Insight into the adaptive role of arachnid genome-wide duplication through chromosome-level genome assembly of the Western black widow spider.

Although spiders are one of the most diverse groups of arthropods, the genetic architecture of their evolutionary adaptations is largely unknown. Specifically, ancient genome-wide duplication occurring during arachnid evolution ~450 mya resulted in a vast assembly of gene families, yet the extent to which selection has shaped this variation is understudied. To aid in comparative genome sequence analyses, we provide a chromosome-level genome of the Western black widow spider (Latrodectus hesperus)-a focus due to its silk properties, venom applications, and as a model for urban adaptation. We used long-read and Hi-C sequencing data, combined with transcriptomes, to assemble 14 chromosomes in a 1.46 Gb genome, with 38,393 genes annotated, and a BUSCO score of 95.3%. Our analyses identified high repetitive gene content and heterozygosity, consistent with other spider genomes, which has led to challenges in genome characterization. Our comparative evolutionary analyses of eight genomes available for species within the Araneoidea group (orb weavers and their descendants) identified 1,827 single-copy orthologs. Of these, 155 exhibit significant positive selection primarily associated with developmental genes, and with traits linked to sensory perception. These results support the hypothesis that several traits unique to spiders emerged from the adaptive evolution of ohnologs-or retained ancestrally duplicated genes-from ancient genome-wide duplication. These comparative spider genome analyses can serve as a model to understand how positive selection continually shapes ancestral duplications in generating novel traits today within and between diverse taxonomic groups.

Differential Gene Expression following DHX36/G4R1 Knockout Is Associated with G-Quadruplex Content and Cancer.

G-quadruplexes (G4s) are secondary DNA and RNA structures stabilized by positive cations in a central channel formed by stacked tetrads of Hoogsteen base-paired guanines. G4s form from G-rich sequences across the genome, whose biased distribution in regulatory regions points towards a gene-regulatory role. G4s can themselves be regulated by helicases, such as DHX36 (aliases: G4R1 and RHAU), which possess the necessary activity to resolve these stable structures. G4s have been shown to both positively and negatively regulate gene expression when stabilized by ligands, or through the loss of helicase activity. Using DHX36 knockout Jurkat cell lines, we identified widespread, although often subtle, effects on gene expression that are associated with the presence or number of observed G-quadruplexes in promoters or gene regions. Genes that significantly change their expression, particularly those that show a significant increase in RNA abundance under DHX36 knockout, are associated with a range of cellular functions and processes, including numerous transcription factors and oncogenes, and are linked to several cancers. Our work highlights the direct and indirect role of DHX36 in the transcriptome of T-lymphocyte leukemia cells and the potential for DHX36 dysregulation in cancer.

Correlation between protein secondary structure and mechanical performance for the ultra-tough dragline silk of Darwin's bark spider.

The spider major ampullate (MA) silk exhibits high tensile strength and extensibility and is typically a blend of MaSp1 and MaSp2 proteins with the latter comprising glycine-proline-glycine-glycine-X repeating motifs that promote extensibility and supercontraction. The MA silk from Darwin's bark spider (Caerostris darwini) is estimated to be two to three times tougher than the MA silk from other spider species. Previous research suggests that a unique MaSp4 protein incorporates proline into a novel glycine-proline-glycine-proline motif and may explain C. darwini MA silk's extraordinary toughness. However, no direct correlation has been made between the silk's molecular structure and its mechanical properties for C. darwini. Here, we correlate the relative protein secondary structure composition of MA silk from C. darwini and four other spider species with mechanical properties before and after supercontraction to understand the effect of the additional MaSp4 protein. Our results demonstrate that C. darwini MA silk possesses a unique protein composition with a lower ratio of helices (31%) and ß-sheets (20%) than other species. Before supercontraction, toughness, modulus and tensile strength correlate with percentages of ß-sheets, unordered or random coiled regions and ß-turns. However, after supercontraction, only modulus and strain at break correlate with percentages of ß-sheets and ß-turns. Our study highlights that additional information including crystal size and crystal and chain orientation is necessary to build a complete structure-property correlation model.

