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.

Rise and fall of a hybrid zone: implications for the roles of aggression, mate choice, and secondary succession.

Hybridization can be an important evolutionary force by generating new species and influencing evolution of parental species in multiple ways, including introgression and the consequences of hybrid vigor. Determining the ecological processes underlying evolution in hybrid zones is difficult however because it requires examining changes in both genotypic frequencies over time and corresponding ecological information, data that are rarely collected together. Here, we describe genetic and ecological aspects of a hybrid zone between the Eastern Fence Lizard, Sceloporus undulatus, and the Florida Scrub Lizard, Sceloporus woodi, occurring over at least 23 generations. The hybrid zone, discovered greater than 35 years ago using morphological characters, originally consisted of nearly even proportions of parental species and hybrids. Now, using genetic markers (species-diagnostic mtDNA sites and 6 nDNA microsatellite loci across a total of n = 117 individuals), we confirm not only that hybridization occurred but also that subsequent backcrossing has resulted in highly introgressed hybrids, with many hybrids containing mitochondrial DNA from one species on a nuclear DNA background of the other. Ecological aspects explaining these shifts in genetic composition include female mate choice, changes in habitat associated with secondary succession, and, most strongly, a hierarchy of male territorial advantage-ecological mechanisms likely to be involved in the emergence and disappearance of many animal hybrid zones. Our results suggest that genetic assimilation is not a significant threat to either species and that rather transient hybrid zones such as this may serve to increase genetic diversity and are candidates for causing genetic discordance in phylogeographic analyses.

Genetic relatedness does not retain spatial pattern across multiple spatial scales: dispersal and colonization in the coral, Pocillopora damicornis.

Patterns of isolation by distance are uncommon in coral populations. Here, we depart from historical trends of large-scale, geographical genetic analyses by scaling down to a single patch reef in Kane'ohe Bay, Hawai'i, USA, and map and genotype all colonies of the coral, Pocillopora damicornis. Six polymorphic microsatellite loci were used to assess population genetic and clonal structure and to calculate individual colony pairwise relatedness values. Our results point to an inbred, highly clonal reef (between 53 and 116 clonal lineages of 2352 genotyped colonies) with a much skewed genet frequency distribution (over 70% of the reef was composed of just seven genotypes). Spatial autocorrelation analyses revealed that corals found close together on the reef were more genetically related than corals further apart. Spatial genetic structure disappears, however, as spatial scale increases and then becomes negative at the largest distances. Stratified, random sampling of three neighbouring reefs confirms that reefs are demographically open and inter-reef genetic structuring was not detected. Attributing process to pattern in corals is complicated by their mixed reproductive strategies. Separate autocorrelation analyses, however, show that the spatial distribution of both clones and nonclones contributes to spatial genetic structure. Overall, we demonstrate genetic structure on an intrareef scale and genetic panmixia on an inter-reef scale indicating that, for P. damicornis, the effect of small- and large-scale dispersal processes on genetic diversity are not the same. By starting from an interindividual, intrareef level before scaling up to an inter-reef level, this study demonstrates that isolation-by-distance patterns for the coral P. damicornis are limited to small scales and highlights the importance of investigating genetic patterns and ecological processes at multiple scales.

Common misconceptions in molecular ecology: echoes of the modern synthesis.

The field of molecular ecology has burgeoned into a large discipline spurred on by technical innovations that facilitate the rapid acquisition of large amounts of genotypic data, by the continuing development of theory to interpret results, and by the availability of computer programs to analyse data sets. As the discipline grows, however, misconceptions have become enshrined in the literature and are perpetuated by routine citations to other articles in molecular ecology. These misconceptions hamper a better understanding of the processes that influence genetic variation in natural populations and sometimes lead to erroneous conclusions. Here, we consider eight misconceptions commonly appearing in the literature: (i) some molecular markers are inherently better than other markers; (ii) mtDNA produces higher F(ST) values than nDNA; (iii) estimated population coalescences are real; (iv) more data are always better; (v) one needs to do a Bayesian analysis; (vi) selective sweeps influence mtDNA data; (vii) equilibrium conditions are critical for estimating population parameters; and (viii) having better technology makes us smarter than our predecessors. This is clearly not an exhaustive list and many others can be added. It is, however, sufficient to illustrate why we all need to be more critical of our own understanding of molecular ecology and to be suspicious of self-evident truths.

