Yosemite toad (Anaxyrus canorus) transcriptome reveals interplay between speciation genes and adaptive introgression.

Genomes are heterogeneous during the early stages of speciation, with small 'islands' of DNA appearing to reflect strong adaptive differences, surrounded by vast seas of relative homogeneity. As species diverge, secondary contact zones between them can act as an interface and selectively filter through advantageous alleles of hybrid origin. Such introgression is another important adaptive process, one that allows beneficial mosaics of recombinant DNA ('rivers') to flow from one species into another. Although genomic islands of divergence appear to be associated with reproductive isolation, and genomic rivers form by adaptive introgression, it is unknown whether islands and rivers tend to be the same or different loci. We examined three replicate secondary contact zones for the Yosemite toad (Anaxyrus canorus) using two genomic data sets and a morphometric data set to answer the questions: (1) How predictably different are islands and rivers, both in terms of genomic location and gene function? (2) Are the adaptive genetic trait loci underlying tadpole growth and development reliably islands, rivers or neither? We found that island and river loci have significant overlap within a contact zone, suggesting that some loci are first islands, and later are predictably converted into rivers. However, gene ontology enrichment analysis showed strong overlap in gene function unique to all island loci, suggesting predictability in overall gene pathways for islands. Genome-wide association study outliers for tadpole development included LPIN3, a lipid metabolism gene potentially involved in climate change adaptation, that is island-like for all three contact zones, but also appears to be introgressing (as a river) across one zone. Taken together, our results suggest that adaptive divergence and introgression may be more complementary forces than currently appreciated.

Using landscape genomics to delineate future adaptive potential for climate change in the Yosemite toad (Anaxyrus canorus).

An essential goal in conservation biology is delineating population units that maximize the probability of species persisting into the future and adapting to future environmental change. However, future-facing conservation concerns are often addressed using retrospective patterns that could be irrelevant. We recommend a novel landscape genomics framework for delineating future "Geminate Evolutionary Units" (GEUs) in a focal species: (1) identify loci under environmental selection, (2) model and map adaptive conservation units that may spawn future lineages, (3) forecast relative selection pressures on each future lineage, and (4) estimate their fitness and likelihood of persistence using geo-genomic simulations. Using this process, we delineated conservation units for the Yosemite toad (Anaxyrus canorus), a U.S. federally threatened species that is highly vulnerable to climate change. We used a genome-wide dataset, redundancy analysis, and Bayesian association methods to identify 24 candidate loci responding to climatic selection (R 2 ranging from 0.09 to 0.52), after controlling for demographic structure. Candidate loci included genes such as MAP3K5, involved in cellular response to environmental change. We then forecasted future genomic response to climate change using the multivariate machine learning algorithm Gradient Forests. Based on all available evidence, we found three GEUs in Yosemite National Park, reflecting contrasting adaptive optima: YF-North (high winter snowpack with moderate summer rainfall), YF-East (low to moderate snowpack with high summer rainfall), and YF-Low-Elevation (low snowpack and rainfall). Simulations under the RCP 8.5 climate change scenario suggest that the species will decline by 29% over 90 years, but the highly diverse YF-East lineage will be least impacted for two reasons: (1) geographically it will be sheltered from the largest climatic selection pressures, and (2) its standing genetic diversity will promote a faster adaptive response. Our approach provides a comprehensive strategy for protecting imperiled non-model species with genomic data alone and has wide applicability to other declining species.

Landscape genetics of a sub-alpine toad: climate change predicted to induce upward range shifts via asymmetrical migration corridors.

Climate change is expected to have a major hydrological impact on the core breeding habitat and migration corridors of many amphibians in the twenty-first century. The Yosemite toad (Anaxyrus canorus) is a species of meadow-specializing amphibian endemic to the high-elevation Sierra Nevada Mountains of California. Despite living entirely on federal lands, it has recently faced severe extirpations, yet our understanding of climatic influences on population connectivity is limited. In this study, we used a previously published double-digest RADseq dataset along with numerous remotely sensed habitat features in a landscape genetics framework to answer two primary questions in Yosemite National Park: (1) Which fine-scale climate, topographic, soil, and vegetation features most facilitate meadow connectivity? (2) How is climate change predicted to influence both the magnitude and net asymmetry of genetic migration? We developed an approach for simultaneously modeling multiple toad migration paths, akin to circuit theory, except raw environmental features can be separately considered. Our workflow identified the most likely migration corridors between meadows and used the unique cubist machine learning approach to fit and forecast environmental models of connectivity. We identified the permuted modeling importance of numerous snowpack-related features, such as runoff and groundwater recharge. Our results highlight the importance of considering phylogeographic structure, and asymmetrical migration in landscape genetics. We predict an upward elevational shift for this already high-elevation species, as measured by the net vector of anticipated genetic movement, and a north-eastward shift in species distribution via the network of genetic migration corridors across the park.

