Genetic homogeneity of the invasive lionfish across the Northwestern Atlantic and the Gulf of Mexico based on Single Nucleotide Polymorphisms.

Despite the devastating impact of the lionfish (Pterois volitans) invasion on NW Atlantic ecosystems, little genetic information about the invasion process is available. We applied Genotyping by Sequencing techniques to identify 1,220 single nucleotide polymorphic sites (SNPs) from 162 lionfish samples collected between 2013 and 2015 from two areas chronologically identified as the first and last invaded areas in US waters: the east coast of Florida and the Gulf of Mexico. We used population genomic analyses, including phylogenetic reconstruction, Bayesian clustering, genetic distances, Discriminant Analyses of Principal Components, and coalescence simulations for detection of outlier SNPs, to understand genetic trends relevant to the lionfish's long-term persistence. We found no significant differences in genetic structure or diversity between the two areas (FST p-values > 0.01, and t-test p-values > 0.05). In fact, our genomic analyses showed genetic homogeneity, with enough gene flow between the east coast of Florida and Gulf of Mexico to erase previous signals of genetic divergence detected between these areas, secondary spreading, and bottlenecks in the Gulf of Mexico. These findings suggest rapid genetic changes over space and time during the invasion, resulting in one panmictic population with no signs of divergence between areas due to local adaptation.

Fine-scale genetic structure due to adaptive divergence among microhabitats.

It has been suggested that adaptive evolution on ecological timescales shapes communities. However, adaptation among environments relies on isolation or large selection coefficients that exceed migration effects. This reliance is tempered if adaptation is polygenic-does not depend on one allele completely replacing another but instead requires small allele frequency changes at many loci. Thus, whether individuals can evolve adaptation to fine-scale habitat variation (for example, microhabitats) is not resolved. Here we analyze the genetic divergence of the teleost fish, Fundulus heteroclitus, among microhabitats that are <200 m apart in three separate saltmarshes using 4741 single-nucleotide polymorphisms (SNPs). Among these SNPs, 1.3-2.3% have large and highly significant differences among microhabitats (mean FST=0.15; false discovery rate ⩽1%). The divergence among microhabitats for these outlier SNPs is larger than that among populations, exceeds neutral expectation and indicates surprising population structure among microhabitats. Thus, we suggest that polygenic selection is surprisingly effective in altering allele frequencies among many different SNPs that share similar biological functions in response to environmental and ecological differences over very small geographic distances. We acknowledge the evolutionary difficulty of large genetic divergence among well-connected habitats. Therefore, these studies are only the first step to discern whether natural selection is responsible and capable of effecting genetic divergence on such a fine scale.

Genomic variation among populations of threatened coral: Acropora cervicornis.

BACKGROUND: Acropora cervicornis, a threatened, keystone reef-building coral has undergone severe declines (>90 %) throughout the Caribbean. These declines could reduce genetic variation and thus hamper the species' ability to adapt. Active restoration strategies are a common conservation approach to mitigate species' declines and require genetic data on surviving populations to efficiently respond to declines while maintaining the genetic diversity needed to adapt to changing conditions. To evaluate active restoration strategies for the staghorn coral, the genetic diversity of A. cervicornis within and among populations was assessed in 77 individuals collected from 68 locations along the Florida Reef Tract (FRT) and in the Dominican Republic. RESULTS: Genotyping by Sequencing (GBS) identified 4,764 single nucleotide polymorphisms (SNPs). Pairwise nucleotide differences (π) within a population are large (~37 %) and similar to π across all individuals. This high level of genetic diversity along the FRT is similar to the diversity within a small, isolated reef. Much of the genetic diversity (>90 %) exists within a population, yet GBS analysis shows significant variation along the FRT, including 300 SNPs with significant FST values and significant divergence relative to distance. There are also significant differences in SNP allele frequencies over small spatial scales, exemplified by the large FST values among corals collected within Miami-Dade county. CONCLUSIONS: Large standing diversity was found within each population even after recent declines in abundance, including significant, potentially adaptive divergence over short distances. The data here inform conservation and management actions by uncovering population structure and high levels of diversity maintained within coral collections among sites previously shown to have little genetic divergence. More broadly, this approach demonstrates the power of GBS to resolve differences among individuals and identify subtle genetic structure, informing conservation goals with evolutionary implications.

