Replicated Landscape Genomics Identifies Evidence of Local Adaptation to Urbanization in Wood Frogs.

Native species that persist in urban environments may benefit from local adaptation to novel selection factors. We used double-digest restriction-side associated DNA (RAD) sequencing to evaluate shifts in genome-wide genetic diversity and investigate the presence of parallel evolution associated with urban-specific selection factors in wood frogs (Lithobates sylvaticus). Our replicated paired study design involved 12 individuals from each of 4 rural and urban populations to improve our confidence that detected signals of selection are indeed associated with urbanization. Genetic diversity measures were less for urban populations; however, the effect size was small, suggesting little biological consequence. Using an FST outlier approach, we identified 37 of 8344 genotyped single nucleotide polymorphisms with consistent evidence of directional selection across replicates. A genome-wide association study analysis detected modest support for an association between environment type and 12 of the 37 FST outlier loci. Discriminant analysis of principal components using the 37 FST outlier loci produced correct reassignment for 87.5% of rural samples and 93.8% of urban samples. Eighteen of the 37 FST outlier loci mapped to the American bullfrog (Rana [Lithobates] catesbeiana) genome, although none were in coding regions. This evidence of parallel evolution to urban environments provides a powerful example of the ability of urban landscapes to direct evolutionary processes.

Genomic signatures of population bottleneck and recovery in Northwest Atlantic pinnipeds.

Population increases over the past several decades provide natural settings in which to study the evolutionary processes that occur during bottleneck, growth, and spatial expansion. We used parallel natural experiments of historical decline and subsequent recovery in two sympatric pinniped species in the Northwest Atlantic, the gray seal (Halichoerus grypus atlantica) and harbor seal (Phoca vitulina vitulina), to study the impact of recent demographic change in genomic diversity. Using restriction site-associated DNA sequencing, we assessed genomic diversity at over 8,700 polymorphic gray seal loci and 3,700 polymorphic harbor seal loci in samples from multiple cohorts collected throughout recovery over the past half-century. Despite significant differences in the degree of genetic diversity assessed in the two species, we found signatures of historical bottlenecks in the contemporary genomes of both gray and harbor seals. We evaluated temporal trends in diversity across cohorts, as well as compared samples from sites at both the center and edge of a recent gray seal range expansion, but found no significant change in genomewide diversity following recovery. We did, however, find that the variance and degree of allele frequency change measured over the past several decades were significantly different from neutral expectations of drift under population growth. These two cases of well-described demographic history provide opportunities for critical evaluation of current approaches to simulating and understanding the genetic effects of historical demographic change in natural populations.

Population structure of Bathymodiolus manusensis, a deep-sea hydrothermal vent-dependent mussel from Manus Basin, Papua New Guinea.

Deep-sea hydrothermal vents in the western Pacific are increasingly being assessed for their potential mineral wealth. To anticipate the potential impacts on biodiversity and connectivity among populations at these vents, environmental baselines need to be established. Bathymodiolus manusensis is a deep-sea mussel found in close association with hydrothermal vents in Manus Basin, Papua New Guinea. Using multiple genetic markers (cytochrome C-oxidase subunit-1 sequencing and eight microsatellite markers), we examined population structure at two sites in Manus Basin separated by 40 km and near a potential mining prospect, where the species has not been observed. No population structure was detected in mussels sampled from these two sites. We also compared a subset of samples with B. manusensis from previous studies to infer broader population trends. The genetic diversity observed can be used as a baseline against which changes in genetic diversity within the population may be assessed following the proposed mining event.

Cost of Tolerance: Physiological Consequences of Evolved Resistance to Inhabit a Polluted Environment in Teleost Fish Fundulus heteroclitus.

Anthropogenic stressors, including pollutants, are key evolutionary drivers. It is hypothesized that rapid evolution to anthropogenic changes may alter fundamental physiological processes (e.g., energy metabolism), compromising an organism's capacity to respond to additional stressors. The Elizabeth River (ER) Superfund site represents a "natural-experiment" to explore this hypothesis in several subpopulations of Atlantic killifish that have evolved a gradation of resistance to a ubiquitous pollutant-polycyclic aromatic hydrocarbons (PAH). We examined bioenergetic shifts and associated consequences in PAH-resistant killifish by integrating genomic, physiological, and modeling approaches. Population genomics data revealed that genomic regions encoding bioenergetic processes are under selection in PAH-adapted fish from the most contaminated ER site and ex vivo studies confirmed altered mitochondrial function in these fish. Further analyses extending to differentially PAH-resistant subpopulations showed organismal level bioenergetic shifts in ER fish that are associated with increased cost of living, decreased performance, and altered metabolic response to temperature stress-an indication of reduced thermal plasticity. A movement model predicted a higher energetic cost for PAH-resistant subpopulations when seeking an optimum habitat. Collectively, we demonstrate that pollution adaption and inhabiting contaminated environments may result in physiological shifts leading to compromised organismal capacity to respond to additional stressors.

Genomewide investigation of adaptation to harmful algal blooms in common bottlenose dolphins (Tursiops truncatus).

