How long have we been mistaken? Multi-tools shedding light into the systematics of the widespread deep-water genus Madrepora Linnaeus, 1758 (Scleractinia).

Deep-water coral reefs are found worldwide and harbor biodiversity levels that are comparable to their shallow-water counterparts. However, the genetic diversity and population structure of deep-water species remain poorly explored, and historical taxonomical issues still need to be resolved. Here we used microsatellite markers as well as ultraconserved elements (UCE) and exons to shed light on the population structure, genetic diversity, and phylogenetic position of the genus Madrepora, which contains M. oculata, one of the most widespread scleractinian species. Population structure of 107 samples from three Southwestern Atlantic sedimentary basins revealed the occurrence of a cryptic species, herein named M. piresae sp. nov. (authored by Kitahara, Capel and Zilberberg), which can be found in sympatry with M. oculata. Phylogeny reconstructions based on 134 UCEs and exon regions corroborated the population genetic data, with the recovery of two well-supported groups, and reinforced the polyphyly of the family Oculinidae. In order to better accommodate the genus Madrepora, while reducing taxonomical confusion associated with the name Madreporidae, we propose the monogeneric family Bathyporidae fam. nov. (authored by Kitahara, Capel, Zilberberg and Cairns). Our findings advance the knowledge on the widespread deep-water genus Madrepora, resolve a long-standing question regarding the phylogenetic position of the genus, and highlight the need of a worldwide review of the genus.

Glacial vicariance and secondary contact shape demographic histories in a freshwater mussel species complex.

Characterizing the mechanisms influencing the distribution of genetic variation in aquatic species can be difficult due to the dynamic nature of hydrological landscapes. In North America's Central Highlands, a complex history of glacial dynamics, long-term isolation, and secondary contact have shaped genetic variation in aquatic species. Although the effects of glacial history have been demonstrated in many taxa, responses are often lineage- or species-specific and driven by organismal ecology. In this study, we reconstruct the evolutionary history of a freshwater mussel species complex using a suite of mitochondrial and nuclear loci to resolve taxonomic and demographic uncertainties. Our findings do not support Pleurobema rubrum as a valid species, which is proposed for listing as threatened under the U.S. Endangered Species Act. We synonymize P. rubrum under Pleurobema sintoxia-a common and widespread species found throughout the Mississippi River Basin. Further investigation of patterns of genetic variation in P. sintoxia identified a complex demographic history, including ancestral vicariance and secondary contact, within the Eastern Highlands. We hypothesize these patterns were shaped by ancestral vicariance driven by the formation of Lake Green and subsequent secondary contact after the last glacial maximum. Our inference aligns with demographic histories observed in other aquatic taxa in the region and mirrors patterns of genetic variation of a freshwater fish species (Erimystax dissimilis) confirmed to serve as a parasitic larval host for P. sintoxia. Our findings directly link species ecology to observed patterns of genetic variation and may have significant implications for future conservation and recovery actions of freshwater mussels.

Testing the depth-differentiation hypothesis in a deepwater octocoral.

The depth-differentiation hypothesis proposes that the bathyal region is a source of genetic diversity and an area where there is a high rate of species formation. Genetic differentiation should thus occur over relatively small vertical distances, particularly along the upper continental slope (200-1000 m) where oceanography varies greatly over small differences in depth. To test whether genetic differentiation within deepwater octocorals is greater over vertical rather than geographical distances, Callogorgia delta was targeted. This species commonly occurs throughout the northern Gulf of Mexico at depths ranging from 400 to 900 m. We found significant genetic differentiation (FST = 0.042) across seven sites spanning 400 km of distance and 400 m of depth. A pattern of isolation by depth emerged, but geographical distance between sites may further limit gene flow. Water mass boundaries may serve to isolate populations across depth; however, adaptive divergence with depth is also a possible scenario. Microsatellite markers also revealed significant genetic differentiation (FST = 0.434) between C. delta and a closely related species, Callogorgia americana, demonstrating the utility of microsatellites in species delimitation of octocorals. Results provided support for the depth-differentiation hypothesis, strengthening the notion that factors covarying with depth serve as isolation mechanisms in deep-sea populations.

Expanding our view of the cold-water coral niche and accounting of the ecosystem services of the reef habitat.

Coral reefs are iconic ecosystems that support diverse, productive communities in both shallow and deep waters. However, our incomplete knowledge of cold-water coral (CWC) niche space limits our understanding of their distribution and precludes a complete accounting of the ecosystem services they provide. Here, we present the results of recent surveys of the CWC mound province on the Blake Plateau off the U.S. east coast, an area of intense human activity including fisheries and naval operations, and potentially energy and mineral extraction. At one site, CWC mounds are arranged in lines that total over 150 km in length, making this one of the largest reef complexes discovered in the deep ocean. This site experiences rapid and extreme shifts in temperature between 4.3 and 10.7 °C, and currents approaching 1 m s-1. Carbon is transported to depth by mesopelagic micronekton and nutrient cycling on the reef results in some of the highest nitrate concentrations recorded in the region. Predictive models reveal expanded areas of highly suitable habitat that currently remain unexplored. Multidisciplinary exploration of this new site has expanded understanding of the cold-water coral niche, improved our accounting of the ecosystem services of the reef habitat, and emphasizes the importance of properly managing these systems.