G-Quadruplex Helicase DHX36/G4R1 Engages Nuclear Lamina Proteins in Quiescent Breast Cancer Cells.

G-quadruplexes (G4s) are nucleic acid structures found enriched within gene regulatory sequences. G4s control fundamental cellular processes, including replication, transcription, and translation. Proto-oncogenes are enriched with G4 sequences, while tumor-suppressor genes are depleted, suggesting roles for G4s in cell survival and proliferation. Specialized helicases participate in G4-mediated gene regulation via enzymatic unwinding activity. One such enzyme, DHX36/G4R1, is the major G4-helicase and is a master regulator of G4-DNAs and mRNAs. G4-resolution promotes the expression of proproliferative genes; as such, DHX36/G4R1 promotes cell proliferation. Little is known about how DHX36/G4R1 itself is regulated in nondividing cells. We hypothesized that DHX36/G4R1 protein binding partners are altered when a cell transitions from a dividing to a quiescent state. We found that DHX36/G4R1 co-purifies with a distinct set of proteins under quiescent conditions, which may represent a novel complex that regulates DHX36/G4R1 during cell cycle transitions and have implications for development and cancer.

Ovarian Transcriptomic Analyses in the Urban Human Health Pest, the Western Black Widow Spider.

Due to their abundance and ability to invade diverse environments, many arthropods have become pests of economic and health concern, especially in urban areas. Transcriptomic analyses of arthropod ovaries have provided insight into life history variation and fecundity, yet there are few studies in spiders despite their diversity within arthropods. Here, we generated a de novo ovarian transcriptome from 10 individuals of the western black widow spider (Latrodectus hesperus), a human health pest of high abundance in urban areas, to conduct comparative ovarian transcriptomic analyses. Biological processes enriched for metabolism-specifically purine, and thiamine metabolic pathways linked to oocyte development-were significantly abundant in L. hesperus. Functional and pathway annotations revealed overlap among diverse arachnid ovarian transcriptomes for highly-conserved genes and those linked to fecundity, such as oocyte maturation in vitellogenin and vitelline membrane outer layer proteins, hormones, and hormone receptors required for ovary development, and regulation of fertility-related genes. Comparative studies across arachnids are greatly needed to understand the evolutionary similarities of the spider ovary, and here, the identification of ovarian proteins in L. hesperus provides potential for understanding how increased fecundity is linked to the success of this urban pest.

Alternative Transcription at Venom Genes and Its Role as a Complementary Mechanism for the Generation of Venom Complexity in the Common House Spider.

The complex composition of venom, a proteinaceous secretion used by diverse animal groups for predation or defense, is typically viewed as being driven by gene duplication in conjunction with positive selection, leading to large families of diversified toxins with selective venom gland expression. Yet, the production of alternative transcripts at venom genes is often overlooked as another potentially important process that could contribute proteins to venom, and requires comprehensive datasets integrating genome and transcriptome sequences together with proteomic characterization of venom to be fully documented. In the common house spider, Parasteatoda tepidariorum, we used RNA sequencing of four tissue types in conjunction with the sequenced genome to provide a comprehensive transcriptome annotation. We also used mass spectrometry to identify a minimum of 99 distinct proteins in P tepidariorum venom, including at least 33 latrotoxins, pore-forming neurotoxins shared with the confamilial black widow. We found that venom proteins are much more likely to come from multiple transcript genes, whose transcripts produced distinct protein sequences. The presence of multiple distinct proteins in venom from transcripts at individual genes was confirmed for eight loci by mass spectrometry, and is possible at 21 others. Alternative transcripts from the same gene, whether encoding or not encoding a protein found in venom, showed a range of expression patterns, but were not necessarily restricted to the venom gland. However, approximately half of venom protein encoding transcripts were found among the 1,318 transcripts with strongly venom gland biased expression. Our findings revealed an important role for alternative transcription in generating venom protein complexity and expanded the traditional model of venom evolution.

The transcriptome of Darwin's bark spider silk glands predicts proteins contributing to dragline silk toughness.

