not associated with habitat type (lake vs. stream), but contributing to genetic divergence between populations. Specific environmental variables related to trophic dynamics, landscape topography and geography were associated with several neutral and outlier loci. These results provide new insights into the morphological divergence and genetic basis of adaptation to differentiated habitats.     

Morphological and genetic divergence of intralacustrine stickleback morphs in Iceland: a case for selective differentiation?

The evolutionary processes involved in population divergence and local adaptation are poorly understood. Theory predicts that divergence of adjacent populations is possible but depends on several factors including gene flow, divergent selection, population size and the number of genes involved in divergence and their distribution on the genome. We analyse variation in neutral markers, markers linked to putative quantitative trait loci and morphological traits in a recent (<10000 years) zone of primary divergence between stickleback morphs in Lake Thingvallavatn, Iceland. Environmental factors, especially predation, are clearly implicated in reducing gene flow between morphs. There is continuous morphological and genetic variation between habitats with a zone centre similar to secondary contact zones. Individual microsatellite loci are implicated as being linked to adaptive variation by direct tests as well as by differences in cline shape. Patterns of linkage disequilibria indicate that the morphs have diverged at several loci. This divergence shows parallels and differences with the well-studied limnetic-benthic stickleback morphs, both in phenotypic divergence and at the genomic level.     

Evolutionary diversification of opercle shape in Cook Inlet threespine stickleback

We investigated the evolution of a large facial bone, the opercle (OP), in lake populations of the threespine stickleback that were founded by anadromous ancestors, in Cook Inlet, Alaska. Recent studies characterized OP variation among marine and lake populations and mapped a quantitative trait locus with a large influence on OP shape. Using populations from diverse environments and independent evolutionary histories, we examined divergence of OP shape from that of the anadromous ancestor. We report preliminary evidence for divergence between benthic and generalist lake ecotypes, necessitating further investigation. Furthermore, rapid divergence of OP shape has occurred in a lake population that was founded by anadromous stickleback in the 1980s, which is consistent with divergence of other phenotypic traits and with OP diversification in other lake populations. By contrast, there has been limited evolution of OP shape in a second lake population that may have experienced a genetic bottleneck early in its history and lacks genetic variation for OP divergence. Taken together, the results obtained from these two populations are consistent with studies of other stickleback phenotypic traits that implicate ancestral variation in postglacial adaptive radiation of threespine stickleback in fresh water.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 832–844.     

DISENTANGLING THE ROLE OF PHENOTYPIC PLASTICITY AND GENETIC DIVERGENCE IN CONTEMPORARY ECOTYPE FORMATION DURING A BIOLOGICAL INVASION

The occurrence of contemporary ecotype formation through adaptive divergence of populations within the range of an invasive species typically requires standing genetic variation but can be facilitated by phenotypic plasticity. The relative contributions of both of these to adaptive trait differentiation have rarely been simultaneously quantified in recently diverging vertebrate populations. Here we study a case of intraspecific divergence into distinct lake and stream ecotypes of threespine stickleback that evolved in the past 140 years within the invasive range in Switzerland. Using a controlled laboratory experiment with full‐sib crosses and treatments mimicking a key feature of ecotypic niche divergence, we test if the phenotypic divergence that we observe in the wild results from phenotypic plasticity or divergent genetic predisposition. Our experimental groups show qualitatively similar phenotypic divergence as those observed among wild adults. The relative contribution of plasticity and divergent genetic predisposition differs among the traits studied, with traits related to the biomechanics of feeding showing a stronger genetic predisposition, whereas traits related to locomotion are mainly plastic. These results implicate that phenotypic plasticity and standing genetic variation interacted during contemporary ecotype formation in this case.     

Contrasting patterns of body shape and neutral genetic divergence in marine and lake populations of threespine sticklebacks

Comparisons of neutral marker and quantitative trait divergence can provide important insights into the relative roles of natural selection and neutral genetic drift in population differentiation. We investigated phenotypic and genetic differentiation among Fennoscandian threespine stickleback (Gasterosteus aculeatus) populations, and found that the highest degree of differentiation occurred between sea and freshwater habitats. Within habitats, morphological divergence was highest among the different freshwater populations. Pairwise phenotypic and neutral genetic distances among populations were positively correlated, suggesting that genetic drift may have contributed to the morphological differentiation among habitats. On the other hand, the degree of phenotypic differentiation (PST) clearly surpassed the neutral expectation set by FST, suggesting a predominant role for natural selection over genetic drift as an explanation for the observed differentiation. However, separate PST/FST comparisons by habitats revealed that body shape divergence between lake and marine populations, and even among marine populations, can be strongly influenced by natural selection. On the other hand, genetic drift can play an important role in the differentiation among lake populations.     

