Phylogenomics of the adaptive radiation of Triturus newts supports gradual ecological niche expansion towards an incrementally aquatic lifestyle.

Newts of the genus Triturus (marbled and crested newts) exhibit substantial variation in the number of trunk vertebrae (NTV) and a higher NTV corresponds to a longer annual aquatic period. Because the Triturus phylogeny has thwarted resolution to date, the evolutionary history of NTV, annual aquatic period, and their potential coevolution has remained unclear. To resolve the phylogeny of Triturus, we generated a c. 6000 transcriptome-derived marker data set using a custom target enrichment probe set, and conducted phylogenetic analyses using: (1) data concatenation with RAxML, (2) gene-tree summary with ASTRAL, and (3) species-tree estimation with SNAPP. All analyses produce the same, highly supported topology, despite cladogenesis having occurred over a short timeframe, resulting in short internal branch lengths. Our new phylogenetic hypothesis is consistent with the minimal number of inferred changes in NTV count necessary to explain the diversity in NTV observed today. Although a causal relationship between NTV, body form, and aquatic ecology has yet to be experimentally established, our phylogeny indicates that these features have evolved together, and suggest that they may underlie the adaptive radiation that characterizes Triturus.

A signature of dynamic biogeography: enclaves indicate past species replacement.

Understanding how species have replaced each other in the past is important to predicting future species turnover. While past species replacement is difficult to detect after the fact, the process may be inferred from present-day distribution patterns. Species with abutting ranges sometimes show a characteristic distribution pattern, where a section of one species range is enveloped by that of the other. Such an enclave could indicate past species replacement: when a species is partly supplanted by a competitor, but a population endures locally while the invading species moves around and past it, an enclave forms. If the two species hybridize and backcross, the receding species is predicted to leave genetic traces within the expanding one under a scenario of species replacement. By screening dozens of genes in hybridizing crested newts, we uncover genetic remnants of the ancestral species, now inhabiting an enclave, in the range of the surrounding invading species. This independent genetic evidence supports the past distribution dynamics we predicted from the enclave. We suggest that enclaves provide a valuable tool in understanding historical species replacement, which is important because a major conservation concern arising from anthropogenic climate change is increased species replacement in the future.

Widespread hybridization within mound-building wood ants in Southern Finland results in cytonuclear mismatches and potential for sex-specific hybrid breakdown.

Hybridization and gene flow between diverging lineages are increasingly recognized as common evolutionary processes, and their consequences can vary from hybrid breakdown to adaptive introgression. We have previously found a population of wood ant hybrids between Formica aquilonia and F. polyctena that shows antagonistic effects of hybridization: females with introgressed alleles show hybrid vigour, whereas males with the same alleles show hybrid breakdown. Here, we investigate whether hybridization is a general phenomenon in this species pair and analyse 647 worker samples from 16 localities in Finland using microsatellite markers and a 1200-bp mitochondrial sequence. Our results show that 27 sampled nests contained parental-like gene pools (six putative F. polyctena and 21 putative F. aquilonia) and all remaining nests (69), from nine localities, contained hybrids of varying degrees. Patterns of genetic variation suggest these hybrids arise from several hybridization events or, instead, have backcrossed to the parental gene pools to varying extents. In contrast to expectations, the mitochondrial haplotypes of the parental species were not randomly distributed among the hybrids. Instead, nests that were closer to parental-like F. aquilonia for nuclear markers preferentially had F. polyctena's mitochondria and vice versa. This systematic pattern suggests there may be underlying selection favouring cytonuclear mismatch and hybridization. We also found a new hybrid locality with strong genetic differences between the sexes similar to those predicted under antagonistic selection on male and female hybrids. Further studies are needed to determine the selective forces that act on male and female genomes in these newly discovered hybrids.

Local adaptation of reproductive performance during thermal stress.

