Population genetics, phylogeography and speciation of Cystodytes (Ascidiacea) in the western Mediterranean Sea

Several morphotypes that so far have been attributed to the allegedly cosmopolitan ascidian Cystodytes dellechiajei occur in the Mediterranean Sea. Colour variation is the difference most frequently reported. In this study, we addressed the genetic structure of this ascidian in relation to geographical location and colour morph. Partial sequences of the gene cytochrome  c oxidase subunit 1 (COI) were obtained from seven populations of the western Mediterranean, encompassing eight colour varieties. All population genetic analyses (exact test, pairwise F _ST, hierarchical analysis of molecular variance, multidimensional scaling, nested clade analysis) indicated clearly that differences between colour morphs are large enough to obscure any geographical differentiation when colours are combined within localities. When variance due to colour divergence was removed, however, a significant geographical variability between localities remained. The genetic divergence between the colour morphs analysed was significant in comparisons of the brown and purple forms with the others, but not among the green, blue, and white morphs. Phylogeographic analyses suggest that population fragmentation and range expansions have shaped the present-day distribution of the haplotypes. Taken together with existing chemotype information, our results indicate that several species are present in the area, and that a thorough revision of the genus is necessary.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 203–214.     

How do morphotypes and chemotypes relate to genotypes? The colonial ascidian Cystodytes (Polycitoridae)

Intraspecies variability is widespread in marine invertebrates. Size, colour, texture, general shape and secondary chemistry can differ quite drastically from one individual to another. Cystodytes dellechiajei (Polycitoridae) is a cosmopolitan colonial ascidian with several morphotypes, most of which differ in colour and spicular composition. New molecular tools enable us to assess the taxonomic status of these morphotypes. To determine whether variation observed in Mediterranean Cystodytes has a genetic basis, we sequenced 45 specimens from eight locations of the western Mediterranean and one from Mayotte (Indian Ocean), and obtained a 617 bp fragment of the mitochondrial gene COI. Fifteen different colour morphs were recorded and four kinds of spicules were found: disk-shaped, sphere-shaped, star-shaped and discoidal, thick spicules with a toothed margin. Zooid morphology was remarkably uniform in the whole sample set. Different tree construction methods (distance-based, parsimony-based, and maximum-likelihood-based) yielded consistent results, and recognized six major clades, which had no correspondence with spicule shape and were only partially consistent with colour morphs. Results are discussed in the light of previous knowledge of the chemistry of blue, green, brown and purple colour morphs. In spite of the different colour patterns and spicular variability we concluded, on the basis of chemical and genetic data, that the morphological traits analysed were not consistent enough to be used to differentiate between Cystodytes species. We point out the importance of genetics and chemistry in assessing the taxonomic status of species with variable morphology.     

Phylogeography of colour polymorphism in the coral reef fish Pseudochromis fuscus, from Papua New Guinea and the Great Barrier Reef

Body colour has played a significant role in the evolution of coral reef fishes, but the phylogenetic level at which colour variation is expressed and the evolutionary processes driving the development and persistence of different colour patterns are often poorly understood. The aim of this study was to examine the genetic relationships between multiple colour morphs of Pseudochromis fuscus (family Pseudochromidae), both within and among geographic locations. Pseudochromis fuscus is currently described as a single species, but exhibits at least six discrete colour morphs throughout its range. In this study, P. fuscus from Papua New Guinea (PNG) and the Great Barrier Reef (GBR), Australia, formed three genetically distinct clades based on mitochondrial DNA (control region) sequence data: (1) yellow and brown morphs from the GBR and southern PNG, as well as an orange morph from southern PNG; (2) a pink morph from southern PNG; and (3) all three morphs (pink, orange and grey) found in Kimbe Bay, northern PNG. The three groups showed deep levels of divergence (d=14.6–25.4%), suggesting that P. fuscus is a complex of polychromatic species, rather than a single widespread species with many different colour morphs. Population genetic analyses indicate that the three clades have experienced unique evolutionary histories, possibly from differential effects of sea level fluctuations, barriers to gene flow and historical biogeography.     