Extreme population subdivision in the crown conch (Melongena corona): historical and contemporary influences.

Organisms with crawl-away larvae are thought to experience highly restricted gene flow. Here, we assess the pattern and magnitude of population subdivision of the direct developing snails in the Melongena corona complex and assess the validity of species and subspecies designations in the genus. A total of 516 individuals from 15 locations were assayed at 8 microsatellite loci. Levels of genetic diversity were moderate and typical of gastropods. There were from 8 to 28 alleles per locus and the average observed per sample heterozygosity ranged from 0.16 to 0.79. Levels of genetic divergence were generally large with all sample pairwise F(ST) values statistically significant and ranging from 0.011 to 0.438 and Jost's D(EST) ranging from 0.028 to 0.731. A Bayesian analysis identified 7 clusters of, usually adjoining, samples. The population subdivision is likely derived from a complex mixture of life-history attributes, frequent short-distance dispersal via swimming larvae, rare short- and long-distance dispersal of rafting larvae and eggs, and a patchwork of adjacent and adjoining habitats. As with a previous study, the current taxonomy is not supported by the genetic results and the complex can be considered as M. corona, a single, albeit clearly geographically genetically structured, species.

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents.

BACKGROUND: Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galápagos Rift. RESULTS: Genetic differentiation (F(ST)) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. CONCLUSIONS: Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events.

Pleistocene population expansions of Antarctic seals.

We sequenced a portion (c. 475 bp) of the mitochondrial control region of three species of Antarctic phocid carnivores (Weddell seal, Leptonychotes weddellii, N = 181; crabeater seal, Lobodon carcinophaga, N = 143; and Ross seal, Ommatophoca rossii, N = 41) that live seasonally or permanently in the fast ice and seasonal pack ice of the western Amundsen and Ross seas of western Antarctica. We resolved 251 haplotypes with a haplotype diversity of 0.98 to 0.99. Bayesian estimates of Theta from the program LAMARC ranged from 0.075 for Weddell seals to 0.576 for crabeater seals. We used the values of theta to estimate female effective population sizes (N(EF)), which were 40,700 to 63,000 for Weddell seals, 44,400 to 97,800 for Ross seals, and 358,500 to 531,900 for crabeater seals. We used mismatch distributions to test for historical population size expansions. Weddell seals and crabeater seals had significant, unimodal mean pairwise difference distributions (P = 0.56 and 0.36, respectively), suggesting that their populations expanded suddenly around 731,000 years ago (Weddell seals) and around 1.6 million years ago (crabeater seals). Both of these expansions occurred during times of intensified glaciations and may have been fostered by expanding pack ice habitat.

Population genetic structure between Yap and Palau for the coral Acropora hyacinthus.

Information on connectivity is becoming increasingly in demand as marine protected areas are being designed as an integral part of a network to protect marine resources at the ecosystem level. Larval dispersal and population structure, however, remain very difficult to assess. Here, we tested the predictions of a detailed oceanographic connectivity model of larval dispersal and coral recruitment within Palau and between Palau and Yap, which was developed to support the review of the existing network of marine protected areas in Palau. We used high throughput microsatellite genotyping of the coral Acropora hyacinthus to characterize population genetic structure. Pairwise F' ST values between Palau and Yap (0.10), Palau and Ngulu (0.09) and Yap and Ngulu (0.09) were all significant and similar to pairwise F' ST values of sites within Palau (0.02-0.12) and within Yap (0.02-0.09) highlighting structure at island scale and indicating that recruitment may be even more localized than previously anticipated. A bottleneck test did not reveal any signs of a founder effect between Yap and Palau. Overall, the data supports the idea that recovery of A. hyacinthus in Palau did not come exclusively from a single source but most likely came from a combination of areas, including sites within Palau. In light of these results there seems to be very little connectivity around the barrier reef and management recommendation would be to increase the number or the size of MPAs within Palau.

Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): analysis of microsatellite loci.