Darwin's bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline's toughness, enabling giant webs. Caerostris darwini's MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin's bark spider, presenting innovative designs for engineering biomaterials.

The relationship between oxidative stress, reproduction, and survival in a bdelloid rotifer.

BACKGROUND: A proposed mediator of trade-offs between survival and reproduction is oxidative stress resistance. Investments in reproduction are associated with increased oxidative stress that reduces lifespan. We used the bdelloid rotifer Adineta vaga to examine baseline patterns of survival, reproduction, and measures of oxidative stress, as well as how these patterns change in the face of treatments known to induce oxidative stress. RESULTS: We discovered that under standard laboratory conditions late-life mortality may be explained by increased levels of oxidative stress induced by reproduction. However, following exposure to the oxidizing agent ionizing radiation, survival was unaffected while reproduction was reduced. CONCLUSIONS: We suggest that under normal environmental conditions, reduced survival is mediated by endogenously generated oxidative stress induced by reproduction, and thus represents a cost of reproduction. Alternatively, the reduced reproduction evident under exogenously applied oxidative stress represents a cost of somatic maintenance. Biochemical analyses designed to assess levels of oxidative stress, oxidative stress resistance, and oxidative damage under normal and oxidizing conditions suggest that varying investments in enzymatic and non-enzymatic based oxidative stress resistance determine whether a cost of reproduction or a cost of somatic maintenance is observed.

Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider silk glands.

Spider silk synthesis is an emerging model for the evolution of tissue-specific gene expression and the role of gene duplication in functional novelty, but its potential has not been fully realized. Accordingly, we quantified transcript (mRNA) abundance in seven silk gland types and three non-silk gland tissues for three cobweb-weaving spider species. Evolutionary analyses based on expression levels of thousands of homologous transcripts and phylogenetic reconstruction of 605 gene families demonstrated conservation of expression for each gland type among species. Despite serial homology of all silk glands, the expression profiles of the glue-forming aggregate glands were divergent from fiber-forming glands. Also surprising was our finding that shifts in gene expression among silk gland types were not necessarily coupled with gene duplication, even though silk-specific genes belong to multi-paralog gene families. Our results challenge widely accepted models of tissue specialization and significantly advance efforts to replicate silk-based high-performance biomaterials.

House spider genome uncovers evolutionary shifts in the diversity and expression of black widow venom proteins associated with extreme toxicity.

BACKGROUND: Black widow spiders are infamous for their neurotoxic venom, which can cause extreme and long-lasting pain. This unusual venom is dominated by latrotoxins and latrodectins, two protein families virtually unknown outside of the black widow genus Latrodectus, that are difficult to study given the paucity of spider genomes. Using tissue-, sex- and stage-specific expression data, we analyzed the recently sequenced genome of the house spider (Parasteatoda tepidariorum), a close relative of black widows, to investigate latrotoxin and latrodectin diversity, expression and evolution. RESULTS: We discovered at least 47 latrotoxin genes in the house spider genome, many of which are tandem-arrayed. Latrotoxins vary extensively in predicted structural domains and expression, implying their significant functional diversification. Phylogenetic analyses show latrotoxins have substantially duplicated after the Latrodectus/Parasteatoda split and that they are also related to proteins found in endosymbiotic bacteria. Latrodectin genes are less numerous than latrotoxins, but analyses show their recruitment for venom function from neuropeptide hormone genes following duplication, inversion and domain truncation. While latrodectins and other peptides are highly expressed in house spider and black widow venom glands, latrotoxins account for a far smaller percentage of house spider venom gland expression. CONCLUSIONS: The house spider genome sequence provides novel insights into the evolution of venom toxins once considered unique to black widows. Our results greatly expand the size of the latrotoxin gene family, reinforce its narrow phylogenetic distribution, and provide additional evidence for the lateral transfer of latrotoxins between spiders and bacterial endosymbionts. Moreover, we strengthen the evidence for the evolution of latrodectin venom genes from the ecdysozoan Ion Transport Peptide (ITP)/Crustacean Hyperglycemic Hormone (CHH) neuropeptide superfamily. The lower expression of latrotoxins in house spiders relative to black widows, along with the absence of a vertebrate-targeting α-latrotoxin gene in the house spider genome, may account for the extreme potency of black widow venom.