Recent speciation and secondary contact in endemic ants

Gene flow is the main force opposing divergent selection, and its effects are greater in populations in close proximity. Thus, complete reproductive isolation between parapatric populations is not expected, particularly in the absence of ecological adaptation and sharp environmental differences. Here, we explore the biogeographical patterns of an endemic ant species, Cataglyphis floricola, for which two colour morphs (black and bicolour) coexist in parapatry throughout continuous sandy habitat in southern Spain. Discriminant analyses of six biometric measurements of male genitalia and 27 cuticular hydrocarbons reveal high differentiation between morphs. Furthermore, the low number of shared alleles derived from nuclear markers (microsatellites) between the morphs at their contact zone suggests the absence of recent gene flow. Mitochondrial DNA (COI) phylogenetic analysis and median‐joining networks show that the black morph is basal to the bicolour morph, with unique haplotypes recovered for each morph. Mismatch distribution analysis and Bayesian skyline plots suggest that they are undergoing different demographic changes, with the bicolour and black morphs at demographic equilibrium and expansion, respectively. Thus, our results show complete reproductive isolation between the two colour morphs as evidenced from genetic, chemical and morphological data. We suggest that these divergence events could be explained by historical vicariance during the Pleistocene, in which reproductive traits experienced strong divergent selection between the morphs initiating or culminating speciation.     

Genetic divergence and assortative mating between colour morphs of the sea urchin Paracentrotus gaimardi

Some species of sea urchins feature large variation in pigmentation. This variability may be the result of phenotypic plasticity or it may be associated with genetic divergence between morphs. Paracentrotus gaimardi exhibits five colour morphs (pink, brown, green, grey and black), which often occur side by side on the same rock. We studied genetic divergence between these morphs in three populations on the coast of Brazil. A fragment of the region encoding the mitochondrial ATPase 8 and 6 mitochondrial genes, a fragment of the intron of a nuclear histone and the entire nuclear gene coding for the sperm protein bindin were analysed. Mitochondrial DNA was differentiated between the pink and all other morphs, but the histone intron was similar in all colour morphs. In bindin, nine codons were found to be under positive selection and significant differences of allelic frequencies were observed in almost all pairwise comparisons between colour morphs. Although the molecular differentiation in bindin is not large enough to suggest reproductive isolation, some degree of assortative mating within morphs seems to be occurring in this species.     

Unexpected patterns of genetic structuring among locations but not colour morphs in Acropora nasuta (Cnidaria; Scleractinia)

Symbiotic relationships have contributed greatly to the evolution and maintenance of biological diversity. On the Great Barrier Reef, species of obligate coral-dwelling fishes (genus Gobiodon) coexist by selectively recruiting to colonies of Acropora nasuta that differ in branch-tip colour. In this study, we investigate genetic variability among sympatric populations of two colour morphs of A. nasuta ('blue-tip' and 'brown-tip') living in symbiosis with two fish species, Gobiodon histrio and G. quinquestrigatus, respectively, to determine whether gobies are selecting between intraspecific colour polymorphisms or cryptic coral species. We also examine genetic differentiation among coral populations containing both these colour morphs that are separated by metres between local sites, tens of kilometres across the continental shelf and hundreds of kilometres along the Great Barrier Reef. We use three nuclear DNA loci, two of which we present here for the first time for Acropora. No significant genetic differentiation was detected between sympatric colour morphs at these three loci. Hence, symbiotic gobies are selecting among colour morphs of A. nasuta, rather than cryptic species. Significant genetic geographical structuring was observed among populations, independent of colour, at regional (i.e. latitudinal separation by < 500 km) and cross-shelf (< 50 km) scales, alongside relative homogeneity between local populations on within reef scales (< 5 km). This contrasts with the reported absence of large-scale genetic structuring in A. valida, which is a member of the same species group as A. nasuta. Apparent differences in biogeographical structuring between species within the A. nasuta group emphasize the need for comparative sampling across both spatial (i.e. within reefs, between reefs and between regions) and taxonomic scales (i.e. within and between closely related species).     

Lack of mitochondrial genetic structure in hamlets (Hypoplectrus spp.): recent speciation or ongoing hybridization?

Species in the genus Hypoplectrus (hamlet fish) have been recognized primarily on the basis of colour morphology, which varies substantially. Limited differentiation in other morphological characters, however, has led to ongoing debate about their taxonomy. Our analysis of mtDNA sequences demonstrates neither reciprocal monophyly nor significant genetic differentiation among hamlet colour morphs. These data are potentially consistent with a model of recent speciation due to sexual selection on colour morphology. The presence within hamlets of two divergent mtDNA clades, however, suggests a longer history during which hybridization and gene flow have prevented the differentiation of hamlet colour morphs, at least in neutral genetic markers.     

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.     

Eight microsatellite loci in Clark's anemonefish,Amphiprion clarkii

To determine the genetic relationship between different colour morphs (orange and black morphs) of Clark's anemonefish (Amphiprion clarkii) in Taiwan, we isolated eight polymorphic microsatellite loci. A large number of alleles (range, 6–30), and high levels of observed heterozygosity (range, 0.1231–0.8358) were resolved in 71 individuals from two populations, indicating that these markers should be useful in assessing the relationship between the two colour morphs of A. clarkii.     