Pleistocene glacial cycles drove lineage diversification and fusion in the Yosemite toad (Anaxyrus canorus).

Species endemic to alpine environments can evolve via steep ecological selection gradients between lowland and upland environments. Additionally, many alpine environments have faced repeated glacial episodes over the past two million years, fracturing these endemics into isolated populations. In this "glacial pulse" model of alpine diversification, cycles of allopatry and ecologically divergent glacial refugia play a role in generating biodiversity, including novel admixed ("fused") lineages. We tested for patterns of glacial pulse lineage diversification in the Yosemite toad (Anaxyrus [Bufo] canorus), an alpine endemic tied to glacially influenced meadow environments. Using double-digest RADseq on populations densely sampled from a portion of the species range, we identified nine distinct lineages with divergence times ranging from 18 to 724 thousand years ago (ka), coinciding with multiple Sierra Nevada glacial events. Three lineages have admixed origins, and demographic models suggest these fused lineages have persisted throughout past glacial cycles. Directionality indices supported the hypothesis that some lineages recolonized Yosemite from east of the ice sheet, whereas other lineages remained in western refugia. Finally, refugial niche reconstructions suggest that low- and high-elevation lineages have convergently adapted to similar climatic niches. Our results suggest glacial cycles and refugia may be important crucibles of adaptive diversity across deep evolutionary time.

Phylogenetics and biogeography of the two-wing flyingfish (Exocoetidae: Exocoetus).

Two-wing flyingfish (Exocoetus spp.) are widely distributed, epipelagic, mid-trophic organisms that feed on zooplankton and are preyed upon by numerous predators (e.g., tunas, dolphinfish, tropical seabirds), yet an understanding of their speciation and systematics is lacking. As a model of epipelagic fish speciation and to investigate mechanisms that increase biodiversity, we studied the phylogeny and biogeography of Exocoetus, a highly abundant holoepipelagic fish taxon of the tropical open ocean. Morphological and molecular data were used to evaluate the phylogenetic relationships, species boundaries, and biogeographic patterns of the five putative Exocoetus species. We show that the most widespread species (E. volitans) is sister to all other species, and we find no evidence for cryptic species in this taxon. Sister relationship between E. monocirrhus (Indo-Pacific) and E. obtusirostris (Atlantic) indicates the Isthmus of Panama and/or Benguela Barrier may have played a role in their divergence via allopatric speciation. The sister species E. peruvianus and E. gibbosus are found in different regions of the Pacific Ocean; however, our molecular results do not show a clear distinction between these species, indicating recent divergence or ongoing gene flow. Overall, our phylogeny reveals that the most spatially restricted species are more recently derived, suggesting that allopatric barriers may drive speciation, but subsequent dispersal and range expansion may affect the distributions of species.

Population Genetic Structure of the Tropical Two-Wing Flyingfish (Exocoetus volitans).

Delineating populations of pantropical marine fish is a difficult process, due to widespread geographic ranges and complex life history traits in most species. Exocoetus volitans, a species of two-winged flyingfish, is a good model for understanding large-scale patterns of epipelagic fish population structure because it has a circumtropical geographic range and completes its entire life cycle in the epipelagic zone. Buoyant pelagic eggs should dictate high local dispersal capacity in this species, although a brief larval phase, small body size, and short lifespan may limit the dispersal of individuals over large spatial scales. Based on these biological features, we hypothesized that E. volitans would exhibit statistically and biologically significant population structure defined by recognized oceanographic barriers. We tested this hypothesis by analyzing cytochrome b mtDNA sequence data (1106 bps) from specimens collected in the Pacific, Atlantic and Indian oceans (n = 266). AMOVA, Bayesian, and coalescent analytical approaches were used to assess and interpret population-level genetic variability. A parsimony-based haplotype network did not reveal population subdivision among ocean basins, but AMOVA revealed limited, statistically significant population structure between the Pacific and Atlantic Oceans (ΦST = 0.035, p<0.001). A spatially-unbiased Bayesian approach identified two circumtropical population clusters north and south of the Equator (ΦST = 0.026, p<0.001), a previously unknown dispersal barrier for an epipelagic fish. Bayesian demographic modeling suggested the effective population size of this species increased by at least an order of magnitude ~150,000 years ago, to more than 1 billion individuals currently. Thus, high levels of genetic similarity observed in E. volitans can be explained by high rates of gene flow, a dramatic and recent population expansion, as well as extensive and consistent dispersal throughout the geographic range of the species.