Considerable evidence exists for local adaptation of critical thermal limits in ectotherms following adult temperature stress, but fewer studies have tested for local adaptation of sublethal heat stress effects across life-history stages. In organisms with complex life cycles, such as holometabolous insects, heat stress during juvenile stages may severely impact gametogenesis, having downstream consequences on reproductive performance that may be mediated by local adaptation, although this is rarely studied. Here, we tested how exposure to either benign or heat stress temperature during juvenile and adult stages, either independently or combined, influences egg-to-adult viability, adult sperm motility and fertility in high- and low-latitude populations of Drosophila subobscura. We found both population- and temperature-specific effects on survival and sperm motility; juvenile heat stress decreased survival and subsequent sperm motility and each trait was lower in the northern population. We found an interaction between population and temperature on fertility following application of juvenile heat stress; although fertility was negatively impacted in both populations, the southern population was less affected. When the adult stage was also subject to heat stress, the southern population exhibited positive carry-over effects whereas the northern population's fertility remained low. Thus, the northern population is more susceptible to sublethal reproductive consequences following exposure to juvenile heat stress. This may be common in other organisms with complex life cycles and current models predicting population responses to climate change, which do not take into account the impact of juvenile heat stress on reproductive performance, may be too conservative.

Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.

Speciation, the evolution of reproductive isolation among populations, is continuous, complex, and involves multiple, interacting barriers. Until it is complete, the effects of this process vary along the genome and can lead to a heterogeneous genomic landscape with peaks and troughs of differentiation and divergence. When gene flow occurs during speciation, barriers restricting gene flow locally in the genome lead to patterns of heterogeneity. However, genomic heterogeneity can also be produced or modified by variation in factors such as background selection and selective sweeps, recombination and mutation rate variation, and heterogeneous gene density. Extracting the effects of gene flow, divergent selection and reproductive isolation from such modifying factors presents a major challenge to speciation genomics. We argue one of the principal aims of the field is to identify the barrier loci involved in limiting gene flow. We first summarize the expected signatures of selection at barrier loci, at the genomic regions linked to them and across the entire genome. We then discuss the modifying factors that complicate the interpretation of the observed genomic landscape. Finally, we end with a road map for future speciation research: a proposal for how to account for these modifying factors and to progress towards understanding the nature of barrier loci. Despite the difficulties of interpreting empirical data, we argue that the availability of promising technical and analytical methods will shed further light on the important roles that gene flow and divergent selection have in shaping the genomic landscape of speciation.

What explains rare and conspicuous colours in a snail? A test of time-series data against models of drift, migration or selection.

It is intriguing that conspicuous colour morphs of a prey species may be maintained at low frequencies alongside cryptic morphs. Negative frequency-dependent selection by predators using search images ('apostatic selection') is often suggested without rejecting alternative explanations. Using a maximum likelihood approach we fitted predictions from models of genetic drift, migration, constant selection, heterozygote advantage or negative frequency-dependent selection to time-series data of colour frequencies in isolated populations of a marine snail (Littorina saxatilis), re-established with perturbed colour morph frequencies and followed for >20 generations. Snails of conspicuous colours (white, red, banded) are naturally rare in the study area (usually <10%) but frequencies were manipulated to levels of ~50% (one colour per population) in 8 populations at the start of the experiment in 1992. In 2013, frequencies had declined to ~15-45%. Drift alone could not explain these changes. Migration could not be rejected in any population, but required rates much higher than those recorded. Directional selection was rejected in three populations in favour of balancing selection. Heterozygote advantage and negative frequency-dependent selection could not be distinguished statistically, although overall the results favoured the latter. Populations varied idiosyncratically as mild or variable colour selection (3-11%) interacted with demographic stochasticity, and the overall conclusion was that multiple mechanisms may contribute to maintaining the polymorphisms.

Ecological speciation in sympatric palms: 2. Pre- and post-zygotic isolation.