Colour variation is incongruent with mitochondrial lineages: cryptic speciation and subsequent diversification in a Gulf of California reef fish (Teleostei: Blennioidei)

The Gulf of California endemic reef fish, Acanthemblemaria crockeri (Blennioidei, Chaenopsidae), reportedly has two colour morphs, one with melanic lateral spots ('Gulf' morph) and one with orange spots ('Cape' morph). In this study, we recorded colour morph in both males and females and collected mitochondrial DNA sequence data for cytochrome c oxidase I (COI) and tRNA-Pro/D-loop of specimens from throughout the Gulf to explore the genetic basis of the colour morphs. Two highly divergent (HKY + I distance = 11.9% for COI), reciprocally monophyletic lineages were identified, consistent with the presence of two parapatric species. A 30-km gap between the distributions of mitochondrial lineages roughly corresponds to a hypothesized former seaway across the Baja California peninsula north of La Paz, although the estimated divergence time (1.84 million years ago) is more recent than the hypothetical seaway (3–4 million years ago). Surprisingly, the distribution of mitochondrial species is not congruent with the distribution of either male or female colour morphs. Our analysis also revealed significant population differentiation within both species and no shared haplotypes among populations. The northern Gulf species includes four populations (NB, CB, NM and CM) corresponding to northern and central Baja and northern and central mainland sites, while the Cape species includes two populations (SB and SM) corresponding to the Baja and mainland sides of the southern Gulf. The NB/CB division corresponds to a hypothesized Plio–Pleistocene mid-peninsular seaway. The level of genetic divergence documented in this lineage is extraordinary for a marine fish with a pelagic larval stage within a semi-enclosed basin.     

Eutrophication and restoration of shallow lakes – the concept of stable equilibria revisited

We studied the genetic structure of populations of the Atlanto-Mediterranean ascidian Clavelina lepadiformis (Müller, 1776). A 369 bp segment of the COI mitochondrial gene was sequenced in Mediterranean and Atlantic populations from inside harbours, marinas and fjords (interior populations), and from the open rocky littoral (exterior populations). Previous work identified genetic differences between C. lepadiformis inhabiting Mediterranean harbours and the Mediterranean rocky littoral, however, the origin of these two clades remained speculative. Here we compared the Mediterranean populations with four Atlantic populations (two interior and two exterior). Gene differentiation and maximum likelihood analyses showed that the Atlantic forms were not divided into interior and exterior clades, and were closely related to the interior clade in the Mediterranean. The results support the hypothesis that both clades evolved allopatrically in the two seas, and that a recent colonisation of Mediterranean marinas from the Atlantic was caused by ship-hull transport. Colonisation of habitats by new genetic variants, morphologically indistinguishable from local populations, may be common among benthic invertebrates, and only genetic tools can uncover these cryptic invasions.     

Variable discrimination and asymmetric preferences in laboratory tests of reproductive isolation between cichlid colour morphs

Mating behaviour affects reproductive isolation and phenotypic differentiation. In Lake Tanganyika, the cichlid fish Tropheus moorii diversified into numerous, currently allopatric colour variants. Allopatric isolation is periodically interrupted by dispersal and secondary contact during lake level fluctuations, making long‐term differentiation partly dependent on assortative mating. Laboratory experiments with two moderately distinct morphs revealed assortative female preferences in one (Nakaku), but random mate choice in the other morph (Mbita). No discrimination was apparent between two subtly differentiated morphs (Chimba and Moliro). Tested against each other in a previous study, the highly distinct Moliro and Nakaku exhibited strong assortative preferences. The correlation between colour pattern similarity and mate discrimination suggests that allopatry and philopatric behaviour are less crucial for the maintenance of differentiation between highly distinct morphs than for more similar morphs. Interestingly, the asymmetric isolation in one pair of morphs is congruent with a pattern of unidirectional mitochondrial introgression between populations.     

