Adam M. Reitzel

Going where traditional markers have not gone before: utility of and promise for RAD sequencing in marine invertebrate phylogeography and population genomics

Characterization of large numbers of single‐nucleotide polymorphisms (SNPs) throughout a genome has the power to refine the understanding of population demographic history and to identify genomic regions under selection in natural populations. To this end, population genomic approaches that harness the power of next‐generation sequencing to understand the ecology and evolution of marine invertebrates represent a boon to test long‐standing questions in marine biology and conservation. We employed restriction‐site‐associated DNA sequencing (RAD‐seq) to identify SNPs in natural populations of the sea anemone Nematostella vectensis, an emerging cnidarian model with a broad geographic range in estuarine habitats in North and South America, and portions of England. We identified hundreds of SNP‐containing tags in thousands of RAD loci from 30 barcoded individuals inhabiting four locations from Nova Scotia to South Carolina. Population genomic analyses using high‐confidence SNPs resulted in a highly‐resolved phylogeography, a result not achieved in previous studies using traditional markers. Plots of locus‐specific FST against heterozygosity suggest that a majority of polymorphic sites are neutral, with a smaller proportion suggesting evidence for balancing selection. Loci inferred to be under balancing selection were mapped to the genome, where 90% were located in gene bodies, indicating potential targets of selection. The results from analyses with and without a reference genome supported similar conclusions, further highlighting RAD‐seq as a method that can be efficiently applied to species lacking existing genomic resources. We discuss the utility of RAD‐seq approaches in burgeoning Nematostella research as well as in other cnidarian species, particularly corals and jellyfishes, to determine phylogeographic relationships of populations and identify regions of the genome undergoing selection.

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

BackgroundCalcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.ResultsThe Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis.ConclusionsThis study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.

Genomic Survey of Candidate Stress-Response Genes in the Estuarine Anemone Nematostella vectensis

Salt marshes are challenging habitats due to natural variability in key environmental parameters including temperature, salinity, ultraviolet light, oxygen, sulfides, and reactive oxygen species. Compounding this natural variation, salt marshes are often heavily impacted by anthropogenic insults including eutrophication, toxic contamination, and coastal development that alter tidal and freshwater inputs. Commensurate with this environmental variability, estuarine animals generally exhibit broader physiological tolerances than freshwater, marine, or terrestrial species. One factor that determines an organisms physiological tolerance is its ability to upregulate “stress-response genes” in reaction to particular stressors. Comparative studies on diverse organisms have identified a number of evolutionarily conserved genes involved in responding to abiotic and biotic stressors. We used homology-based scans to survey the sequenced genome of Nematostella vectensis, the starlet sea anemone, an estuarine specialist, to identify genes involved in the response to three kinds of insult—physiochemical insults, pathogens, and injury. Many components of the stress-response networks identified in triploblastic animals have clear orthologs in the sea anemone, meaning that they must predate the cnidarian-triploblast split (e.g., xenobiotic receptors, biotransformative genes, ATP-dependent transporters, and genes involved in responding to reactive oxygen species, toxic metals, osmotic shock, thermal stress, pathogen exposure, and wounding). However, in some instances, stress-response genes known from triploblasts appear to be absent from the Nematostella genome (e.g., many metal-complexing genes). This is the first comprehensive examination of the genomic stress-response repertoire of an estuarine animal and a member of the phylum Cnidaria. The molecular markers of stress response identified in Nematostella may prove useful in monitoring estuary health and evaluating coastal conservation efforts. These data may also inform conservation efforts on other cnidarians, such as the reef-building corals.

Heat shock protein expression during stress and diapause in the marine copepod Calanus finmarchicus

Calanoid copepods, such as Calanus finmarchicus, are a key component of marine food webs. C. finmarchicus undergo a facultative diapause during juvenile development, which profoundly affects their seasonal distribution and availability to their predators. The current ignorance of how copepod diapause is regulated limits understanding of copepod population dynamics, distribution, and ecosystem interactions. Heat shock proteins (Hsps) are a superfamily of molecular chaperones characteristically upregulated in response to stress conditions and frequently associated with diapause in other taxa. In this study, 8 heat shock proteins were identified in C. finmarchicus C5 copepodids (Hsp21, Hsp22, p26, Hsp90, and 4 forms of Hsp70), and expression of these transcripts was characterized in response to handling stress and in association with diapause. Hsp21, Hsp22, and Hsp70A (cytosolic subfamily) were induced by handling stress. Expression of Hsp70A was also elevated in shallow active copepodids relative to deep diapausing copepodids, which may reflect induction of this gene by varied stressors in active animals. In contrast, expression of Hsp22 was elevated in deep diapausing animals; Hsp22 may play a role both in short-term stress responses and in protecting proteins from degradation during diapause. Expression of most of the Hsps examined did not vary in response to diapause, perhaps because the diapause of C. finmarchicus is not associated with the extreme environmental conditions (e.g., freezing and desiccation) experienced by many other taxa, such as overwintering insects or Artemia cysts.