Population Genomics of the Euryhaline Teleost Poecilia latipinna.

Global climate change and increases in sea levels will affect coastal marine communities. The conservation of these ecologically important areas will be a challenge because of their wide geographic distribution, ecological diversity and species richness. To address this problem, we need to better understand how the genetic variation of the species in these communities is distributed within local populations, among populations and between distant regions. In this study we apply genotyping by sequencing (GBS) and examine 955 SNPs to determine Sailfin molly (Poecilia latipinna) genetic diversity among three geographically close mangrove salt marsh flats in the Florida Keys compared to populations in southern and northern Florida. The questions we are asking are whether there is sufficient genetic variation among isolated estuarine fish within populations and whether there are significant divergences among populations. Additionally, we want to know if GBS approaches agree with previous studies using more traditional molecular approaches. We are able to identify large genetic diversity within each saltmarsh community (π ≈ 36%). Additionally, among the Florida Key populations and the mainland or between southern and northern Florida regions, there are significant differences in allele frequencies seen in population structure and evolutionary relationships among individuals. Surprisingly, even though the cumulative FST value using all 955 SNPs within the three Florida Key populations is small, there are 29 loci with significant FST values, and 11 of these were outliers suggestive of adaptive divergence. These data suggest that among the salt marsh flats surveyed here, there is significant genetic diversity within each population and small but significant differences among populations. Much of the genetic variation within and among populations found here with GBS is very similar to previous studies using allozymes and microsatellites. However, the meaningful difference between GBS and these previous measures of genetic diversity is the number of loci examined, which allows more precise delineations of population structure as well as facilitates identifying loci with excessive FST values that could indicate adaptive divergence.

Genomic approaches with natural fish populations.

Natural populations v. inbred stocks provide a much richer resource for identifying the effects of nucleotide substitutions because natural populations have greater polymorphism. Additionally, natural populations offer an advantage over most common research organisms because they are subject to natural selection, and analyses of these adaptations can be used to identify biologically important changes. Among fishes, these analyses are enhanced by having a wide diversity of species (>28 000 species, more than any other group of vertebrates) living in a huge range of environments (from below freezing to > 46 degrees C, in fresh water to salinities >40 ppt.). Moreover, fishes exhibit many different life-history and reproductive strategies and have many different phenotypes and social structures. Although fishes provide numerous advantages over other vertebrate models, there is still a dearth of available genomic tools for fishes. Fishes make up approximately half of all known vertebrate species, yet <0.2% of fish species have significant genomic resources. Nonetheless, genomic approaches with fishes have provided some of the first measures of individual variation in gene expression and insights into environmental and ecological adaptations. Thus, genomic approaches with natural fish populations have the potential to revolutionize fundamental studies of diverse fish species that offer myriad ecological and evolutionary questions.

Utility of natural populations for microarray analyses: isolation of genes necessary for functional genomic studies.

How much variation is there in gene expression? How is this variation partitioned within and among populations? How much variation is biologically important? That is, how much of this variation affects longevity, reproductive fitness, or probability of survival? Microarray analyses can be used to accurately quantify the expression of most, if not all, genes expressed in a tissue and thus address the first question. The latter questions can be investigated by examining the patterns of variation within and among natural populations of Fundulus. These populations are large and affected by historical, demographic, and selective constraints, providing a framework for the partition of variation in gene expression within and among populations. Additionally, the well established, phylogenetic relationship among Fundulus species can be used to discern adaptive change. A phylogenetic perspective reveals changes that are produced by natural selection and therefore indicates whether this variation affects longevity, reproductive fitness, or probability of survival, i.e., whether the variation is biologically important. However, a Fundulus microarray requires DNAs encoding specific Fundulus genes. This paper provides information on the production, isolation, and characterization of 4440 Fundulus cDNAs used in microarrays. Our approach was to pick random colonies from a normalized cDNA library and then PCR amplify and sequence these genes in a 96-well format. Periodically, the isolated and sequenced cDNAs were subtracted from the normalized library. Normalization reduced the number of redundant genes from 33% to 11%, increasing the effectiveness of this screening process. From 4440 sequenced cDNAs, 49% (2173) had a match in GenBank using BlastX searches. Of these, 53% were nonredundant, yielding 1149 identified genes. These data suggest that cDNAs necessary for microarray analyses can be produced effectively from most organisms.