Harmful algal blooms (HABs), which can be lethal in marine species and cause illness in humans, are increasing worldwide. In the Gulf of Mexico, HABs of Karenia brevis produce neurotoxic brevetoxins that cause large-scale marine mortality events. The long history of such blooms, combined with the potentially severe effects of exposure, may have produced a strong selective pressure for evolved resistance. Advances in next-generation sequencing, in particular genotyping-by-sequencing, greatly enable the genomic study of such adaptation in natural populations. We used restriction site-associated DNA (RAD) sequencing to investigate brevetoxicosis resistance in common bottlenose dolphins (Tursiops truncatus). To improve our understanding of the epidemiology and aetiology of brevetoxicosis and the potential for evolved resistance in an upper trophic level predator, we sequenced pools of genomic DNA from dolphins sampled from both coastal and estuarine populations in Florida and during multiple HAB-associated mortality events. We sequenced 129 594 RAD loci and analysed 7431 single nucleotide polymorphisms (SNPs). The allele frequencies of many of these polymorphic loci differed significantly between live and dead dolphins. Some loci associated with survival showed patterns suggesting a common genetic-based mechanism of resistance to brevetoxins in bottlenose dolphins along the Gulf coast of Florida, but others suggested regionally specific mechanisms of resistance or reflected differences among HABs. We identified candidate genes that may be the evolutionary target for brevetoxin resistance by searching the dolphin genome for genes adjacent to survival-associated SNPs.

Comparative population structure of two deep-sea hydrothermal-vent-associated decapods (Chorocaris sp. 2 and Munidopsis lauensis) from southwestern Pacific back-arc basins.

Studies of genetic connectivity and population structure in deep-sea chemosynthetic ecosystems often focus on endosymbiont-hosting species that are directly dependent on chemical energy extracted from vent effluent for survival. Relatively little attention has been paid to vent-associated species that are not exclusively dependent on chemosynthetic ecosystems. Here we assess connectivity and population structure of two vent-associated invertebrates--the shrimp Chorocaris sp. 2 and the squat lobster Munidopsis lauensis--that are common at deep-sea hydrothermal vents in the western Pacific. While Chorocaris sp. 2 has only been observed at hydrothermal vent sites, M. lauensis can be found throughout the deep sea but occurs in higher abundance around the periphery of active vents We sequenced mitochondrial COI genes and deployed nuclear microsatellite markers for both species at three sites in Manus Basin and either North Fiji Basin (Chorocaris sp. 2) or Lau Basin (Munidopsis lauensis). We assessed genetic differentiation across a range of spatial scales, from approximately 2.5 km to more than 3000 km. Population structure for Chorocaris sp. 2 was comparable to that of the vent-associated snail Ifremeria nautilei, with a single seemingly well-mixed population within Manus Basin that is genetically differentiated from conspecifics in North Fiji Basin. Population structure for Munidopsis lauensis was more complex, with two genetically differentiated populations in Manus Basin and a third well-differentiated population in Lau Basin. The unexpectedly high level of genetic differentiation between M. lauensis populations in Manus Basin deserves further study since it has implications for conservation and management of diversity in deep-sea hydrothermal vent ecosystems.

Combining genetic and demographic information to prioritize conservation efforts for anadromous alewife and blueback herring.

A major challenge in conservation biology is the need to broadly prioritize conservation efforts when demographic data are limited. One method to address this challenge is to use population genetic data to define groups of populations linked by migration and then use demographic information from monitored populations to draw inferences about the status of unmonitored populations within those groups. We applied this method to anadromous alewife (Alosa pseudoharengus) and blueback herring (Alosa aestivalis), species for which long-term demographic data are limited. Recent decades have seen dramatic declines in these species, which are an important ecological component of coastal ecosystems and once represented an important fishery resource. Results show that most populations comprise genetically distinguishable units, which are nested geographically within genetically distinct clusters or stocks. We identified three distinct stocks in alewife and four stocks in blueback herring. Analysis of available time series data for spawning adult abundance and body size indicate declines across the US ranges of both species, with the most severe declines having occurred for populations belonging to the Southern New England and the Mid-Atlantic Stocks. While all alewife and blueback herring populations deserve conservation attention, those belonging to these genetic stocks warrant the highest conservation prioritization.

Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species.

Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0-8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.

Factors affecting harp seal (Pagophilus groenlandicus) strandings in the Northwest Atlantic.