Phylogeography of the poison frog Mantella viridis (Amphibia: Mantellidae) reveals chromatic and genetic differentiation across ecotones in northern Madagascar

Mantella viridis is a threatened poison frog species endemic to the ecologically very heterogeneous northern region of Madagascar. The existence of several colour morphs within M. viridis and its very low genetic differentiation to the allopatrically distributed Mantella ebenaui raise questions about the processes driving the differentiation between these poison frog populations and about their taxonomic status. Using a DNA fragment of 476 nucleotides of the mitochondrial cytochrome b gene from 240 individuals of this species complex, we investigated the genetic variability of all known colour morphs of M. viridis, sampling this species throughout its known range, as well as several populations of M. ebenaui. Our genetic results confirm that M. viridis and M. ebenaui are closely related but reveal that no haplotype sharing occurs between these two taxa. Further, our molecular analyses provided evidence for barriers to gene flow among some of the colour morphs. Estimates of overlap of bioclimatic envelopes as assessed by ecological niche modelling also suggest a distinct bioclimatic niche of some of the lineages studied.     

Increased inter-colony fusion rates are associated with reduced COI haplotype diversity in an invasive colonial ascidian Didemnum vexillum

Considerable progress in our understanding of the population genetic changes associated with biological invasions has been made over the past decade. Using selectively neutral loci, it has been established that reductions in genetic diversity, reflecting founder effects, have occurred during the establishment of some invasive populations. However, some colonial organisms may actually gain an ecological advantage from reduced genetic diversity because of the associated reduction in inter-colony conflict. Here we report population genetic analyses, along with colony fusion experiments, for a highly invasive colonial ascidian, Didemnum vexillum. Analyses based on mitochondrial cytochrome oxidase I (COI) partial coding sequences revealed two distinct D. vexillum clades. One COI clade appears to be restricted to the probable native region (i.e., north-west Pacific Ocean), while the other clade is present in widely dispersed temperate coastal waters around the world. This clade structure was supported by 18S ribosomal DNA (rDNA) sequence data, which revealed a one base-pair difference between the two clades. Recently established populations of D. vexillum in New Zealand displayed greatly reduced COI genetic diversity when compared with D. vexillum in Japan. In association with this reduction in genetic diversity was a significantly higher inter-colony fusion rate between randomly paired New Zealand D. vexillum colonies (80%, standard deviation ±18%) when compared with colonies found in Japan (27%, standard deviation ±15%). The results of this study add to growing evidence that for colonial organisms reductions in population level genetic diversity may alter colony interaction dynamics and enhance the invasive potential of newly colonizing species.     

No barriers to gene flow among sympatric polychromatic 'small'Telmatherina antoniae from Lake Matano, Indonesia

Genetic divergence, assortative courtship and intermale aggression were assessed between sympatric colour morphs of the sailfin silverside Telmatherina antoniae , endemic to Lake Matano, Indonesia. Genetic analysis using microsatellite markers showed no barriers to gene flow among T. antoniae primary colour morphs (blue and yellow) within sampling sites, sympatric populations or at the lake-wide level. Low but significant genetic differentiation was found between yellow morphs and mixed (blue–yellow) morphs. Behavioural surveys indicated assortative courtship does occur along primary colour lines; however, intermale aggression among paired and intruding male morphs appeared equal with respect to male colour. These observations support the hypothesis that males view other males as threats to their courtship regardless of their colour. This study supports recent work suggesting that assortative mating is present in T. antoniae despite a lack of reproductive isolation among colour morphs.     

Convergence and divergence in lizard colour polymorphisms

Colour polymorphic species are model systems for examining the evolutionary processes that generate and maintain discrete phenotypic variation in natural populations. Lizards have repeatedly evolved strikingly similar polymorphic sexual signals in distantly related lineages, providing an opportunity to examine convergence and divergence in colour polymorphism, correlated traits and associated evolutionary processes. Herein, we synthesise the extensive literature on lizard colour polymorphisms in both sexes, including recent advances in understanding of the underlying biochemical, cellular and genetic mechanisms, and correlated behavioural, physiological and life-history traits. Male throat, head or ventral colour morphs generally consist of red/orange, yellow and white/blue morphs, and sometimes mixed morphs with combinations of two colours. Despite these convergent phenotypes, there is marked divergence in correlated behavioural, physiological and life-history traits. We discuss the need for coherence in morph classification, particularly in relation to ‘mixed’ morphs. We highlight future research directions such as the genetic basis of convergent phenotypes and the role of environmental variation in the maintenance of polymorphism. Research in this very active field promises to continue to provide novel insights with broad significance to evolutionary biologists.     