We assessed the degree of population subdivision among global populations of green sea turtles, Chelonia mydas, using four microsatellite loci. Previously, a single-copy nuclear DNA study indicated significant male-mediated gene flow among populations alternately fixed for different mitochondrial DNA haplotypes and that genetic divergence between populations in the Atlantic and Pacific Oceans was more common than subdivisions among populations within ocean basins. Even so, overall levels of variation at single-copy loci were low and inferences were limited. Here, the markedly more variable microsatellite loci confirm the presence of male-mediated gene flow among populations within ocean basins. This analysis generally confirms the genetic divergence between the Atlantic and Pacific. As with the previous study, phylogenetic analyses of genetic distances based on the microsatellite loci indicate a close genetic relationship among eastern Atlantic and Indian Ocean populations. Unlike the single-copy study, however, the results here cannot be attributed to an artifact of general low variability and likely represent recent or ongoing migration between ocean basins. Sequence analyses of regions flanking the microsatellite repeat reveal considerable amounts of cryptic variation and homoplasy and significantly aid in our understanding of population connectivity. Assessment of the allele frequency distributions indicates that at least some of the loci may not be evolving by the stepwise mutation model.

Depth as an organizing force in Pocillopora damicornis: intra-reef genetic architecture.

Relative to terrestrial plants, and despite similarities in life history characteristics, the potential for corals to exhibit intra-reef local adaptation in the form of genetic differentiation along an environmental gradient has received little attention. The potential for natural selection to act on such small scales is likely increased by the ability of coral larval dispersal and settlement to be influenced by environmental cues. Here, we combine genetic, spatial, and environmental data for a single patch reef in Kane'ohe Bay, O'ahu, Hawai'i, USA in a landscape genetics framework to uncover environmental drivers of intra-reef genetic structuring. The genetic dataset consists of near-exhaustive sampling (n = 2352) of the coral, Pocillopora damicornis at our study site and six microsatellite genotypes. In addition, three environmental parameters - depth and two depth-independent temperature indices - were collected on a 4 m grid across 85 locations throughout the reef. We use ordinary kriging to spatially interpolate our environmental data and estimate the three environmental parameters for each colony. Partial Mantel tests indicate a significant correlation between genetic relatedness and depth while controlling for space. These results are also supported by multi-model inference. Furthermore, spatial Principle Component Analysis indicates a statistically significant genetic cline along a depth gradient. Binning the genetic dataset based on size-class revealed that the correlation between genetic relatedness and depth was significant for new recruits and increased for larger size classes, suggesting a possible role of larval habitat selection as well as selective mortality in structuring intra-reef genetic diversity. That both pre- and post-recruitment processes may be involved points to the adaptive role of larval habitat selection in increasing adult survival. The conservation importance of uncovering intra-reef patterns of genetic diversity is discussed.

The origins of tropical marine biodiversity.

Recent phylogeographic studies have overturned three paradigms for the origins of marine biodiversity. (i) Physical (allopatric) isolation is not the sole avenue for marine speciation: many species diverge along ecological boundaries. (ii) Peripheral habitats such as oceanic archipelagos are not evolutionary graveyards: these regions can export biodiversity. (iii) Speciation in marine and terrestrial ecosystems follow similar processes but are not the same: opportunities for allopatric isolation are fewer in the oceans, leaving greater opportunity for speciation along ecological boundaries. Biodiversity hotspots such as the Caribbean Sea and the Indo-Pacific Coral Triangle produce and export species, but can also accumulate biodiversity produced in peripheral habitats. Both hotspots and peripheral ecosystems benefit from this exchange in a process dubbed biodiversity feedback.

Microsatellites for next-generation ecologists: a post-sequencing bioinformatics pipeline.

Microsatellites are the markers of choice for a variety of population genetic studies. The recent advent of next-generation pyrosequencing has drastically accelerated microsatellite locus discovery by providing a greater amount of DNA sequencing reads at lower costs compared to other techniques. However, laboratory testing of PCR primers targeting potential microsatellite markers remains time consuming and costly. Here we show how to reduce this workload by screening microsatellite loci via bioinformatic analyses prior to primer design. Our method emphasizes the importance of sequence quality, and we avoid loci associated with repetitive elements by screening with repetitive sequence databases available for a growing number of taxa. Testing with the Yellowstripe Goatfish Mulloidichthys flavolineatus and the marine planktonic copepod Pleuromamma xiphias we show higher success rate of primers selected by our pipeline in comparison to previous in silico microsatellite detection methodologies. Following the same pipeline, we discover and select microsatellite loci in nine additional species including fishes, sea stars, copepods and octopuses.