Effects of Gene Duplication, Positive Selection, and Shifts in Gene Expression on the Evolution of the Venom Gland Transcriptome in Widow Spiders.

Gene duplication and positive selection can be important determinants of the evolution of venom, a protein-rich secretion used in prey capture and defense. In a typical model of venom evolution, gene duplicates switch to venom gland expression and change function under the action of positive selection, which together with further duplication produces large gene families encoding diverse toxins. Although these processes have been demonstrated for individual toxin families, high-throughput multitissue sequencing of closely related venomous species can provide insights into evolutionary dynamics at the scale of the entire venom gland transcriptome. By assembling and analyzing multitissue transcriptomes from the Western black widow spider and two closely related species with distinct venom toxicity phenotypes, we do not find that gene duplication and duplicate retention is greater in gene families with venom gland biased expression in comparison with broadly expressed families. Positive selection has acted on some venom toxin families, but does not appear to be in excess for families with venom gland biased expression. Moreover, we find 309 distinct gene families that have single transcripts with venom gland biased expression, suggesting that the switching of genes to venom gland expression in numerous unrelated gene families has been a dominant mode of evolution. We also find ample variation in protein sequences of venom gland-specific transcripts, lineage-specific family sizes, and ortholog expression among species. This variation might contribute to the variable venom toxicity of these species.

Gene structure, regulatory control, and evolution of black widow venom latrotoxins.

Black widow venom contains α-latrotoxin, infamous for causing intense pain. Combining 33 kb of Latrodectus hesperus genomic DNA with RNA-Seq, we characterized the α-latrotoxin gene and discovered a paralog, 4.5 kb downstream. Both paralogs exhibit venom gland specific transcription, and may be regulated post-transcriptionally via musashi-like proteins. A 4 kb intron interrupts the α-latrotoxin coding sequence, while a 10 kb intron in the 3' UTR of the paralog may cause non-sense-mediated decay. Phylogenetic analysis confirms these divergent latrotoxins diversified through recent tandem gene duplications. Thus, latrotoxin genes have more complex structures, regulatory controls, and sequence diversity than previously proposed.

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BACKGROUND: Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution. RESULTS: We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression. CONCLUSIONS: Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.

Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit.

BACKGROUND: Spiders (Order Araneae) are essential predators in every terrestrial ecosystem largely because they have evolved potent arsenals of silk and venom. Spider silks are high performance materials made almost entirely of proteins, and thus represent an ideal system for investigating genome level evolution of novel protein functions. However, genomic level resources remain limited for spiders. RESULTS: We de novo assembled a transcriptome for the Western black widow (Latrodectus hesperus) from deeply sequenced cDNAs of three tissue types. Our multi-tissue assembly contained ~100,000 unique transcripts, of which > 27,000 were annotated by homology. Comparing transcript abundance among the different tissues, we identified 647 silk gland-specific transcripts, including the few known silk fiber components (e.g. six spider fibroins, spidroins). Silk gland specific transcripts are enriched compared to the entire transcriptome in several functions, including protein degradation, inhibition of protein degradation, and oxidation-reduction. Phylogenetic analyses of 37 gene families containing silk gland specific transcripts demonstrated novel gene expansions within silk glands, and multiple co-options of silk specific expression from paralogs expressed in other tissues. CONCLUSIONS: We propose a transcriptional program for the silk glands that involves regulating gland specific synthesis of silk fiber and glue components followed by protecting and processing these components into functional fibers and glues. Our black widow silk gland gene repertoire provides extensive expansion of resources for biomimetic applications of silk in industry and medicine. Furthermore, our multi-tissue transcriptome facilitates evolutionary analysis of arachnid genomes and adaptive protein systems.

Population genetics of a trochid gastropod broadens picture of Caribbean Sea connectivity.