Molecular phylogeny of the Greek populations of the genus Ligidium (Isopoda, Oniscidea) using three mtDNA gene segments

The phylogeny of Greek populations of the terrestrial isopod genus Ligidium is reconstructed based on three mtDNA gene segments: 12S rRNA, 16S rRNA and COI. Two widely distributed European species, as well as three outgroups belonging to different isopod genera, were also included in the analyses. The samples used represent almost all Ligidium species known to occur in Greece, as well as several populations of unknown specific status plus some new records. Phylogenetic analyses of the combined data set were performed using Bayesian inference and maximum parsimony. The two main sister clades with good support indicate the sympatric differentiation of two lineages in southern continental Greece (Peloponnisos), where Ligidium populations exhibit a mosaic distribution of sibling species. The insular populations of the Aegean Islands show increased genetic divergence and form separate clades. The presence of a third lineage of Asiatic origin is strongly suggested by both the molecular phylogeny and morphology. The only presumably valid diagnostic morphological character exhibits only partial correspondence to well supported clades of the molecular phylogeny. Genetic differentiation between populations is very high, a fact that can be attributed to the strict ecological specialization of these animals that leads to increased levels of isolation even between populations that are in close proximity. As a consequence, Greek Ligidium populations, especially those present on islands, are unique genetic pools and extremely vulnerable to extinction.     

Floral size variation causes differentiation of pollinators and genetic parameters in Alpinia nieuwenhuizii, a flexistylous ginger (Zingiberaceae)

Floral size dimorphism, pollination, and genetic variation of Alpinia nieuwenhuizii (Zingiberaceae), a flexitylous ginger, were studied. This study revealed that floral size differed among habitats (i.e., roadsides/riversides vs. forest floors). The effective pollinators of small-flowered populations of the species on a forest floor were different from those of large-flowered populations along roadsides/riversides. Using inter-simple sequence repeat (ISSR) PCR, considerable genetic differentiation was detected between small- and large-flowered populations. These results indicate that reproductive isolation in A. nieuwenhuizii owing to the differentiation of pollen vectors between two floral size morphs may lead to genetic differentiation between the two morphs.     

Genetic and Ecological Characterisation of Colour Dimorphism in a Coral Reef Fish

Synopsis Many recognised species of coral reef fishes exhibit two or more colour variants, but it is unknown whether these represent genetically identical phenotypes, genetic polymorphisms or closely related species. We tested between these alternatives for two colour morphs of the coral reef fish, Pseudochromis fuscus, from Lizard Island (Great Barrier Reef). A molecular analysis using mtDNA did not detect any genetic differentiation between co-occurring ‘yellow’ and ‘brown’ colour morphs. A previous study proposed that these two colour morphs are aggressive mimics of yellow and brown damselfishes. Here, a manipulative field experiment was used to evaluate whether the colour dimorphism in P. fuscus is a phenotypic response to the presence of two different model species. Colonies of either yellow or brown damselfish species were established on different patch reefs, and each of the two different P. fuscus morphs was then placed on the different reefs. Contrary to expectations, all yellow individuals that stayed on the reefs changed to brown, regardless of the model species. No brown individuals changed to the yellow colouration. However, P. fuscus were more likely to emigrate from, or suffer higher mortality on, patch reefs where they were not matched with similarly coloured models. Clearly, yellow and brown P. fuscus are members of a single species with sufficient phenotypic plasticity to switch from yellow to brown colouration.     

Evolution of adaptive diversity and genetic connectivity in Arctic charr (Salvelinus alpinus) in Iceland

The ecological theory of adaptive radiation predicts that the evolution of phenotypic diversity within species is generated by divergent natural selection arising from different environments and competition between species. Genetic connectivity among populations is likely also to have an important role in both the origin and maintenance of adaptive genetic diversity. Our goal was to evaluate the potential roles of genetic connectivity and natural selection in the maintenance of adaptive phenotypic differences among morphs of Arctic charr, Salvelinus alpinus, in Iceland. At a large spatial scale, we tested the predictive power of geographic structure and phenotypic variation for patterns of neutral genetic variation among populations throughout Iceland. At a smaller scale, we evaluated the genetic differentiation between two morphs in Lake Thingvallavatn relative to historically explicit, coalescent-based null models of the evolutionary history of these lineages. At the large spatial scale, populations are highly differentiated, but weakly structured, both geographically and with respect to patterns of phenotypic variation. At the intralacustrine scale, we observe modest genetic differentiation between two morphs, but this level of differentiation is nonetheless consistent with strong reproductive isolation throughout the Holocene. Rather than a result of the homogenizing effect of gene flow in a system at migration-drift equilibrium, the modest level of genetic differentiation could equally be a result of slow neutral divergence by drift in large populations. We conclude that contemporary and recent patterns of restricted gene flow have been highly conducive to the evolution and maintenance of adaptive genetic variation in Icelandic Arctic charr.