Gene Coexpression Networks Drive and Predict Reproductive Effects in Daphnia in Response to Environmental Disturbances.

Increasing effects of anthropogenic stressors and those of natural origin on aquatic ecosystems have intensified the need for predictive and functional models of their effects. Here, we use gene expression patterns in combination with weighted gene coexpression networks and generalized additive models to predict effects on reproduction in the aquatic microcrustacean Daphnia. We developed models to predict effects on reproduction upon exposure to different cyanobacteria, different insecticides and binary mixtures of cyanobacteria and insecticides. Models developed specifically for groups of stressors (e.g., either cyanobacteria or insecticides) performed better than general models developed on all data. Furthermore, models developed using in silico generated mixture gene expression profiles from single stressor data were able to better predict effects on reproduction compared to models derived from the mixture exposures themselves. Our results highlight the potential of gene expression data to quantify effects of complex exposures at higher level organismal effects without prior mechanistic knowledge or complex exposure data.

Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism.

This corrects the article DOI: 10.1038/hdy.2015.73.

Genomic Appraisal of the Multifactorial Basis for In Vitro Acquisition of Miltefosine Resistance in Leishmania donovani.

Protozoan parasites of the Leishmania donovani complex are the causative agents of visceral leishmaniasis (VL), the most severe form of leishmaniasis, with high rates of mortality if left untreated. Leishmania parasites are transmitted to humans through the bite of infected female sandflies (Diptera: Phlebotominae), and approximately 500,000 new cases of VL are reported each year. In the absence of a safe human vaccine, chemotherapy, along with vector control, is the sole tool with which to fight the disease. Miltefosine (hexadecylphosphatidylcholine [HePC]), an antitumoral drug, is the only successful oral treatment for VL. In the current study, we describe the phenotypic traits of L. donovani clonal lines that have acquired resistance to HePC. We performed whole-genome and RNA sequencing of these resistant lines to provide an inclusive overview of the multifactorial acquisition of experimental HePC resistance, circumventing the challenge of identifying changes in membrane-bound proteins faced by proteomics. This analysis was complemented by assessment of the in vitro infectivity of HePC-resistant parasites. Our work underscores the importance of complementary "omics" to acquire the most comprehensive insight for multifaceted processes, such as HePC resistance.

Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism.

Convergent evolution of tetrodotoxin (TTX) resistance, at both the phenotypic and genetic levels, characterizes coevolutionary arms races between amphibians and their snake predators around the world, and reveals remarkable predictability in the process of adaptation. Here we examine the repeatability of the evolution of TTX resistance in an undescribed predator-prey relationship between TTX-bearing Eastern Newts (Notophthalmus viridescens) and Eastern Hog-nosed Snakes (Heterodon platirhinos). We found that that local newts contain levels of TTX dangerous enough to dissuade most predators, and that Eastern Hog-nosed Snakes within newt range are highly resistant to TTX. In fact, these populations of Eastern Hog-nosed Snakes are so resistant to TTX that the potential for current reciprocal selection might be limited. Unlike all other cases of TTX resistance in vertebrates, H. platirhinos lacks the adaptive amino acid substitutions in the skeletal muscle sodium channel that reduce TTX binding, suggesting that physiological resistance in Eastern Hog-nosed Snakes is conferred by an alternate genetic mechanism. Thus, phenotypic convergence in this case is not due to parallel molecular evolution, indicating that there may be more than one way for this adaptation to arise, even among closely related species.

Triangulating the genetic basis of adaptation to multifarious selection.

Understanding how natural populations adapt to their local environments is a major research theme for ecological genomics. This endeavour begins by sleuthing for shared genetic similarities among unrelated natural populations sharing adaptive traits to documented selective pressures. When the selective pressures have low dimensionality, and the genetic response is localized to a few genes of major effect, this detective work is relatively straightforward. However, in the real world, populations face a complex mixture of selective pressures and many adaptive responses are the result of changes in quantitative traits that have a polygenic genetic basis. This complex relationship between environment and adaptation presents a significant challenge. How can we begin to identify drivers of adaptation in natural settings? In this issue of Molecular Ecology, Orsini et al. (2012) take advantage of the biological attributes of the freshwater microcrustacean Daphnia (Fig. 1) to disentangle multidimensional selection's signature on the genome of populations that have repeatedly evolved adaptive responses to isolated selective pressures including predation, parasitism and anthropogenic changes in land use. Orsini et al. (2012) leverage a powerful combination of spatially structured populations in a geographic mosaic of environmental stressors, the historical archive of past genotypes preserved in lake-bottom sediments and selection experiments to identify sets of candidate genomic regions associated with adaptation in response to these three environmental stressors. This study provides a template for future investigation in ecological genomics, combining multiple experimental approaches with the genomic investigation of a well-studied ecological model species.

Landscape-scale genetic variation in a forest outbreak species, the mountain pine beetle (Dendroctonus ponderosae).