The effect of multiple paternity on the genetically effective size of a population.

With the availability of highly variable microsatellite loci, many previously elusive aspects of the lives of animals have been revealed. One important finding is that multiple paternity (MP) appears to be somewhat common throughout the metazoa. Frequently, along with the discovery of MP are assertions that it can increase the genetically effective size of the population (N(E)). I argue that MP is not likely to have a positive effect on N(E) because it increases the variance in male reproductive success. Published studies suggesting the contrary have implicitly or explicitly included other changes to the breeding system, and these additions are likely responsible for the presumed increase in N(E).

Sexing pinnipeds with ZFX and ZFY loci.

We developed and tested a protocol for determining the sex of individual pinnipeds using the sex-chromosome-specific genes ZFX and ZFY. We screened a total of 368 seals (168 crabeater, Lobodon carcinophaga; 159 Weddell, Leptonychotes weddellii; and 41 Ross, Ommatophoca rossii) of known or unknown sex and compared the molecular sex to the sex assigned at the time of biopsy sample collection in the Ross and Amundsen seas, Antarctica. We also screened 6 captive northern elephant seals (Mirounga angustirostris) and 2 captive California sea lions (Zalophus californianus) of known sex. The assigned sex and genetic sex agreed for virtually all seals. Indeed, discrepancies ranged from 0.0% to 6.7% among species. It is not clear, however, if the few mis-assignments of sex occurred in situ or in the laboratory. The assigned morphological and molecular sex might both be correct with the discrepancies owing perhaps to developmental effects of environmental pollution. A subset of individuals sequenced at both loci revealed no intraspecific sequence variation. There was, however, sequence variation among species at both loci, which allowed them to be uniquely identified with as few as 2 and as many as 31 nucleotides.

Multiple paternity and breeding system in the gopher tortoise, Gopherus polyphemus.

Little is known about the reproductive behaviors and the actual outcomes of mating attempts in the gopher tortoise (Gopherus polyphemus). We examined the mating system and reproductive behaviors of a population of gopher tortoises in central Florida. Using microsatellite markers, we assigned fathers to the offspring of seven clutches and determined that multiple fathers were present in two of the seven clutches examined. We found that gopher tortoises exhibited a promiscuous mating system with larger males fertilizing the majority of clutches. The advantage of larger males over smaller males in fertilizing females may be a result of larger males winning access to females in aggressive bouts with other males or larger males may be more attractive to females. Clutches produced by larger females tended to be sired by a single male, whereas clutches of smaller females tended to be sired by multiple males.

Genetics and morphology in a Borrichia frutescens and B. arborescens (Asteraceae) hybrid zone.

Interspecific plant hybridization is a common and evolutionarily important phenomenon. Here, the results of a study of hybridization in the Florida Keys between two species of sea oxeye daisy, Borrichia frutescens and B. arborescens, are reported. Nuclear and chloroplast genetic loci, log-likelihood assignment tests, and maximum likelihood estimates of genealogical class frequencies were used to identify hybrid and parent genotypes, to investigate the utility of leaf and flower morphology for hybrid identification, and to study symmetry and degree of introgression between the species. Genetic analyses confirmed the identity of the hybrid and parent plants that were used for the morphological studies. Together, leaf and flower morphology can be used to identify hybrid and parental types with moderate accuracy (4% error rate). Population genetic analyses indicate that, in spite of a significant level of hybridization, pure B. frutescens and B. arborescens are persisting in the hybrid zone. Of the nonparentals, about 18% appear to be F(1) hybrids, over 50% F(2) hybrids, and the remainder backcrossed individuals but only with the B. frutescens parent. It is postulated that the hybrid zone in the Florida Keys is being maintained by a combination of positive assortative mating and clonal reproduction.