Genetic and experimental evidence for a mixed-age, mixed-origin bank of kelp microscopic stages in southern California.

Laboratory studies have demonstrated that the microscopic stages of kelps can rapidly resume development from a delayed state. Like terrestrial seeds or aquatic resting eggs, banks of delayed kelp stages may supplement population recovery after periods of stress, playing an important role for kelp populations that experience adult sporophyte absences due to seasonal or interannual disturbances. We found that removing the microscopic stages from natural rock substratum could prevent the appearance of juvenile kelp sporophytes for three months and the establishment of a diverse kelp assemblage for over four months within a southern California kelp forest. Juveniles were observed within one month in plots where microscopic stages were left intact, which may confer an advantage for the resulting sporophytes as they attain larger sizes before later recruiting neighbors. Microsatellite diversity was high (expected heterozygosity HE approximately 0.9) for juveniles and adults within our sites. Using a microsatellite-based parentage analysis for the dominant kelp, Macrocystis pyrifera, we estimated that a portion of the new M. pyrifera sporophyte recruits had originated from their parents at least seven months after their parents had disappeared. Similar delay durations have been demonstrated in recent laboratory studies. Additionally, our results suggest that zoospore dispersal distances > 50 m may be supported by including additional microsatellite loci in the analysis. We propose a mixed-age and, potentially, a mixed-origin bank of M. pyrifera gametophytes promotes maximal genetic diversity in recovering populations and reduces population genetic subdivision and self-fertilization rates for intact populations by promoting the survival of zoospores dispersed > 10 m and during inhospitable environmental conditions.

Genetic signature of rapid IHHNV (infectious hypodermal and hematopoietic necrosis virus) expansion in wild Penaeus shrimp populations.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89 Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39x10(-4) substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks.

Parallelization and optimization of genetic analyses in isolation by distance web service.

BACKGROUND: The Isolation by Distance Web Service (IBDWS) is a user-friendly web interface for analyzing patterns of isolation by distance in population genetic data. IBDWS enables researchers to perform a variety of statistical tests such as Mantel tests and reduced major axis regression (RMA), and returns vector based graphs. The more than 60 citations since 2005 confirm the popularity and utility of this website. Despite its usefulness, the data sets with over 65 populations can take hours or days to complete due to the computational intensity of the statistical tests. This is especially troublesome for web-based software analysis, since users tend to expect real-time results on the order of seconds, or at most, minutes. Moreover, as genetic data continue to increase and diversify, so does the demand for more processing power. In order to increase the speed and efficiency of IBDWS, we first determined which aspects of the code were most time consuming and whether they might be amenable to improvements by parallelization or algorithmic optimization. RESULTS: Runtime tests uncovered two areas of IBDWS that consumed significant amounts of time: randomizations within the Mantel test and the RMA calculations. We found that these sections of code could be restructured and parallelized to improve efficiency. The code was first optimized by combining two similar randomization routines, implementing a Fisher-Yates shuffling algorithm, and then parallelizing those routines. Tests of the parallelization and Fisher-Yates algorithmic improvements were performed on a variety of data sets ranging from 10 to 150 populations. All tested algorithms showed runtime reductions and a very close fit to the predicted speedups based on time-complexity calculations. In the case of 150 populations with 10,000 randomizations, data were analyzed 23 times faster. CONCLUSION: Since the implementation of the new algorithms in late 2007, datasets have continued to increase substantially in size and many exceed the largest population sizes we used in our test sets. The fact that the website has continued to work well in "real-world" tests, and receives a considerable number of new citations provides the strongest testimony to the effectiveness of our improvements. However, we soon expect the need to upgrade the number of nodes in our cluster significantly as dataset sizes continue to expand. The parallel implementation can be found at http://ibdws.sdsu.edu/.