The effects of climate change on high latitude regions are becoming increasingly evident, particularly in the rapid decline of sea ice cover in the Arctic. Many high latitude species dependent on sea ice are being forced to adapt to changing habitats. Harp seals (Pagophilus groenlandicus) are an indicator species for changing high-latitude ecosystems. This study analyzed multiple factors including ice cover, demographics, and genetic diversity, which could affect harp seal stranding rates along the eastern coast of the United States. Ice cover assessments were conducted for the month of February in the Gulf of St. Lawrence whelping region from 1991-2010 using remote sensing data, and harp seal stranding data were collected over the same time period. Genetic diversity, which may affect how quickly species can adapt to changing climates, was assessed using ten microsatellite markers to determine mean d (2) in a subset of stranded and by-caught (presumably healthy) seals sampled along the northeast U.S. coast. Our study found a strong negative correlation (R (2) = 0.49) between ice cover in the Gulf of St. Lawrence and yearling harp seal strandings, but found no relationship between sea ice conditions and adult strandings. Our analysis revealed that male seals stranded more frequently than females during the study period and that this relationship was strongest during light ice years. In contrast, we found no significant difference in mean d (2) between stranded and by-caught harp seals. The results demonstrate that sea ice cover and demographic factors have a greater influence on harp seal stranding rates than genetic diversity, with only a little of the variance in mean d (2) among stranded seals explained by ice cover. Any changes in these factors could have major implications for harp seals, and these findings should be considered in the development of future management plans for the Arctic that incorporate climate variability.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

The spatial scale of genetic subdivision in populations of Ifremeria nautilei, a hydrothermal-vent gastropod from the southwest Pacific.

BACKGROUND: Deep-sea hydrothermal vents provide patchy, ephemeral habitats for specialized communities of animals that depend on chemoautotrophic primary production. Unlike eastern Pacific hydrothermal vents, where population structure has been studied at large (thousands of kilometres) and small (hundreds of meters) spatial scales, population structure of western Pacific vents has received limited attention. This study addresses the scale at which genetic differentiation occurs among populations of a western Pacific vent-restricted gastropod, Ifremeria nautilei. RESULTS: We used mitochondrial and DNA microsatellite markers to infer patterns of gene flow and population subdivision. A nested sampling strategy was employed to compare genetic diversity in discrete patches of Ifremeria nautilei separated by a few meters within a single vent field to distances as great as several thousand kilometres between back-arc basins that encompass the known range of the species. No genetic subdivisions were detected among patches, mounds, or sites within Manus Basin. Although I. nautilei from Lau and North Fiji Basins (~1000 km apart) also exhibited no evidence for genetic subdivision, these populations were genetically distinct from the Manus Basin population. CONCLUSIONS: An unknown process that restricts contemporary gene flow isolates the Manus Basin population of Ifremeria nautilei from widespread populations that occupy the North Fiji and Lau Basins. A robust understanding of the genetic structure of hydrothermal vent populations at multiple spatial scales defines natural conservation units and can help minimize loss of genetic diversity in situations where human activities are proposed and managed.

The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth.

The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana. Here we identify a protein complex (called the evening complex)--composed of the proteins encoded by EARLY FLOWERING 3 (ELF3), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO (LUX; also known as PHYTOCLOCK 1)--that directly regulates plant growth. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6) under diurnal conditions. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4-ELF3-LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis.

The temporal control of CONSTANS (CO) expression and activity is a key mechanism in photoperiodic flowering in Arabidopsis. FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) protein regulates CO transcription, although the molecular mechanism is unknown. We demonstrate here that FKF1 controls the stability of a Dof transcription factor, CYCLING DOF FACTOR 1 (CDF1). FKF1 physically interacts with CDF1, and CDF1 protein is more stable in fkf1 mutants. Plants with elevated levels of CDF1 flower late and have reduced expression of CO. CDF1 and CO are expressed in the same tissues, and CDF1 binds to the CO promoter. Thus, FKF1 controls daily CO expression in part by degrading CDF1, a repressor of CO transcription.

LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms.

In higher plants, the circadian clock orchestrates fundamental processes such as light signaling and the transition to flowering. We isolated mutants of the circadian clock from an Arabidopsis thaliana mutagenized reporter line by screening for seedlings with long hypocotyl phenotypes and subsequently assaying for abnormal clock-regulated CAB2::LUC expression. This screen identified five mutant alleles of a clock gene, LUX ARRHYTHMO (LUX), that significantly affect amplitude and robustness of rhythms in both constant white light and dark conditions. In addition, the transition from vegetative to floral development is accelerated and hypocotyl elongation is accentuated in these mutants under light:dark cycles. We genetically mapped the mutations by bulk segregant analysis with high-density oligonucleotide array genotyping to a small putative Myb transcription factor related to other clock components and response regulators in Arabidopsis. The negative arm of the Arabidopsis circadian clock, CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), is repressed in the lux mutants, whereas TIMING OF CAB2 EXPRESSION (TOC1) is activated. We demonstrate that CCA1 and LHY bind to the evening element motif in the LUX promoter, which strongly suggests that these proteins repress LUX expression, as they do TOC1. The data are also consistent with LUX being necessary for activation of CCA1 and LHY expression.

Rapid array mapping of circadian clock and developmental mutations in Arabidopsis.

Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F(2) individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION 1, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays.

Circadian clocks in daily and seasonal control of development.

The rotation of the earth results in regular changes in the light environment, and organisms have evolved a molecular oscillator that allows them to anticipate these changes. This daily molecular oscillator, known as the circadian clock, regulates a diverse array of physiologies across a wide variety of organisms. This review highlights a few of the insights we have into circadian clock regulation of development, in both plants and animals. A common thread linking plants and animals is the use of the circadian clock to sense changes in day length and to mediate a diverse number of photoperiodic responses.