Does plasticity enhance or dampen phenotypic parallelism? A test with three lake–stream stickleback pairs

Parallel (and convergent) phenotypic variation is most often studied in the wild, where it is difficult to disentangle genetic vs. environmentally induced effects. As a result, the potential contributions of phenotypic plasticity to parallelism (and nonparallelism) are rarely evaluated in a formal sense. Phenotypic parallelism could be enhanced by plasticity that causes stronger parallelism across populations in the wild than would be expected from genetic differences alone. Phenotypic parallelism could be dampened if site‐specific plasticity induced differences between otherwise genetically parallel populations. We used a common‐garden study of three independent lake–stream stickleback population pairs to evaluate the extent to which adaptive divergence has a genetic or plastic basis, and to investigate the enhancing vs. dampening effects of plasticity on phenotypic parallelism. We found that lake–stream differences in most traits had a genetic basis, but that several traits also showed contributions from plasticity. Moreover, plasticity was much more prevalent in one watershed than in the other two. In most cases, plasticity enhanced phenotypic parallelism, whereas in a few cases, plasticity had a dampening effect. Genetic and plastic contributions to divergence seem to play a complimentary, likely adaptive, role in phenotypic parallelism of lake–stream stickleback. These findings highlight the value of formally comparing wild‐caught and laboratory‐reared individuals in the study of phenotypic parallelism.     

Ontogenetic shifts in phenotype–environment associations in Nile perch,Lates niloticus (Perciformes: Latidae) from Lake Nabugabo,Uganda

Habitat‐associated trait divergence may vary across ontogeny if there are strong size‐related shifts in selection pressures. We quantified patterns of phenotypic divergence in Nile perch (Lates niloticus) from ecologically distinct wetland edge and forest edge habitats in Lake Nabugabo, Uganda, and we compared patterns of divergence across three size classes to determine whether trends are consistent through Nile perch ontogeny. We predicted that inter‐habitat variation in biotic (e.g. vegetation structure) and abiotic (e.g. dissolved oxygen concentration) variables may create divergent selective regimes. We compared body morphology using geometric morphometrics and found substantial differences between habitats, although not all trends were consistent across size classes. The most striking aspects of divergence in small Nile perch were in mouth orientation, head size, and development of the caudal region. Medium‐sized Nile perch also showed differences in mouth orientation. Differences in large individuals were related to eye size and orientation, as well as caudal length. The observed patterns of divergence are consistent with functional morphological predictions for fish across divergent trophic regimes, high and low predation environments, and complex and simple habitats. Although this suggests adaptive divergence, the source of phenotypic variation is unknown and may reflect phenotypic plasticity and/or genetic differences. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 449–465.     

Natural selection drives patterns of lake-stream divergence in stickleback foraging morphology

To what extent are patterns of biological diversification determined by natural selection? We addressed this question by exploring divergence in foraging morphology of threespine stickleback fish inhabiting lake and stream habitats within eight independent watersheds. We found that lake fish generally displayed more developed gill structures and had more streamlined bodies than did stream fish. Diet analysis revealed that these morphological differences were associated with limnetic vs. benthic foraging modes, and that the extent of morphological divergence within watersheds reflected differences in prey resources utilized by lake and stream fish. We also found that patterns of divergence were unrelated to patterns of phenotypic trait (co)variance within populations (i.e. the ‘line of least resistance’). Instead, phenotypic (co)variances were more likely to have been shaped by adaptation to lake vs. stream habitats. Our study thus implicates natural selection as a strong deterministic force driving morphological diversification in lake–stream stickleback. The strength of this inference was obtained by complementing a standard analysis of parallel divergence in means between discrete habitat categories (lake vs. stream) with quantitative estimates of selective forces and information on trait (co)variances.     

