An enhancer of Agouti contributes to parallel evolution of cryptically colored beach mice.

Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida's Gulf and Atlantic coasts as camouflage from predators. To facilitate genomic analyses, we first generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus. Next, in a uniquely variable mainland population (Peromyscus polionotus albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that pigment variation is strongly associated with an ∼2-kb region ∼5 kb upstream of the Agouti signaling protein coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000 km. Together, our results suggest that this identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there, has spread to and been selected in two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.

On the prospect of identifying adaptive loci in recently bottlenecked populations.

Identifying adaptively important loci in recently bottlenecked populations - be it natural selection acting on a population following the colonization of novel habitats in the wild, or artificial selection during the domestication of a breed - remains a major challenge. Here we report the results of a simulation study examining the performance of available population-genetic tools for identifying genomic regions under selection. To illustrate our findings, we examined the interplay between selection and demography in two species of Peromyscus mice, for which we have independent evidence of selection acting on phenotype as well as functional evidence identifying the underlying genotype. With this unusual information, we tested whether population-genetic-based approaches could have been utilized to identify the adaptive locus. Contrary to published claims, we conclude that the use of the background site frequency spectrum as a null model is largely ineffective in bottlenecked populations. Results are quantified both for site frequency spectrum and linkage disequilibrium-based predictions, and are found to hold true across a large parameter space that encompasses many species and populations currently under study. These results suggest that the genomic footprint left by selection on both new and standing variation in strongly bottlenecked populations will be difficult, if not impossible, to find using current approaches.

Evidence of adaptation from ancestral variation in young populations of beach mice.

To understand how organisms adapt to novel habitats, which involves both demographic and selective events, we require knowledge of the evolutionary history of populations and also selected alleles. There are still few cases in which the precise mutations (and hence, defined alleles) that contribute to adaptive change have been identified in nature; one exception is the genetic basis of camouflaging pigmentation of oldfield mice (Peromyscus polionotus) that have colonized the sandy dunes of Florida's Gulf Coast. To quantify the genomic impact of colonization as well as the signature of selection, we resequenced 5000 1.5-kb noncoding loci as well as a 160-kb genomic region surrounding the melanocortin-1 receptor (Mc1r), a gene that contributes to pigmentation differences, in beach and mainland populations. Using a genome-wide phylogenetic approach, we recovered a single monophyletic group comprised of beach mice, consistent with a single colonization event of the Gulf Coast. We also found evidence of a severe founder event, estimated to have occurred less than 3000 years ago. In this demographic context, we show that all beach subspecies share a single derived light Mc1r allele, which was likely selected from standing genetic variation that originated in the mainland. Surprisingly, we were unable to identify a clear signature of selection in the Mc1r region, despite independent evidence that this locus contributes to adaptive coloration. Nonetheless, these data allow us to reconstruct and compare the evolutionary history of populations and alleles to better understand how adaptive evolution, following the colonization of a novel habitat, proceeds in nature.

The developmental role of Agouti in color pattern evolution.

Animal color patterns can affect fitness in the wild; however, little is known about the mechanisms that control their formation and subsequent evolution. We took advantage of two locally camouflaged populations of Peromyscus mice to show that the negative regulator of adult pigmentation, Agouti, also plays a key developmental role in color pattern evolution. Genetic and functional analyses showed that ventral-specific embryonic expression of Agouti establishes a prepattern by delaying the terminal differentiation of ventral melanocytes. Moreover, a skin-specific increase in both the level and spatial domain of Agouti expression prevents melanocyte maturation in a regionalized manner, resulting in a novel and adaptive color pattern. Thus, natural selection favors late-acting, tissue-specific changes in embryonic Agouti expression to produce large changes in adult color pattern.

Convergence in pigmentation at multiple levels: mutations, genes and function.

Convergence--the independent evolution of the same trait by two or more taxa--has long been of interest to evolutionary biologists, but only recently has the molecular basis of phenotypic convergence been identified. Here, we highlight studies of rapid evolution of cryptic coloration in vertebrates to demonstrate that phenotypic convergence can occur at multiple levels: mutations, genes and gene function. We first show that different genes can be responsible for convergent phenotypes even among closely related populations, for example, in the pale beach mice inhabiting Florida's Gulf and Atlantic coasts. By contrast, the exact same mutation can create similar phenotypes in distantly related species such as mice and mammoths. Next, we show that different mutations in the same gene need not be functionally equivalent to produce similar phenotypes. For example, separate mutations produce divergent protein function but convergent pale coloration in two lizard species. Similarly, mutations that alter the expression of a gene in different ways can, nevertheless, result in similar phenotypes, as demonstrated by sister species of deer mice. Together these studies underscore the importance of identifying not only the genes, but also the precise mutations and their effects on protein function, that contribute to adaptation and highlight how convergence can occur at different genetic levels.

Genetic divergence in the Atlantic-Mediterranean Montagu's blenny, Coryphoblennius galerita (Linnaeus 1758) revealed by molecular and morphological characters.