Genetic diversity and connectivity of chemosynthetic cold seep mussels from the U.S. Atlantic margin.

BACKGROUND: Deep-sea mussels in the subfamily Bathymodiolinae have unique adaptations to colonize hydrothermal-vent and cold-seep environments throughout the world ocean. These invertebrates function as important ecosystem engineers, creating heterogeneous habitat and promoting biodiversity in the deep sea. Despite their ecological significance, efforts to assess the diversity and connectivity of this group are extremely limited. Here, we present the first genomic-scale diversity assessments of the recently discovered bathymodioline cold-seep communities along the U.S. Atlantic margin, dominated by Gigantidas childressi and Bathymodiolus heckerae. RESULTS: A Restriction-site Associated DNA Sequencing (RADSeq) approach was used on 177 bathymodiolines to examine genetic diversity and population structure within and between seep sites. Assessments of genetic differentiation using single-nucleotide polymorphism (SNP) data revealed high gene flow among sites, with the shallower and more northern sites serving as source populations for deeper occurring G. childressi. No evidence was found for genetic diversification across depth in G. childressi, likely due to their high dispersal capabilities. Kinship analyses indicated a high degree of relatedness among individuals, and at least 10-20% of local recruits within a particular site. We also discovered candidate adaptive loci in G. childressi and B. heckerae that suggest differences in developmental processes and depth-related and metabolic adaptations to chemosynthetic environments. CONCLUSIONS: These results highlight putative source communities for an important ecosystem engineer in the deep sea that may be considered in future conservation efforts. Our results also provide clues into species-specific adaptations that enable survival and potential speciation within chemosynthetic ecosystems.

Genetic and morphological characterization of the freshwater mussel clubshell species complex (Pleurobema clava and Pleurobema oviforme) to inform conservation planning.

The shell morphologies of the freshwater mussel species Pleurobema clava (federally endangered) and Pleurobema oviforme (species of concern) are similar, causing considerable taxonomic confusion between the two species over the last 100 years. While P. clava was historically widespread throughout the Ohio River basin and tributaries to the lower Laurentian Great Lakes, P. oviforme was confined to the Tennessee and the upper Cumberland River basins. We used two mitochondrial DNA (mtDNA) genes, 13 novel nuclear DNA microsatellite markers, and shell morphometrics to help resolve this taxonomic confusion. Evidence for a single species was apparent in phylogenetic analyses of each mtDNA gene, revealing monophyletic relationships with minimal differentiation and shared haplotypes. Analyses of microsatellites showed significant genetic structuring, with four main genetic clusters detected, respectively, in the upper Ohio River basin, the lower Ohio River and Great Lakes, and upper Tennessee River basin, and a fourth genetic cluster, which included geographically intermediate populations in the Ohio and Tennessee river basins. While principal components analysis (PCA) of morphometric variables (i.e., length, height, width, and weight) showed significant differences in shell shape, only 3% of the variance in shell shape was explained by nominal species. Using Linear Discriminant and Random Forest (RF) analyses, correct classification rates for the two species' shell forms were 65.5% and 83.2%, respectively. Random Forest classification rates for some populations were higher; for example, for North Fork Holston (HOLS), it was >90%. While nuclear DNA and shell morphology indicate that the HOLS population is strongly differentiated, perhaps indicative of cryptic biodiversity, we consider the presence of a single widespread species the most likely biological scenario for many of the investigated populations based on our mtDNA dataset. However, additional sampling of P. oviforme populations at nuclear loci is needed to corroborate this finding.

Palaeoclimate ocean conditions shaped the evolution of corals and their skeletons through deep time.

Identifying how past environmental conditions shaped the evolution of corals and their skeletal traits provides a framework for predicting their persistence and that of their non-calcifying relatives under impending global warming and ocean acidification. Here we show that ocean geochemistry, particularly aragonite-calcite seas, drives patterns of morphological evolution in anthozoans (corals, sea anemones) by examining skeletal traits in the context of a robust, time-calibrated phylogeny. The lability of skeletal composition among octocorals suggests a greater ability to adapt to changes in ocean chemistry compared with the homogeneity of the aragonitic skeleton of scleractinian corals. Pulses of diversification in anthozoans follow mass extinctions and reef crises, with sea anemones and proteinaceous corals filling empty niches as tropical reef builders went extinct. Changing environmental conditions will likely diminish aragonitic reef-building scleractinians, but the evolutionary history of the Anthozoa suggests other groups will persist and diversify in their wake.