We evaluated reproductive isolation in two species of palms (Howea) that have evolved sympatrically on Lord Howe Island (LHI, Australia). We estimated the strength of some pre- and post-zygotic mechanisms in maintaining current species boundaries. We found that flowering time displacement between species is consistent across in and ex situ common gardens and is thus partly genetically determined. On LHI, pre-zygotic isolation due solely to flowering displacement was 97% for Howea belmoreana and 80% for H. forsteriana; this asymmetry results from H. forsteriana flowering earlier than H. belmoreana and being protandrous. As expected, only a few hybrids (here confirmed by genotyping) at both juvenile and adult stages could be detected in two sites on LHI, in which the two species grow intermingled (the Far Flats) or adjacently (Transit Hill). Yet, the distribution of hybrids was different between sites. At Transit Hill, we found no hybrid adult trees, but 13.5% of younger palms examined there were of late hybrid classes. In contrast, we found four hybrid adult trees, mostly of late hybrid classes, and only one juvenile F1 hybrid in the Far Flats. This pattern indicates that selection acts against hybrids between the juvenile and adult stages. An in situ reciprocal seed transplant between volcanic and calcareous soils also shows that early fitness components (up to 36 months) were affected by species and soil. These results are indicative of divergent selection in reproductive isolation, although it does not solely explain the current distribution of the two species on LHI.

Ecological speciation in sympatric palms: 1. Gene expression, selection and pleiotropy.

Ecological speciation requires divergent selection, reproductive isolation and a genetic mechanism to link the two. We examined the role of gene expression and coding sequence evolution in this process using two species of Howea palms that have diverged sympatrically on Lord Howe Island, Australia. These palms are associated with distinct soil types and have displaced flowering times, representing an ideal candidate for ecological speciation. We generated large amounts of RNA-Seq data from multiple individuals and tissue types collected on the island from each of the two species. We found that differentially expressed loci as well as those with divergent coding sequences between Howea species were associated with known ecological and phenotypic differences, including response to salinity, drought, pH and flowering time. From these loci, we identified potential 'ecological speciation genes' and further validate their effect on flowering time by knocking out orthologous loci in a model plant species. Finally, we put forward six plausible ecological speciation loci, providing support for the hypothesis that pleiotropy could help to overcome the antagonism between selection and recombination during speciation with gene flow.

Selection on outlier loci and their association with adaptive phenotypes in Littorina saxatilis contact zones.

A fundamental issue in speciation research is to evaluate phenotypic variation and the genomics driving the evolution of reproductive isolation between sister taxa. Above all, hybrid zones are excellent study systems for researchers to examine the association of genetic differentiation, phenotypic variation and the strength of selection. We investigated two contact zones in the marine gastropod Littorina saxatilis and utilized landmark-based geometric morphometric analysis together with amplified fragment length polymorphism (AFLP) markers to assess phenotypic and genomic divergence between ecotypes under divergent selection. From genetic markers, we calculated the cline width, linkage disequilibrium and the average effective selection on a locus. Additionally, we conducted an association analysis linking the outlier loci and phenotypic variation between ecotypes and show that a proportion of outlier loci are associated with key adaptive phenotypic traits.

The environmental genomics of metazoan thermal adaptation.

Continued and accelerating change in the thermal environment places an ever-greater priority on understanding how organisms are going to respond. The paradigm of 'move, adapt or die', regarding ways in which organisms can respond to environmental stressors, stimulates intense efforts to predict the future of biodiversity. Assuming that extinction is an unpalatable outcome, researchers have focussed attention on how organisms can shift in their distribution to stay in the same thermal conditions or can stay in the same place by adapting to a changing thermal environment. How likely these respective outcomes might be depends on the answer to a fundamental evolutionary question, namely what genetic changes underpin adaptation to the thermal environment. The increasing access to and decreasing costs of next-generation sequencing (NGS) technologies, which can be applied to both model and non-model systems, provide a much-needed tool for understanding thermal adaptation. Here we consider broadly what is already known from non-NGS studies about thermal adaptation, then discuss the benefits and challenges of different NGS methodologies to add to this knowledge base. We then review published NGS genomics and transcriptomics studies of thermal adaptation to heat stress in metazoans and compare these results with previous non-NGS patterns. We conclude by summarising emerging patterns of genetic response and discussing future directions using these increasingly common techniques.

What can aquatic gastropods tell us about phenotypic plasticity? A review and meta-analysis.