Evidence of Adaptive Evolutionary Divergence during Biological Invasion

Rapid phenotypic diversification during biological invasions can either arise by adaptation to alternative environments or by adaptive phenotypic plasticity. Where experimental evidence for adaptive plasticity is common, support for evolutionary diversification is rare. Here, we performed a controlled laboratory experiment using full-sib crosses between ecologically divergent threespine stickleback populations to test for a genetic basis of adaptation. Our populations are from two very different habitats, lake and stream, of a recently invaded range in Switzerland and differ in ecologically relevant morphological traits. We found that in a lake-like food treatment lake fish grow faster than stream fish, resembling the difference among wild type individuals. In contrast, in a stream-like food treatment individuals from both populations grow similarly. Our experimental data suggest that genetically determined diversification has occurred within less than 140 years after the arrival of stickleback in our studied region.     

Does predation drive morphological differentiation among Adriatic populations of the three‐spined stickleback?

Morphometric differentiation among freshwater fish populations is a commonplace occurrence, although the underlying causes for this divergence often remain elusive. We analysed the degree and patterns of morphological differentiation among nine freshwater three‐spined stickleback (Gasterosteus aculeatus) populations inhabiting isolated karst rivers of the Adriatic Sea basin, to characterise the phenotypic diversity and differentiation in these populations. The analyses revealed marked and significant morphometric differentiation – especially in traits related to predator defence amongst most populations – even among those located within close geographic proximity in the same catchment system. Accordingly, the degree of morphometric and neutral genetic differentiation, as assessed from variability in 15 microsatellite loci from a parallel study, were uncorrelated across the populations. However, P_ST/F_ST comparisons revealed that the degree of phenotypic differentiation (P_ST) among populations exceeded that to be expected from genetic drift alone, suggesting a possible adaptive basis for the observed differentiation. In fact, avian predation pressure and several physiochemical environmental variables were identified as the main putative drivers of the observed differentiation, particularly in the dorsal spines, ascending process and lateral plates. Hence, the high degree of morphometric differentiation among Adriatic three‐spined stickleback populations appears to reflect adaptation to local ecological conditions. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 219–240.     

Historical and ecological correlates of body shape in the brook stickleback, Culaea inconstans

Using geometric morphometric methods, we evaluated the correlation between phenotypic variation and available historical and habitat information for two genetically differentiated, allopatric lineages of a widespread North American species, the brook stickleback ( Culaea inconstans ). The results obtained revealed strong patterns of structured phenotypic differentiation across the species range with extreme phenotypes occurring at the northwest and southeast range boundaries. Shape variation was broadly congruent with the distribution of two mitochondrial DNA lineages; a deep-bodied eastern form (Atlantic refugium) and a slim-bodied western form (Mississippian refugium); however, the two forms were not lineage-specific and phenotypic cladistic diversification is likely to be an artefact of underlying clinal variation associated with longitudinal and latitudinal gradients. In addition, we found little evidence of diagnosable lake and river forms across North America. Taken together, large-scale patterns of phenotypic diversity observed in C. inconstans suggest that relatively recent factors, such as continually varying natural selection across the range and/or potential local gene flow, may substantially mitigate the effects of historical separation or a generalized adaptive response to alternative habitats.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 769–783.     

Phenotypic divergence among threespine stickleback that differ in nuptial coloration

By studying systems in their earliest stages of differentiation, we can learn about the evolutionary forces acting within and among populations and how those forces could contribute to reproductive isolation. Such an understanding would help us to better discern and predict how selection leads to the maintenance of multiple morphs within a species, rather than speciation. The postglacial adaptive radiation of the threespine stickleback (Gasterosteus aculeatus) is one of the best‐studied cases of evolutionary diversification and rapid, repeated speciation. Following deglaciation, marine stickleback have continually invaded freshwater habitats across the northern hemisphere and established resident populations that diverged innumerable times from their oceanic ancestors. Independent freshwater colonization events have yielded broadly parallel patterns of morphological differences in freshwater and marine stickleback. However, there is also much phenotypic diversity within and among freshwater populations. We studied a lesser‐known freshwater “species pair” found in southwest Washington, where male stickleback in numerous locations have lost the ancestral red sexual signal and instead develop black nuptial coloration. We measured phenotypic variation in a suite of traits across sites where red and black stickleback do not overlap in distribution and at one site where they historically co‐occurred. We found substantial phenotypic divergence between red and black morphs in noncolor traits including shape and lateral plating, and additionally find evidence that supports the hypothesis of sensory drive as the mechanism responsible for the evolutionary switch in color from red to black. A newly described third “mixed” morph in Connor Creek, Washington, differs in head shape and size from the red and black morphs, and we suggest that their characteristics are most consistent with hybridization between anadromous and freshwater stickleback. These results lay the foundation for future investigation of the underlying genetic basis of this phenotypic divergence as well as the evolutionary processes that may drive, maintain, or limit divergence among morphs.     