Molecular characterization of Bathymodiolus mussels and gill symbionts associated with chemosynthetic habitats from the U.S. Atlantic margin.

Mussels of the genus Bathymodiolus are among the most widespread colonizers of hydrothermal vent and cold seep environments, sustained by endosymbiosis with chemosynthetic bacteria. Presumed species of Bathymodiolus are abundant at newly discovered cold seeps on the Mid-Atlantic continental slope, however morphological taxonomy is challenging, and their phylogenetic affinities remain unestablished. Here we used mitochondrial sequence to classify species found at three seep sites (Baltimore Canyon seep (BCS; ~400m); Norfolk Canyon seep (NCS; ~1520m); and Chincoteague Island seep (CTS; ~1000m)). Mitochondrial COI (N = 162) and ND4 (N = 39) data suggest that Bathymodiolus childressi predominates at these sites, although single B. mauritanicus and B. heckerae individuals were detected. As previous work had suggested that methanotrophic and thiotrophic interactions can both occur at a site, and within an individual mussel, we investigated the symbiont communities in gill tissues of a subset of mussels from BCS and NCS. We constructed metabarcode libraries with four different primer sets spanning the 16S gene. A methanotrophic phylotype dominated all gill microbial samples from BCS, but sulfur-oxidizing Campylobacterota were represented by a notable minority of sequences from NCS. The methanotroph phylotype shared a clade with globally distributed Bathymodiolus spp. symbionts from methane seeps and hydrothermal vents. Two distinct Campylobacterota phylotypes were prevalent in NCS samples, one of which shares a clade with Campylobacterota associated with B. childressi from the Gulf of Mexico and the other with Campylobacterota associated with other deep-sea fauna. Variation in chemosynthetic symbiont communities among sites and individuals has important ecological and geochemical implications and suggests shifting reliance on methanotrophy. Continued characterization of symbionts from cold seeps will provide a greater understanding of the ecology of these unique environments as well and their geochemical footprint in elemental cycling and energy flux.

The complete maternal mitochondrial genome sequences of two imperiled North American freshwater mussels: Alasmidonta heterodon and Alasmidonta varicosa (Bivalvia: Unionoida: Unionidae).

The freshwater mussels Alasmidonta heterodon and A. varicosa historically inhabited rivers along the North American Atlantic coast from the Carolinas, U.S.A., to New Brunswick, CA. However, many populations have been extirpated, and A. heterodon is now federally listed in the U.S.A. as endangered, and both A. heterodon and A. varicosa are listed as vulnerable on the IUCN Red List. To facilitate genetic study of these species, we sequenced the complete female mitochondrial genomes of A. heterodon (15,909 bp; GenBank accession no. MG905826), and A. varicosa (15,693 bp; GenBank accession no. MG938673). Both mitogenomes contained 14 protein coding genes, 2 rRNA genes, and 22 tRNAs with the same gene order as reported for other members of the subfamily Anodontinae. When these two genomes were put into a phylogenetic context with other members of the Unionidae, they clustered together with other species in the subfamily Anodontinae, Tribe Anodontini.

Comparison of microbiomes of cold-water corals Primnoa pacifica and Primnoa resedaeformis, with possible link between microbiome composition and host genotype.

Cold-water corals provide critical habitats for a multitude of marine species, but are understudied relative to tropical corals. Primnoa pacifica is a cold-water coral prevalent throughout Alaskan waters, while another species in the genus, Primnoa resedaeformis, is widely distributed in the Atlantic Ocean. This study examined the V4-V5 region of the 16S rRNA gene after amplifying and pyrosequencing bacterial DNA from samples of these species. Key differences between the two species' microbiomes included a robust presence of bacteria belonging to the Chlamydiales order in most of the P. pacifica samples, whereas no more than 2% of any microbial community from P. resedaeformis comprised these bacteria. Microbiomes of P. resedaeformis exhibited higher diversity than those of P. pacifica, and the two species largely clustered separately in a principal coordinate analysis. Comparison of P. resedaeformis microbiomes from samples collected in two submarine canyons revealed a significant difference between locations. This finding mirrored significant genetic differences among the P. resedaeformis from the two canyons based upon population genetic analysis of microsatellite loci. This study presents the first report of microbiomes associated with these two coral species.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis.

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Characterization of 13 microsatellite loci for the deep-sea coral, Lophelia pertusa (Linnaeus 1758), from the western North Atlantic Ocean and Gulf of Mexico.