There have been few attempts to synthesise the growing body of literature on phenotypic plasticity to reveal patterns and generalities about the extent and magnitude of plastic responses. Here, we conduct a review and meta-analysis of published literature on phenotypic plasticity in aquatic (marine and freshwater) gastropods, a common system for studying plasticity. We identified 96 studies, using pre-determined search terms, published between 1985 and November 2013. The literature was dominated by studies of predator-induced shell form, snail growth rates and life history parameters of a few model taxa, accounting for 67% of all studies reviewed. Meta-analyses indicated average plastic responses in shell thickness, shell shape, and growth and fecundity of freshwater species was at least three times larger than in marine species. Within marine gastropods, species with planktonic development had similar average plastic responses to species with benthic development. We discuss these findings in the context of the role of costs and limits of phenotypic plasticity and environmental heterogeneity as important constraints on the evolution of plasticity. We also consider potential publication biases and discuss areas for future research, indicating well-studied areas and important knowledge gaps.

The genetic architecture of sexually selected traits in two natural populations of Drosophila montana.

We investigated the genetic architecture of courtship song and cuticular hydrocarbon traits in two phygenetically distinct populations of Drosophila montana. To study natural variation in these two important traits, we analysed within-population crosses among individuals sampled from the wild. Hence, the genetic variation analysed should represent that available for natural and sexual selection to act upon. In contrast to previous between-population crosses in this species, no major quantitative trait loci (QTLs) were detected, perhaps because the between-population QTLs were due to fixed differences between the populations. Partitioning the trait variation to chromosomes suggested a broadly polygenic genetic architecture of within-population variation, although some chromosomes explained more variation in one population compared with the other. Studies of natural variation provide an important contrast to crosses between species or divergent lines, but our analysis highlights recent concerns that segregating variation within populations for important quantitative ecological traits may largely consist of small effect alleles, difficult to detect with studies of moderate power.

Do the same genes underlie parallel phenotypic divergence in different Littorina saxatilis populations?

Parallel patterns of adaptive divergence and speciation are cited as powerful evidence for the role of selection driving these processes. However, it is often not clear whether parallel phenotypic divergence is underlain by parallel genetic changes. Here, we asked about the genetic basis of parallel divergence in the marine snail Littorina saxatilis, which has repeatedly evolved coexisting ecotypes adapted to either crab predation or wave action. We sequenced the transcriptome of snails of both ecotypes from three distant geographical locations (Spain, Sweden and United Kingdom) and mapped the reads to the L. saxatilis reference genome. We identified genomic regions potentially under divergent selection between ecotypes within each country, using an outlier approach based on F(ST) values calculated per locus. In line with previous studies indicating that gene reuse is generally common, we expected to find extensive sharing of outlier loci due to recent shared ancestry and gene flow between at least two of the locations in our study system. Contrary to our expectations, we found that most outliers were country specific, suggesting that much of the genetic basis of divergence is not shared among locations. However, we did find that more outliers were shared than expected by chance and that differentiation of shared outliers is often generated by the same SNPs. We discuss two mechanisms potentially explaining the limited amount of sharing we observed. First, a polygenic basis of divergent traits might allow for multiple distinct molecular mechanisms generating the same phenotypic patterns. Second, additional, location-specific axes of selection that we did not focus on in this study may produce distinct patterns of genetic divergence within each site.

Genital divergence in sympatric sister snails.

A pattern of greater divergence in mating traits between sister-species pairs with overlapping ranges than between allopatric species pairs is expected if reinforcement commonly contributes to speciation. Few large-scale comparative analyses have addressed this prediction, especially for genital form. Here, we show that penial morphology follows the predicted pattern in 40 robustly identified sister-species pairs in the marine gastropod subfamily Littorininae. Further work is needed to exclude other processes that may contribute to genital divergence between sympatric species, but the clear pattern we observe strongly suggests a role for genital form in reproductive isolation in this large clade.

A comparative analysis of the mechanisms underlying speciation on Lord Howe Island.