Coryphoblennius galerita is a small intertidal fish with a wide distribution and limited dispersal ability, occurring in the northeastern Atlantic and Mediterranean. In this study, we examined Atlantic and Mediterranean populations of C. galerita to assess levels of genetic divergence across populations and to elucidate historical and contemporary factors underlying the distribution of the genetic variability. We analyse three mitochondrial and one nuclear marker and 18 morphological measurements. The combined dataset clearly supports the existence of two groups of C. galerita: one in the Mediterranean and another in the northeastern Atlantic. The latter group is subdivided in two subgroups: Azores and the remaining northeastern Atlantic locations. Divergence between the Atlantic and the Mediterranean can be the result of historical isolation between the populations of the two basins during the Pleistocene glaciations. Present-day barriers such as the Gibraltar Strait or the 'Almeria-Oran jet' are also suggested as responsible for this isolation. Our results show no signs of local extinctions during the Pleistocene glaciations, namely at the Azores, and contrast with the biogeographical pattern that has been observed for Atlantic-Mediterranean warm-water species, in which two groups of populations exist, one including the Mediterranean and the Atlantic coast of western Europe, and another encompassing the western tropical coast of Africa and the Atlantic islands of the Azores, Madeira and Canaries. Species like C. galerita that tolerate cooler waters, may have persisted during the Pleistocene glaciations in moderately affected locations, thus being able to accumulate genetic differences in the more isolated locations such as the Azores and the Mediterranean. This study is one of the first to combine morphological and molecular markers (mitochondrial and nuclear) with variable rates of molecular evolution to the study of the relationships of the Atlantic and Mediterranean populations of a cool-water species.

Historical population dynamics and demography of the Eastern Atlantic pomacentrid Chromis limbata (Valenciennes, 1833).

Recent studies have focused on the relationship between the marine fauna of the Eastern Atlantic and the Mediterranean Sea, but within the Atlantic, little is known about genetic relationships between populations of the Macaronesian islands. In this study, we tested whether the paleo-climatology and paleo-oceanography of the region could predict the genetic relationships among three Eastern Atlantic populations (Azores, Madeira, and Canaries) of a damselfish, Chromis limbata, and compared our results with its Mediterranean and adjacent Atlantic sister species, Chromis chromis. We combined phylogeographic and coalescent approaches using the fast evolving mitochondrial control region gene. No population structure was found for the three archipelagos. The coalescence time estimated for C. limbata (0.857-1.17 Mya) was much greater than that estimated for C. chromis. We propose that this difference reflects differences in glaciating extents in the Northeastern Atlantic and the Mediterranean. Diversity indexes (Hd and genetic distances) together with historical demographic parameters of C. limbata (Theta and g) revealed a more stable population history when compared to C. chromis. Our results suggest that the Macaronesian populations of C. limbata have probably been less affected by the last glaciation than the Mediterranean populations of C. chromis. Migration across the three archipelagos was estimated and a prevailing northwest trend was detected. This result supports the idea of a colonization of the Azores by warm water fish from Madeira or the westernmost Canary islands which acted as major glacial refugia for the tropical and subtropical marine fauna during the glaciations.

Historical colonization and demography of the Mediterranean damselfish, Chromis chromis.

The desiccation of the Mediterranean Sea during the Messinian Salinity Crisis 6.0-5.3 million years ago (Ma), caused a major extinction of the marine ichthyofauna of the Mediterranean. This was followed by an abrupt replenishment of the Mediterranean from the Atlantic after the opening of the Strait of Gibraltar. In this study, we combined demographic and phylogeographic approaches using mitochondrial and nuclear DNA markers to test the alternative hypotheses of where (Atlantic or Mediterranean) and when (before or after the Messinian Salinity Crisis) speciation occurred in the Mediterranean damselfish, Chromis chromis. The closely related geminate transisthmian pair Chromis multilineata and Chromis atrilobata was used as a way of obtaining an internally calibrated molecular clock. We estimated C. chromis speciation timing both by determining the time of divergence between C. chromis and its Atlantic sister species Chromis limbata (0.93-3.26 Ma depending on the molecular marker used, e.g. 1.23-1.39 Ma for the control region), and by determining the time of coalescence for C. chromis based on mitochondrial control region sequences (0.14-0.21 Ma). The time of speciation of C. chromis was always posterior to the replenishment of the Mediterranean basin, after the Messinian Salinity Crisis. Within the Mediterranean, C. chromis population structure and demographic characteristics revealed a genetic break at the Peloponnese, Greece, with directional and eastbound gene flow between western and eastern groups. The eastern group was found to be more recent and with a faster growing population (coalescent time = 0.09-0.13 Ma, growth = 485.3) than the western group (coalescent time = 0.13-0.20 Ma, growth = 325.6). Our data thus suggested a western origin of C. chromis, most likely within the Mediterranean. Low sea water levels during the glacial periods, the hydrographic regime of the Mediterranean and dispersal restriction during the short pelagic larval phase of C. chromis (18-19 days) have probably played an important role in C. chromis historical colonization.