A suite of 13 polymorphic tri- and tetranucleotide microsatellite loci were isolated from the ahermatypic deep-sea coral, Lophelia pertusa. Among 51 individuals collected from three disjunct oceanic regions, allelic diversity ranged from six to 38 alleles and averaged 9.1 alleles per locus. Observed heterozygosity ranged from 9.1 to 96.8% and averaged 62.3% in the Gulf of Mexico population. For some loci, amplification success varied among collections, suggesting regional variation in priming site sequences. Four loci showed departures from Hardy-Weinberg equilibrium in certain collections which may reflect nonrandom mating.

Wild and aquaculture populations of the eastern oyster compared using microsatellites.

Five new microsatellite markers were developed for the eastern oyster (Crassostrea virginica), and allelic variability was compared between a wild Chesapeake Bay population (James River) and a hatchery strain (DEBY). All loci amplified readily and demonstrated allelic variability with the number of alleles ranging from 16 to 36 in the wild population and from 11 to 19 in the DEBY strain. Average observed and expected heterozygosities were estimated at 0.66 and 0.80 in the hatchery sample. The corresponding estimates were 0.91 and 0.75 in the wild sample. Results indicated lower genetic variability in the DEBY strain and significant genetic differentiation between the wild population and hatchery strain. These microsatellite loci will prove valuable for future population genetic studies and in tracking of hatchery strains used in restoration.

Comprehensive genetic analyses reveal evolutionary distinction of a mouse (Zapus hudsonius preblei) proposed for delisting from the US Endangered Species Act.

Zapus hudsonius preblei, listed as threatened under the US Endangered Species Act (ESA), is one of 12 recognized subspecies of meadow jumping mice found in North America. Recent morphometric and phylogenetic comparisons among Z. h. preblei and neighbouring conspecifics questioned the taxonomic status of selected subspecies, resulting in a proposal to delist the Z. h. preblei from the ESA. We present additional analyses of the phylogeographic structure within Z. hudsonius that calls into question previously published data (and conclusions) and confirms the original taxonomic designations. A survey of 21 microsatellite DNA loci and 1380 base pairs from two mitochondrial DNA (mtDNA) regions (control region and cytochrome b) revealed that each Z. hudsonius subspecies is genetically distinct. These data do not support the null hypothesis of a homogeneous gene pool among the five subspecies found within the southwestern portion of the species' range. The magnitude of the observed differentiation was considerable and supported by significant findings for nearly every statistical comparison made, regardless of the genome or the taxa under consideration. Structuring of nuclear multilocus genotypes and subspecies-specific mtDNA haplotypes corresponded directly with the disjunct distributions of the subspecies investigated. Given the level of correspondence between the observed genetic population structure and previously proposed taxonomic classification of subspecies (based on the geographic separation and surveys of morphological variation), we conclude that the nominal subspecies surveyed in this study do not warrant synonymy, as has been proposed for Z. h. preblei, Z. h. campestris, and Z. h. intermedius.

Phylogenetic evidence for an ancient rapid radiation of Caribbean sponge-dwelling snapping shrimps (Synalpheus).

A common challenge in reconstructing phylogenies involves a high frequency of short internal branches, which makes basal relationships difficult to resolve. Often it is not clear whether this pattern results from insufficient or inappropriate data, versus from a rapid evolutionary radiation. The snapping shrimp genus Synalpheus, which contains in excess of 100 species and is a prominent component of coral-reef faunas worldwide, provides an example. Its taxonomy has long been problematic due to the subtlety of diagnostic characters and apparently widespread variability within species. Here we use partial mt COI and 16S rRNA sequences and morphological characters to reconstruct relationships among 31 species in the morphologically well-defined gambarelloides species group, a putative clade of obligate sponge associates that is mostly endemic to the Caribbean and contains the only known eusocial marine animals. Analysis of the combined data produced a single tree with good support for many terminal clades and for relationships with outgroups, but poor support for branches near the base of the gambarelloides group. Most basal branches are extremely short and terminal branches are long, suggesting a relatively ancient, but rapid radiation of the gambarelloides group. This hypothesis is supported by significant departure from a null model of temporally random cladogenesis. Calibration of divergence times among gambarelloides-group species using data from three geminate pairs of Synalpheus species separated by the isthmus of Panamá suggests a major radiation between approximately 5 and 7 Mya, a few My before final closure of the Panamanian seaway during a period of spreading carbonate environments in the Caribbean; a second, smaller radiation occurred approximately 4 Mya. This molecular evidence for a rapid radiation among Caribbean marine organisms in the late Miocene/early Pliocene is strikingly similar to patterns documented from fossil data for several other Caribbean reef-associated invertebrate taxa. The similar patterns and timing of cladogenesis evidenced by molecular and fossil data for different Caribbean and East Pacific taxa suggests that the radiation involved a wide range of organisms, and strengthens the case that poor basal resolution in the gambarelloides group of Synalpheus reflects a real evolutionary phenomenon. The rapid radiation also helps explain the historical difficulty of diagnosing species in Synalpheus.