On Lord Howe Island, speciation is thought to have taken place in situ in a diverse array of distantly related plant taxa (Metrosideros, Howea and Coprosma; Proc. Natl Acad. Sci. USA 108, 2011, 13188). We now investigate whether the speciation processes were driven by divergent natural selection in each genus by examining the extent of ecological and genetic divergence. We present new and extensive, ecological and genetic data for all three genera. Consistent with ecologically driven speciation, outlier loci were detected using genome scan methods. This mechanism is supported by individual-based analyses of genotype-environment correlations within species, demonstrating that local adaptation is currently widespread on the island. Genetic analyses show that prezygotic isolating barriers within species are currently insufficiently strong to allow further population differentiation. Interspecific hybridization was found in both Howea and Coprosma, and species distribution modelling indicates that competitive exclusion may result in selection against admixed individuals. Colonization of new niches, partly fuelled by the rapid generation of new adaptive genotypes via hybridization, appears to have resulted in the adaptive radiation in Coprosma - supporting the 'Syngameon hypothesis'.

Hybridization and speciation.

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near-instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky-Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock-on effects on speciation both within and outside regions of hybridization.

Fewer invited talks by women in evolutionary biology symposia.

Lower visibility of female scientists, compared to male scientists, is a potential reason for the under-representation of women among senior academic ranks. Visibility in the scientific community stems partly from presenting research as an invited speaker at organized meetings. We analysed the sex ratio of presenters at the European Society for Evolutionary Biology (ESEB) Congress 2011, where all abstract submissions were accepted for presentation. Women were under-represented among invited speakers at symposia (15% women) compared to all presenters (46%), regular oral presenters (41%) and plenary speakers (25%). At the ESEB congresses in 2001-2011, 9-23% of invited speakers were women. This under-representation of women is partly attributable to a larger proportion of women, than men, declining invitations: in 2011, 50% of women declined an invitation to speak compared to 26% of men. We expect invited speakers to be scientists from top ranked institutions or authors of recent papers in high-impact journals. Considering all invited speakers (including declined invitations), 23% were women. This was lower than the baseline sex ratios of early-mid career stage scientists, but was similar to senior scientists and authors that have published in high-impact journals. High-quality science by women therefore has low exposure at international meetings, which will constrain Evolutionary Biology from reaching its full potential. We wish to highlight the wider implications of turning down invitations to speak, and encourage conference organizers to implement steps to increase acceptance rates of invited talks.

Population structure of the mosquito Aedes aegypti (Stegomyia aegypti) in Pakistan.

Eleven microsatellite markers were used to determine the genetic population structure and spread of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) in Pakistan using mosquitoes collected from 13 different cities. There is a single genetic cluster of Ae. aegypti in Pakistan with a pattern of isolation by distance within the population. The low level of isolation by distance suggests the long-range passive dispersal of this mosquito, which may be facilitated by the tyre trade in Pakistan. A decrease in genetic diversity from south to north suggests a recent spread of this mosquito from Karachi. A strong negative correlation between genetic distance and the quality of road connections shows that populations in cities connected by better road networks are less differentiated, which suggests the human-aided passive dispersal of Ae. aegypti in Pakistan. Dispersal on a large spatial scale may facilitate the strategy of introducing transgenic Ae. aegypti or intracellular bacteria such as Wolbachia to control the spread of dengue disease in Pakistan, but it also emphasizes the need for simple measures to control container breeding sites.

A review of dengue as an emerging disease in Pakistan.

The presence of dengue virus has been detected using neutralization and haemagglutination inhibition antibodies in local populations in Pakistan since the 1960s. However, the first epidemic was not reported until 1994. This was followed by some cases in 1995, but the disease was confined to the port city of Karachi. Since 2006, dengue epidemics have occurred every year and the range has extended to most cities in Pakistan. Dengue now affects thousands of people and has caused hundreds of deaths. It has become a major health problem in Pakistan, and it is likely to become an even greater health problem in the coming years. This review gives an insight into the dengue situation from the early 1960s to the most recent epidemics in Pakistan, and also describes the primary vector of this disease (Aedes aegypti) in Pakistan. As such, it provides the first comprehensive review of the emergence of this important public health problem.