BACKGROUND: Regional genetic connectivity models are critical for successful conservation and management of marine species. Even though rocky shore invertebrates have been used as model systems to understand genetic structure in some marine environments, our understanding of connectivity in Caribbean communities is based overwhelmingly on studies of tropical fishes and corals. In this study, we investigate population connectivity and diversity of Cittarium pica, an abundant rocky shore trochid gastropod that is commercially harvested across its natural range, from the Bahamas to Venezuela. METHODOLOGY/PRINCIPAL FINDINGS: We tested for genetic structure using DNA sequence variation at the mitochondrial COI and 16S loci, AMOVA and distance-based methods. We found substantial differentiation among Caribbean sites. Yet, genetic differentiation was associated only with larger geographic scales within the Caribbean, and the pattern of differentiation only partially matched previous assessments of Caribbean connectivity, including those based on larval dispersal from hydrodynamic models. For instance, the Bahamas, considered an independent region by previous hydrodynamic studies, showed strong association with Eastern Caribbean sites in our study. Further, Bonaire (located in the east and close to the meridional division of the Caribbean basin) seems to be isolated from other Eastern sites. CONCLUSIONS/SIGNIFICANCE: The significant genetic structure and observed in C. pica has some commonalities in pattern with more commonly sampled taxa, but presents features, such as the differentiation of Bonaire, that appear unique. Further, the level of differentiation, together with regional patterns of diversity, has important implications for the application of conservation and management strategies in this commercially harvested species.

Effects of selection and mutation on mitochondrial variation and inferences of historical population expansion in a Caribbean reef fish.

Mitochondrial sequence data has contributed to the understanding of historical demography through the application of neutrality tests and coalescent estimators of population growth rates. Characteristics of the mitochondrial genome, such as high mutation rate and lack of recombination, render it particularly suitable for these types of studies. However, selection can also affect patterns of mitochondrial variation. Furthermore, conclusions based on single mitochondrial loci can be sensitive to differences among mitochondrial genes in mutation rate and pattern and levels of homoplasy. We tested the contributions of these factors to patterns of mitochondrial variation in the Caribbean reef fish Halichoeres bivittatus using a multilocus sequence-based approach. Mitochondrial protein-coding loci deviated strongly from a neutral model of evolution and indicated high rates of estimated population growth. In contrast, the mitochondrial control region and a nuclear intron showed little or no deviation from neutrality and low estimated growth rates. The level of variation among loci is inconsistent with a demographic explanation and likely stems from the influence of mutation and selection on the mitochondrial genome. In H. bivittatus, a finding of high rates of population growth is likely an artifact of selection on mitochondrial proteins. This result suggests caution in the interpretation of variation at single mitochondrial loci, and highlights the importance of the use of unlinked nuclear loci to test demographic inferences made from mitochondrial DNA.

Phylogeny disambiguates the evolution of heat-shock cis-regulatory elements in Drosophila.

Heat-shock genes have a well-studied control mechanism for their expression that is mediated through cis-regulatory motifs known as heat-shock elements (HSEs). The evolution of important features of this control mechanism has not been investigated in detail, however. Here we exploit the genome sequencing of multiple Drosophila species, combined with a wealth of available information on the structure and function of HSEs in D. melanogaster, to undertake this investigation. We find that in single-copy heat shock genes, entire HSEs have evolved or disappeared 14 times, and the phylogenetic approach bounds the timing and direction of these evolutionary events in relation to speciation. In contrast, in the multi-copy gene Hsp70, the number of HSEs is nearly constant across species. HSEs evolve in size, position, and sequence within heat-shock promoters. In turn, functional significance of certain features is implicated by preservation despite this evolutionary change; these features include tail-to-tail arrangements of HSEs, gapped HSEs, and the presence or absence of entire HSEs. The variation among Drosophila species indicates that the cis-regulatory encoding of responsiveness to heat and other stresses is diverse. The broad dimensions of variation uncovered are particularly important as they suggest a substantial challenge for functional studies.