Light entrained rhythmic gene expression in the sea anemone Nematostella vectensis : the evolution of the animal circadian clock

Background Circadian rhythms in behavior and physiology are the observable phenotypes from cycles in expression of, interactions between, and degradation of the underlying molecular components. In bilaterian animals, the core molecular components include Timeless-Timeout, photoreceptive cryptochromes, and several members of the basic-loop-helix-Per-ARNT-Sim (bHLH-PAS) family. While many of core circadian genes are conserved throughout the Bilateria, their specific roles vary among species. Here, we identify and experimentally study the rhythmic gene expression of conserved circadian clock members in a sea anemone in order to characterize this gene network in a member of the phylum Cnidaria and to infer critical components of the clockwork used in the last common ancestor of cnidarians and bilaterians. Methodology/Principal Findings We identified homologs of circadian regulatory genes in the sea anemone Nematostella vectensis, including a gene most similar to Timeout, three cryptochromes, and several key bHLH-PAS transcription factors. We then maintained N. vectensis either in complete darkness or in a 12 hour light: 12 hour dark cycle in three different light treatments (blue only, full spectrum, blue-depleted). Gene expression varied in response to light cycle and light treatment, with a particularly strong pattern observed for NvClock. The cryptochromes more closely related to the light-sensitive clade of cryptochromes were upregulated in light treatments that included blue wavelengths. With co-immunoprecipitation, we determined that heterodimerization between CLOCK and CYCLE is conserved within N. vectensis. Additionally, we identified E-box motifs, DNA sequences recognized by the CLOCK:CYCLE heterodimer, upstream of genes showing rhythmic expression. Conclusions/Significance This study reveals conserved molecular and functional components of the circadian clock that were in place at the divergence of the Cnidaria and Bilateria, suggesting the animal circadian clockwork is more ancient than previous data suggest. Characterizing circadian regulation in a cnidarian provides insight into the early origins of animal circadian rhythms and molecular regulation of environmentally cued behaviors.

Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome.

Gene families, which encode toxins, are found in many poisonous animals, yet there is limited understanding of their evolution at the nucleotide level. The release of the genome draft sequence for the sea anemone Nematostella vectensis enabled a comprehensive study of a gene family whose neurotoxin products affect voltage-gated sodium channels. All gene family members are clustered in a highly repetitive approximately 30-kb genomic region and encode a single toxin, Nv1. These genes exhibit extreme conservation at the nucleotide level which cannot be explained by purifying selection. This conservation greatly differs from the toxin gene families of other animals (e.g., snakes, scorpions, and cone snails), whose evolution was driven by diversifying selection, thereby generating a high degree of genetic diversity. The low nucleotide diversity at the Nv1 genes is reminiscent of that reported for DNA encoding ribosomal RNA (rDNA) and 2 hsp70 genes from Drosophila, which have evolved via concerted evolution. This evolutionary pattern was experimentally demonstrated in yeast rDNA and was shown to involve unequal crossing-over. Through sequence analysis of toxin genes from multiple N. vectensis populations and 2 other anemone species, Anemonia viridis and Actinia equina, we observed that the toxin genes for each sea anemone species are more similar to one another than to those of other species, suggesting they evolved by manner of concerted evolution. Furthermore, in 2 of the species (A. viridis and A. equina) we found genes that evolved under diversifying selection, suggesting that concerted evolution and accelerated evolution may occur simultaneously.

Regional population structure of a widely introduced estuarine invertebrate: Nematostella vectensis Stephenson in New England

Nematostella vectensis is an infaunal anemone occurring in salt marshes, lagoons and other estuarine habitats in North America and the United Kingdom. Although it is considered rare and receives protection in England, it is widely distributed and abundant in the United States, particularly along the Atlantic coast. Recent studies suggest that both anthropogenic dispersal and reproductive plasticity may significantly influence the genetic structure of N. vectensis populations. Amplified fragment length polymorphism (AFLP) fingerprinting of individuals from nine populations in the northeastern United States indicates that stable populations are maintained by both asexual and sexual reproduction; in some cases asexually reproducing lineages exist within sexually reproducing populations. F statistics reveal extraordinarily high degrees of genetic differentiation between populations, even those separated by very short distances (less than 100 m). Genetic distances show little to no correlation with geographical distances, consistent with a role for sporadic, geographically discontinuous dispersal coupled with limited gene flow. No single genotype was found at more than one site, despite apparent homogeneity of habitat. In contrast with reported genotypic distributions for Nematostella in the United Kingdom, where a single clonal genotype dominates at multiple sites through southern England, our data thus fail to support the hypothesis of a general‐purpose genotype in the northeastern United States. However, they are consistent with important roles for reproductive plasticity, sporadic introductions and complex local population dynamics in determining the global and regional distribution of this species.