The mountain pine beetle Dendroctonus ponderosae is a native species currently experiencing large-scale outbreaks in western North American pine forests. We sought to describe the pattern of genetic variation across the range of this species, to determine whether there were detectable genetic differences between D. ponderosae occupying different host trees in common localities, and to determine whether there was molecular evidence for a past demographic expansion. Using a combination of amplified fragment length polymorphism (AFLP) and mitochondrial sequencing analyses, we found evidence of genetic structuring among populations that followed a broad isolation-by-distance pattern. Our results suggest that the geographical pattern of gene flow follows the core distribution of the principal D. ponderosae host species, around rather than across the Great Basin and Mojave Deserts. Patterns of haplotype diversity and divergence were consistent with a range-wide population expansion. This signal was particularly pronounced in the northern part of the species' range, where outbreak activity is currently increasing. Using AFLP markers, we were unable to detect significant differences among groups of insects sampled from different host trees in common locations. Incidentally, we found that a large proportion of the polymorphic AFLP markers were gender-specific, occurring only in males. While we did not include these markers in our analyses, this finding warrants further investigation.

Characterization of Pacific golden chanterelle (Cantharellus formosus) genet size using co-dominant microsatellite markers.

We characterized five co-dominant microsatellite markers and used them to study Pacific golden chanterelle (Cantharellus formosus) genet size and its relation to forest age and disturbance. Fruit-bodies were mapped in and collected from nine replicate study plots in old-growth, recently thinned, and unthinned 40-60-year-old second-growth stands dominated by Douglas fir (Pseudotsuga menziesii). Information from microsatellite loci, combined with random fragment length polymorphism analysis of the nuclear DNA internal transcribed spacer indicates that putative 'C. formosus' fruit-body collections may include a cryptic chanterelle species. Small genets were characterized for both genetic types with mean maximum widths of 3.2 +/- 3.6 m for C. formosus and 1.5 +/- 1.7 m for the alternative genetic group. Variance in genet size was high and some multilocus genotypes were observed on multiple plots separated by 0.3 km or more, indicating that genets were not fully resolved by the loci described here. There was no evidence that genet size differed across the three disturbance treatments.

Patterns of genetic architecture for life-history traits and molecular markers in a subdivided species.

Understanding the utility and limitations of molecular markers for predicting the evolutionary potential of natural populations is important for both evolutionary and conservation genetics. To address this issue, the distribution of genetic variation for quantitative traits and molecular markers is estimated within and among 14 permanent lake populations of Daphnia pulicaria representing two regional groups from Oregon. Estimates of population subdivision for molecular and quantitative traits are concordant, with QST generally exceeding GST. There is no evidence that microsatellites loci are less informative about subdivision for quantitative traits than are allozyme loci. Character-specific comparison of QST and GST support divergent selection pressures among populations for the majority of life-history traits in both coast and mountain regions. The level of within-population variation for molecular markers is uninformative as to the genetic variation maintained for quantitative traits. In D. pulicaria, regional differences in the frequency of sex may contribute to variation in the maintenance of expressed within-population quantitative-genetic variation without substantially impacting diversity at the genic level. These data are compared to an identical dataset for 17 populations of the temporary-pond species, D. pulex.

The adaptive landscape as a conceptual bridge between micro- and macroevolution.

An adaptive landscape concept outlined by G.G. Simpson constitutes the major conceptual bridge between the fields of micro- and macroevolutionary study. Despite some important theoretical extensions since 1944, this conceptual bridge has been ignored in many empirical studies. In this article, we review the status of theoretical work and emphasize the importance of models for peak movement. Although much theoretical work has been devoted to evolution on stationary, unchanging landscapes, an important new development is a focus on the evolution of the landscape itself. We also sketch an agenda of empirical issues that is inspired by theoretical developments.

Quantitative genetic variation in Daphnia: temporal changes in genetic architecture.

Nonadditive genetic variation and genetic disequilibrium are two important factors that influence the evolutionary trajectory of natural populations. We assayed quantitative genetic variation in a temporary-pond-dwelling population of Daphnia pulex over a full season to examine the role of nonadditive genetic variation and genetic disequilibrium in determining the short-term evolutionary trajectory of a cyclic parthenogen. Quantitative traits were influenced by three factors: (1) clonal selection significantly changed the population mean phenotype during the course of the growing season; (2) sexual reproduction and recombination led to significant changes in life-history trait means and the levels of expressed genetic variation, implying the presence of substantial nonadditive genetic variation and genetic disequilibrium; and (3) Egg-bank effects were found to be an important component of the realized year-to-year change. Additionally, we examined the impact of genetic disequilibria induced by clonal selection on the genetic (co)variance structure with a common principal components model. Clonal selection caused significant changes in the (co)variance structure that were eliminated by a single bout of random mating, suggesting that a build-up of disequilibria was the primary source of changes in the (co)variance structure. The results of this study highlight the complexity of natural selection operating on populations that undergo alternating phases of sexual and asexual reproduction.

Multi-locus genetic evidence for rapid ecologically based speciation in Daphnia.