Repeated and predictable patterns of ecotypic differentiation during a biological invasion: lake–stream divergence in parapatric Swiss stickleback

The relative importance of ecological selection and geographical isolation in promoting and constraining genetic and phenotypic differentiation among populations is not always obvious. Interacting with divergent selection, restricted opportunity for gene flow may in some cases be as much a cause as a consequence of adaptation, with the latter being a hallmark of ecological speciation. Ecological speciation is well studied in parts of the native range of the three‐spined stickleback. Here, we study this process in a recently invaded part of its range. Switzerland was colonized within the past 140 years from at least three different colonization events involving different stickleback lineages. They now occupy diverse habitats, ranging from small streams to the pelagic zone of large lakes. We use replicated systems of parapatric lake and stream populations, some of which trace their origins to different invasive lineages, to ask (i) whether phenotypic divergence occurred among populations inhabiting distinct habitats, (ii) whether trajectories of phenotypic divergence follow predictable parallel patterns and (iii) whether gene flow constrains divergent adaptation or vice versa. We find consistent phenotypic divergence between populations occupying distinct habitats. This involves parallel evolution in several traits with known ecological relevance in independent evolutionary lineages. Adaptive divergence supersedes homogenizing gene flow even at a small spatial scale. We find evidence that adaptive phenotypic divergence places constraints on gene flow over and above that imposed by geographical distance, signalling the early onset of ecological speciation.     

Drift versus selection as drivers of phenotypic divergence at small spatial scales: The case of Belgjarskógur threespine stickleback

Divergence in phenotypic traits is facilitated by a combination of natural selection, phenotypic plasticity, gene flow, and genetic drift, whereby the role of drift is expected to be particularly important in small and isolated populations. Separating the components of phenotypic divergence is notoriously difficult, particularly for multivariate phenotypes. Here, we assessed phenotypic divergence of threespine stickleback (Gasterosteus aculeatus) across 19 semi‐interconnected ponds within a small geographic region (~7.5 km²) using comparisons of multivariate phenotypic divergence (PST), neutral genetic (FST), and environmental (EST) variation. We found phenotypic divergence across the ponds in a suite of functionally relevant phenotypic traits, including feeding, defense, and swimming traits, and body shape (geometric morphometric). Comparisons of PSTs with FSTs suggest that phenotypic divergence is predominantly driven by neutral processes or stabilizing selection, whereas phenotypic divergence in defensive traits is in accordance with divergent selection. Comparisons of population pairwise PSTs with ESTs suggest that phenotypic divergence in swimming traits is correlated with prey availability, whereas there were no clear associations between phenotypic divergence and environmental difference in the other phenotypic groups. Overall, our results suggest that phenotypic divergence of these small populations at small geographic scales is largely driven by neutral processes (gene flow, drift), although environmental determinants (natural selection or phenotypic plasticity) may play a role.     

Intraspecific vicariant history and the evolution of adaptive morphological diversity in a fish species (Osmerus mordax)

Vicariant geographic isolation and resource partitioning have long been independently identified as processes contributing to the morphological divergence of closely-related species. However, little is known about the extent to which vicariant history influences the adaptive ecological divergence associated with resource partitioning and trophic specialization within species. The present study thus quantified the contribution of vicariant historical genetic divergence to the adaptive contemporary morphological divergence of intraspecific feeding specialists in the Rainbow smelt (Pisces: Osmerus mordax ). This species is characterized by the polyphyletic origin of two lacustrine feeding specialists originating in two intraspecific lineages associated with independent glacial refuges. The historical genetic segregation was initiated approximately 350 000 years ago, whereas the lacustrine trophic segregation arose within the past 10 000 years. Wild caught lacustrine smelt populations were grouped a priori based on known historical genetic identities (Acadian and Atlantic mitochondrial DNA clades) and contemporary feeding specializations (microphageous and macrophageous morphotypes). The present study demonstrated that independent suites of correlated morphological traits are associated with either vicariant history or contemporary feeding specializations. Second, functionally-similar feeding specialists exhibit distinct morphologies resulting largely from vicariant historical processes. Although, the evolutionary processes producing historical phenotypes remains unknown, the results obtained demonstrate how adaptive radiation associated with ecological resource partitioning and feeding specializations can be strongly influenced by intraspecific phenotypic diversification resulting from relatively recent vicariant histories.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 140–151.     