Global population genetic structure of the starlet anemone Nematostella vectensis: multiple introductions and implications for conservation policy

Distinguishing natural versus anthropogenic dispersal of organisms is essential for determining the native range of a species and implementing an effective conservation strategy. For cryptogenic species with limited historical records, molecular data can help to identify introductions. Nematostella vectensis is a small, burrowing estuarine sea anemone found in tidally restricted salt marsh pools. This species’ current distribution extends over three coast lines: (i) the Atlantic coast of North America from Nova Scotia to Georgia, (ii) the Pacific coast of North America from Washington to central California, and (iii) the southeast coast of England. The 1996 IUCN Red List designates N.xa0vectensis as “vulnerable” in England. Amplified fragment length polymorphism (AFLP) fingerprinting of 516 individuals from 24 N.xa0vectensis populations throughout its range and mtDNA sequencing of a subsample of these individuals strongly suggest that anthropogenic dispersal has played a significant role in its current distribution. Certain western Atlantic populations of N.xa0vectensis exhibit greater genetic similarity to Pacific populations or English populations than to other western Atlantic populations. At the same time, F-statistics showing high degrees of genetic differentiation between geographically proximate populations support a low likelihood for natural dispersal between salt marshes. Furthermore, the western Atlantic harbors greater genetic diversity than either England or the eastern Pacific. Collectively, these data clearly imply that N.xa0vectensis is native to the Atlantic coast of North America and that populations along the Pacific coast and in England are cases of successful introduction.

Nuclear receptor complement of the cnidarian Nematostella vectensis: phylogenetic relationships and developmental expression patterns

BackgroundNuclear receptors are a superfamily of metazoan transcription factors that regulate diverse developmental and physiological processes. Sequenced genomes from an increasing number of bilaterians have provided a more complete picture of duplication and loss of nuclear receptors in protostomes and deuterostomes but have left open the question of which nuclear receptors were present in the cnidarian-bilaterian ancestor. In addition, nuclear receptor expression and function are largely uncharacterized within cnidarians, preventing determination of conserved and novel nuclear receptor functions in the context of animal evolution.ResultsHere we report the first complete set of nuclear receptors from a cnidarian, the starlet sea anemone Nematostella vectensis. Genomic searches using conserved DNA- and ligand-binding domains revealed seventeen nuclear receptors in N. vectensis. Phylogenetic analyses support N. vectensis orthologs of bilaterian nuclear receptors in four nuclear receptor subfamilies within nuclear receptor family 2 (COUP-TF, TLL, HNF4, TR2/4) and one putative ortholog of GCNF (nuclear receptor family 6). Other N. vectensis genes grouped well with nuclear receptor family 2 but represented lineage-specific duplications somewhere within the cnidarian lineage and were not clear orthologs of bilaterian genes. Three nuclear receptors were not well-supported within any particular nuclear receptor family. The seventeen nuclear receptors exhibited distinct developmental expression patterns, with expression of several nuclear receptors limited to a subset of developmental stages.ConclusionN. vectensis contains a diverse complement of nuclear receptors including orthologs of several bilaterian nuclear receptors. Novel nuclear receptors in N. vectensis may be ancient genes lost from triploblastic lineages or may represent cnidarian-specific radiations. Nuclear receptors exhibited distinct developmental expression patterns, which are consistent with diverse regulatory roles for these genes. Understanding the evolutionary relationships and developmental expression of the N. vectensis nuclear receptor complement provides insight into the evolution of the nuclear receptor superfamily and a foundation for mechanistic characterization of cnidarian nuclear receptor function.

Two Alleles of NF-κB in the Sea Anemone Nematostella vectensis Are Widely Dispersed in Nature and Encode Proteins with Distinct Activities

Background NF-κB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-κB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain. Methods/Principal Findings We characterized two NF-κB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-κB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-κB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-κB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-κB proteins affect DNA binding as in human NF-κB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant. Conclusions/Significance These results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-κB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.