Understanding the genetic effects of recent habitat fragmentation in the context of evolutionary history: phylogeography and landscape genetics of a southern California endemic Jerusalem cricket (Orthoptera: Stenopelmatidae: Stenopelmatus).

Habitat loss and fragmentation due to urbanization are the most pervasive threats to biodiversity in southern California. Loss of habitat and fragmentation can lower migration rates and genetic connectivity among remaining populations of native species, reducing genetic variability and increasing extinction risk. However, it may be difficult to separate the effects of recent anthropogenic fragmentation from the genetic signature of prehistoric fragmentation due to previous natural geological and climatic changes. To address these challenges, we examined the phylogenetic and population genetic structure of a flightless insect endemic to cismontane southern California, Stenopelmatus'mahogani' (Orthoptera: Stenopelmatidae). Analyses of mitochondrial DNA sequence data suggest that diversification across southern California began during the Pleistocene, with most haplotypes currently restricted to a single population. Patterns of genetic divergence correlate with contemporary urbanization, even after correcting for (geographical information system) GIS-based reconstructions of fragmentation during the Pleistocene. Theoretical simulations confirm that contemporary patterns of genetic structure could be produced by recent urban fragmentation using biologically reasonable assumptions about model parameters. Diversity within populations was positively correlated with current fragment size, but not prehistoric fragment size, suggesting that the effects of increased drift following anthropogenic fragmentation are already being seen. Loss of genetic connectivity and diversity can hinder a population's ability to adapt to ecological perturbations commonly associated with urbanization, such as habitat degradation, climatic changes and introduced species. Consequently, our results underscore the importance of preserving and restoring landscape connectivity for long-term persistence of low vagility native species.

Isolation by distance, web service.

BACKGROUND: The population genetic pattern known as "isolation by distance" results from spatially limited gene flow and is a commonly observed phenomenon in natural populations. However, few software programs exist for estimating the degree of isolation by distance among populations, and they tend not to be user-friendly. RESULTS: We have created Isolation by Distance Web Service (IBDWS) a user-friendly web interface for determining patterns of isolation by distance. Using this site, population geneticists can perform a variety of powerful statistical tests including Mantel tests, Reduced Major Axis (RMA) regression analysis, as well as calculate FST between all pairs of populations and perform basic summary statistics (e.g., heterozygosity). All statistical results, including publication-quality scatter plots in Postscript format, are returned rapidly to the user and can be easily downloaded. CONCLUSION: IBDWS population genetics analysis software is hosted at http://phage.sdsu.edu/~jensen/ and documentation is available at http://www.bio.sdsu.edu/pub/andy/IBD.html. The source code has been made available on Source Forge at http://sourceforge.net/projects/ibdws/.

Population structure of the pumpkin fruit fly Bactrocera depressa (Tephritidae) in Korea and Japan: Pliocene allopatry or recent invasion?

Because of their widespread agricultural impact and rapid range expansions, true fruit flies (Tephritidae) are the subject of quarantine and control efforts worldwide. Among these flies, the pumpkin fruit fly Bactrocera depressa, which infests squash and other cucurbitaceous plants in Korea, Japan and Taiwan, was recently isolated from produce shipments entering Japan and identified as a regulatory target. This species was described in 1933 from collections in Japan and discovered in 1974 in Korea, suggesting that it may have recently invaded mainland Asia. We analysed the genetic structure of Asian populations of B. depressa using sequence variation for mitochondrial gene cytochrome-oxidase I and three nuclear loci: elongation factor 1alpha, tubulinbeta1 and tubulinbeta3, using frequency-based approaches, nested clade analysis and assignment tests. Contrary to the hypothesis of recent invasion, high levels of genetic subdivision were found among five Korean and three Japanese populations. Nested clade analysis suggested a variety of processes operating over different time scales, including ancient isolation between Korea and Japan and more recent range expansions within each country. Contrary to a priori expectations, the results also suggested the recent introduction of a mitochondrial haplotype into Yokohama, Japan that is related closely to a widespread haplotype found throughout Korea. Assignment tests also supported these conclusions. The combination of a genealogical approach and probabilistic assignments of individuals to populations of origin was able to provide statistical support for the identification of cryptic introductions within an otherwise widespread indigenous species.