Identification, functional characterization, and regulation of a new cytochrome P450 subfamily, the CYP2Ns.

The screening of liver and heart cDNA libraries from the teleost Fundulus heteroclitus with degenerate oligonucleotide probes to conserved alpha-helical regions in mammalian P450s resulted in the identification of two cDNAs that together represent a novel P450 subfamily, the CYP2Ns. Northern analysis demonstrated that CYP2N1 transcripts are most abundant in liver and intestine, whereas CYP2N2 mRNAs are most abundant in heart and brain. CYP2N1 and CYP2N2 proteins were co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells, and their ability to metabolize arachidonic acid and xenobiotic substrates was examined. Both CYP2N1 and CYP2N2 metabolize arachidonic acid to epoxyeicosatrienoic acids. Epoxidation is highly regio- and enantioselective with preferential formation of (8R,9S)-epoxyeicosatrienoic acid (optical purities are 91 and 90% for CYP2N1 and CYP2N2, respectively) and (11R, 12S)-epoxyeicosatrienoic acid (optical purities are 92 and 70% for CYP2N1 and CYP2N2, respectively). CYP2N1 and CYP2N2 also catalyze the formation of a variety of hydroxyeicosatetraenoic acids. Both P450s have benzphetamine N-demethylase activities but show minimal alkoxyresorufin O-dealkylase activities. To investigate factors affecting CYP2N expression in vivo, CYP2N transcripts were examined following starvation and/or treatment with 12-O-tetradecanoyl phorbol-13-acetate. Intestinal CYP2N1 mRNAs decrease in starved and/or phorbol ester-treated fish, whereas intestinal CYP2N2 transcripts decrease only following phorbol ester treatment. Interestingly, cardiac CYP2N2 expression decreases following phorbol ester treatment but increases following starvation. These results demonstrate that members of this novel P450 subfamily encode early vertebrate forms of arachidonic acid catalysts that are widely expressed and are regulated by environmental factors. Given the wealth of information on the functional role of P450-derived arachidonate metabolites in mammals, we postulate that CYP2N1 and CYP2N2 products have similar biological functions in early vertebrates. The identity of the mammalian orthologue(s) of the CYP2Ns remains unknown.

Cytochromes P450 (CYP) in tropical fishes: catalytic activities, expression of multiple CYP proteins and high levels of microsomal P450 in liver of fishes from Bermuda.

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with beta-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 beta- and 16 beta-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.

Identification of cytochrome P-450 1A (CYP1A) genes from two teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and phylogenetic analysis of CYP1A genes.

Cytochrome P-450-mediated responses to environmental challenges are well known in diverse animal taxa, but the evolution of the complex gene superfamily coding for these enzymes is poorly understood. Here we report a phylogenetic analysis of the cytochrome P-450 1A (CYP1A) genes including two new sequences determined from teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops). Degenerate PCR primers were used to amplify a 1.2 kbp fragment from liver cDNA. The toadfish PCR product was used as a probe to identify a full-length CYP1A clone from a toadfish liver cDNA library. The entire coding region of the scup CYP1A was obtained by rapid amplification of cDNA ends (RACE) using specific primers based on the sequence of the partial PCR product. The predicted protein sequences for toadfish and scup CYP1A shared 78% and 83% amino acid identity with rainbow trout CYP1A1 respectively. Amino acid identity with mammalian CYP1A proteins ranged from 51 to 60% for 505 aligned positions. Phylogenetic analysis of four teleost fish CYP1A genes (trout, toadfish, scup and plaice) and 12 mammalian CYP1A genes suggests a monophyletic origin of the teleost genes, with the trout gene being most divergent, and indicates three distinct groupings: mammalian 1A1, mammalian 1A2, and fish 1A. This supports the idea that the gene duplication event which gave rise to CYP1A1 and CYP1A2 occurred after the divergence of the lines leading to mammals and fish. These results establish a molecular phylogeny within the CYP1A subfamily, the first such detailed phylogenetic analysis within a cytochrome P-450 family.

The molecular cloning and expression of two CRABP cDNAs from human skin.

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.