Contrasting patterns of transposable element insertions in Drosophila heat-shock promoters.

The proximal promoter regions of heat-shock genes harbor a remarkable number of P transposable element (TE) insertions relative to both positive and negative control proximal promoter regions in natural populations of Drosophila melanogaster. We have screened the sequenced genomes of 12 species of Drosophila to test whether this pattern is unique to these populations. In the 12 species' genomes, transposable element insertions are no more abundant in promoter regions of single-copy heat-shock genes than in promoters with similar or dissimilar architecture. Also, insertions appear randomly distributed across the promoter region, whereas insertions clustered near the transcription start site in promoters of single-copy heat-shock genes in D. melanogaster natural populations. Hsp70 promoters exhibit more TE insertions per promoter than all other genesets in the 12 species, similarly to in natural populations of D. melanogaster. Insertions in the Hsp70 promoter region, however, cluster away from the transcription start site in the 12 species, but near it in natural populations of D. melanogaster. These results suggest that D. melanogaster heat-shock promoters are unique in terms of their interaction with transposable elements, and confirm that Hsp70 promoters are distinctive in TE insertions across Drosophila.

The comparative phylogeography of east coast estuarine fishes in formerly glaciated sites: Persistence versus recolonization in Cyprinodon variegatus ovinus and Fundulus heteroclitus macrolepidotus.

Species distributions may be dramatically affected by climatic variability, such as occurred during Pleistocene glaciation. Populations of coastal organisms could have been affected directly by ice movement or through sea level change. Response could involve shifts in distribution southwards or persistence through the full range or in limited high-latitude refugia. Comparative studies of the response of ecologically similar species can provide a useful complement to those examining response across disparate species in defining what parameters influence persistence. Patterns of mitochondrial genetic variation in 2 estuarine fish subspecies from the Northwest Atlantic, Fundulus heteroclitus macrolepidotus and Cyprinodon variegatus ovinus, indicate that ecological similarity does not necessarily predict propensity for glacial persistence. Fundulus heteroclitus macrolepidotus is highly diverse in glaciated regions, with isolated populations whose origins predate the last glacial maximum and may have recently expanded it's range to the south from New England. However, within glaciated regions, signals of population growth and distributional shifts indicate a dynamic Pleistocene history for F. h. macrolepidotus, in contrast with recent studies involving microsatellites. A different pattern is found in C. v. ovinus, which is depauperate in formerly glaciated sites, with a clear signal of recent recolonization of glaciated regions from the south. Genetic differentiation in glaciated areas is consistent with isolation after glacial withdrawal. In C. v. ovinus, rapidly evolving microsatellite loci show a similar pattern to mitochondrial DNA but may be reaching equilibrium on small spatial scales. These contrasting patterns of variation illustrate how ecologically similar species can respond to large-scale environmental change in distinct ways.

The Pleistocene history of the sheepshead minnow (Cyprinodon variegatus): Non-equilibrium evolutionary dynamics within a diversifying species complex.

The sheepshead minnow, Cyprinodon variegatus, is a widespread fish species that typically inhabits coastal tidal marsh and mangrove swamp environments, ranging from Cape Cod, Massaschusetts to northern Mexico and into the Caribbean. This wide range crosses several biogeographic boundaries which are coincident with genetic structuring within numerous species originating in the Pleistocene. In addition, the more northerly reaches of this species range have been further subject to the evolutionary consequences of Pleistocene glaciation due to local extinction and recolonization of formerly glaciated sites. C. variegatus thus provides an excellent vertebrate model system within which to test the extent of genetic differentiation among populations in a dominant coastal ecosystem and examine patterns of historical demography in populations distributed along a latitudinal gradient. Using mitochondrial control region and ND2 sequence data, we discovered monophyletic clades within C. variegatus with divergence times within the Pleistocene, and very low gene flow between most sites. Intraspecific genetic breaks appear to correspond broadly to biogeographic or oceanic boundaries. Pleistocene climate change appears to have had dramatic impacts on the size and distribution of populations within and near the glacial margins, but has also affected populations far from formerly glaciated regions.