The process of speciation involves the divergence of two or more subpopulations of a parent species into independent evolutionary trajectories. To study this process in natural populations requires a detailed knowledge of the genetic and ecological characteristics of the parent species and an understanding of how its populations can lose evolutionary cohesion. The cosmopolitan and speciose genus Daphnia provides many of these features by existing in multiple freshwater habitat types, particularly permanent lakes and temporary ponds, each of which presents distinct ecological challenges. We assayed the genetic composition of 20 temporary pond populations of members of the Daphnia pulex species complex in north-western Oregon and compared them to published data on related lake and pond populations. We collected molecular genetic data from 13 allozyme loci, from six microsatellite loci, and from the control region of the mitochondrial DNA. By assaying over 400 individual Daphnia for these data, we were able to compile composite genotypes not only of individual Daphnia but of each pond population as a whole. In these ponds, we discovered two distinct genotypic constellations, one which bears resemblance to the lake-dwelling taxon D. pulicaria, and one which bears resemblance to the pond-dwelling taxon, D. pulex. Using published genetic data from these and other species as a frame of reference, we characterized 13 of these ponds as being 'pond-like', three as being 'lake-like', and four as being 'mixed'. Unlike studies performed elsewhere, however, these ponds do not exhibit high probabilities of interspecific hybridization. Over 95% of all individuals have either a lake-like or a pond-like genotype at all three genetic systems, suggesting the two forms do not represent hybridized vs. nonhybridized genotypes. Because both types can be found in the same ponds at the same time in gametic disequilibrium, we also discount the possibility that they are two extremes of a single species that is highly genetically subdivided. With these genetic data, and with supporting life-history and ecological data previously gathered on these pond populations, we conclude that the most likely description of this system is of a taxon caught in the act of speciating, with new pond-adapted populations periodically stemming from lake-adapted sources during river flooding events.

Mitochondrial DNA variation, phylogeography and population structure of the Asian elephant.

We report the first genetic analysis of free-ranging Asian elephants (Elephas maximus). We sampled 118 elephants from Sri Lanka, Bhutan/North India, and Laos/Vietnam by extracting DNA from dung, PCR amplifying and sequencing 630 nucleotides of mitochondrial DNA, including part of the variable left domain of the control region. Comparison with African elephant (Loxodonta africana) sequences indicated a relatively slow molecular clock in the Proboscidea with a sequence divergence of approximately 1%/Myr. Genetic diversity within Asian elephants was low, suggesting a small long-term effective population size. Seventeen haplotypes were identified within Asian elephants, which clustered into two well-differentiated assemblages with an estimated Pliocene divergence of 2.5-3.5 million years ago. The two assemblages showed incomplete geographical partitioning, suggesting allopatric divergence and secondary admixture. On the mainland, little genetic differentiation was observed between elephant populations of Bhutan and India or Laos and Vietnam. A significant difference in haplotype frequencies but relatively weak subdivision was observed between the regions Bhutan-India and Laos-Vietnam. Significant genetic differentiation was observed between the mainland and Sri Lanka, and between northern, mid-latitude and southern regions in Sri Lanka.

Patterns in the geographical range sizes of ectotherms in North America.

The distributions of homeothermic mammals and birds in continental North America show a distinct pattern in the configuration of their geographical ranges. Smaller ranges tend to be elongated north-south while larger ranges tend to be elongated east-west. To examine the generality of this pattern in ectotherms, we analyzed the distribution on continental North America of 139 species of mosquitoes, 164 amphibians, and 221 reptiles. Unlike birds and mammals, small ranges of ectotherms were not elongated north-south and the small ranges of snakes were elongated east-west. The distribution of ectotherms with small ranges does not appear to be affected by the major topographic features of North America which tend to run north-south. Like birds and mammals, large ranges of mosquitoes and reptiles but not amphibians are elongated east-west. The east-west orientation of mosquitoes with large ranges is not attributable to the three largest genera in North America taken singly, Aedes, Culex, or Anopheles, but appears only when all genera are pooled. The east-west orientation of reptiles with large ranges is attributable to turtles and snakes but not lizards. Climatic zones may thus affect the distribution of mosquitoes, turtles, and snakes with large ranges but are not the major determinants of range dimensions among ectotherms in general.

A hierarchical molecular phylogeny within the genus Daphnia.

The nucleotide sequences of two mitochondrial DNA regions were used to determine phylogenetic relationships in the genus Daphnia (water fleas), a group for which systematics are historically unstable. A portion of the small ribosomal RNA was used to reconstruct higher-level relationships among species, while a portion of the more rapidly evolving control region was used to reconstruct lower-level relationships among populations. Two unexpected results were obtained. First, the subgeneric status of Ctenodaphnia falls into uncertainty on the basis of the failure of the 12S rRNA sequences to support these species as comprising an outgroup to the remainder of Daphnia species. Second, the high similarity of 12S rRNA sequences of Daphnia pulex and Daphnia pulicaria samples, coupled with the dual paraphyly of these samples as reconstructed from control-region analysis, suggests that they are two clonotype constellations within the same species complex. A combination of a variety of ecological influences has apparently resulted in the evolution of sets of Daphnia genotypes that are genetically cohesive despite their phenotypic divergence.