Quantifying the constraining influence of gene flow on adaptive divergence in the lake-stream threespine stickleback system

The constraining effect of gene flow on adaptive divergence is often inferred but rarely quantified. We illustrate ways of doing so using stream populations of threespine stickleback (Gasterosteus aculeatus) that experience different levels of gene flow from a parapatric lake population. In the Misty Lake watershed (British Columbia, Canada), the inlet stream population is morphologically divergent from the lake population, and presumably experiences little gene flow from the lake. The outlet stream population, however, shows an intermediate phenotype and may experience more gene flow from the lake. We first used microsatellite data to demonstrate that gene flow from the lake is low into the inlet but high into the outlet, and that gene flow from the lake remains relatively constant with distance along the outlet. We next combined gene flow data with morphological and habitat data to quantify the effect of gene flow on morphological divergence. In one approach, we assumed that inlet stickleback manifest well-adapted phenotypic trait values not constrained by gene flow. We then calculated the deviation between the observed and expected phenotypes for a given habitat in the outlet. In a second approach, we parameterized a quantitative genetic model of adaptive divergence. Both approaches suggest a large impact of gene flow, constraining adaptation by 80-86% in the outlet (i.e., only 14-20% of the expected morphological divergence in the absence of gene flow was observed). Such approaches may be useful in other taxa to estimate how important gene flow is in constraining adaptive divergence in nature.     

Microhabitat contributes to microgeographic divergence in threespine stickleback

Since the New Synthesis, most migration-selection balance theory has predicted that there should be negligible differentiation over small spatial scales (relative to dispersal), because gene flow should erode any effect of divergent selection. Nevertheless, there are classic examples of microgeographic divergence, which theory suggests can arise under specific conditions: exceptionally strong selection, phenotypic plasticity in philopatric individuals, or nonrandom dispersal. Here, we present evidence of microgeographic morphological variation within lake and stream populations of threespine stickleback (Gasterosteus aculeatus). It seems reasonable to assume that a given lake or stream population of fish is well-mixed. However, we found this assumption to be untenable. We examined trap-to-trap variation in 34 morphological traits measured on stickleback from 16 lakes and 16 streams. Most traits varied appreciably among traps within populations. Both between-trap distance and microhabitat characteristics such as depth and substrate explained some of the within-population morphological variance. Microhabitat was also associated with genotype at particular loci but there was no genetic isolation by distance, implying that heritable habitat preferences may contribute to microgeographic variation. Our study adds to growing evidence that microgeographic divergence can occur across small spatial scales within individuals’ daily dispersal neighborhood where gene flow is expected to be strong.     

Evidence for asymmetric migration load in a pair of ecologically divergent stickleback populations

Gene flow between ecologically divergent populations can prevent local adaptation, resulting in lower mean fitness and directional selection within a population. Such maladaptation should tend to be stronger in populations receiving a relatively larger fraction of immigrants. We test this expectation by comparing the strength of selection in a pair of three-spine stickleback populations in adjoining but unequal-sized lake basins in British Columbia. A larger deeper basin is connected to a smaller shallower basin by a short channel that allows extensive migration between populations. The two basins contain distinct habitats and prey communities, and stickleback stomach contents and isotope ratios differ accordingly. Trophic morphology is correlated with diet, so we would expect these ecological differences to be accompanied by morphological divergence. However, high gene flow appears to constrain adaptive divergence: microsatellites indicate that the two basins represent a single panmictic gene pool, and phenotypic divergence is very subtle. As a result, fish in the smaller lake basin are subject to persistent directional selection towards a more benthic phenotype, whereas the larger population exhibits no significant selection. The results illustrate the potentially asymmetrical effect of migration-selection balance, and its effect on fitness within populations.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 273–287.