Genetic structure and historic demography of endangered unarmoured threespine stickleback at southern latitudes signals a potential new management approach.

Habitat loss, flood control infrastructure, and drought have left most of southern California and northern Baja California's native freshwater fish near extinction, including the endangered unarmoured threespine stickleback (Gasterosteus aculeatus williamsoni). This subspecies, an unusual morph lacking the typical lateral bony plates of the G. aculeatus complex, occurs at arid southern latitudes in the eastern Pacific Ocean and survives in only three inland locations. Managers have lacked molecular data to answer basic questions about the ancestry and genetic distinctiveness of unarmoured populations. These data could be used to prioritize conservation efforts. We sampled G. aculeatus from 36 localities and used microsatellites and whole genome data to place unarmoured populations within the broader evolutionary context of G. aculeatus across southern California/northern Baja California. We identified three genetic groups with none consisting solely of unarmoured populations. Unlike G. aculeatus at northern latitudes, where Pleistocene glaciation has produced similar historical demographic profiles across populations, we found markedly different demographics depending on sampling location, with inland unarmoured populations showing steeper population declines and lower heterozygosity compared to low armoured populations in coastal lagoons. One exception involved the only high elevation population in the region, where the demography and alleles of unarmoured fish were similar to low armoured populations near the coast, exposing one of several cases of artificial translocation. Our results suggest that the current "management-by-phenotype" approach, based on lateral plates, is incidentally protecting the most imperilled populations; however, redirecting efforts toward evolutionary units, regardless of phenotype, may more effectively preserve adaptive potential.

Mechanisms of cnidocyte development in the moon jellyfish Aurelia.

Stinging cells called cnidocytes are a defining trait of the cnidarians (sea anemones, corals, jellyfish, and their relatives). In hydrozoan cnidarians such as Hydra, cnidocytes develop from interstitial stem cells set aside in the ectoderm. It is less clear how cnidocytes develop outside the Hydrozoa, as other cnidarians appear to lack interstitial stem cells. We addressed this question by studying cnidogenesis in the moon jellyfish (Aurelia) through the visualization of minicollagen-a protein associated with cnidocyte development-as well as transmission electron microscopy. We discovered that developing cnidoblasts are rare or absent in feeding structures rich in mature cnidocytes, such as tentacles and lappets. Using transmission electron microscopy, we determined that the progenitors of cnidocytes have traits consistent with epitheliomuscular cells. Our data suggests a dynamic where cnidocytes develop at high concentrations in the epithelium of more proximal regions, and subsequently migrate to more distal regions where they exhibit high usage and turnover. Similar to some anthozoans, cnidocytes in Aurelia do not appear to be generated by interstitial stem cells; instead, epitheliomuscular cells appear to be the progenitor cell type. This observation polarizes the evolution of cnidogenesis, and raises the question of how interstitial stem cells came to regulate cnidogenesis in hydrozoans.

How sea level change mediates genetic divergence in coastal species across regions with varying tectonic and sediment processes.

Plate tectonics and sediment processes control regional continental shelf topography. We examine the genetic consequences of how glacial-associated sea level change interacted with variable nearshore topography since the last glaciation. We reconstructed the size and distribution of areas suitable for tidal estuary formation from the last glacial maximum, ~20 thousand years ago, to present from San Francisco, California, USA (~38°N) to Reforma, Sinaloa, Mexico (~25°N). We assessed range-wide genetic structure and diversity of three codistributed tidal estuarine fishes (California Killifish, Shadow Goby, Longjaw Mudsucker) along ~4,600 km using mitochondrial control region and cytB sequence, and 16-20 microsatellite loci from a total of 524 individuals. Results show that glacial-associated sea level change limited estuarine habitat to few, widely separated refugia at glacial lowstand, and present-day genetic clades were sourced from specific refugia. Habitat increased during postglacial sea level rise and refugial populations admixed in newly formed habitats. Continental shelves with active tectonics and/or low sediment supply were steep and hosted fewer, smaller refugia with more genetically differentiated populations than on broader shelves. Approximate Bayesian computation favoured the refuge-recolonization scenarios from habitat models over isolation by distance and seaway alternatives, indicating isolation at lowstand is a major diversification mechanism among these estuarine (and perhaps other) coastal species. Because sea level change is a global phenomenon, we suggest this top-down physical control of extirpation-isolation-recolonization may be an important driver of genetic diversification in coastal taxa inhabiting other topographically complex coasts globally during the Mid- to Late Pleistocene and deeper timescales.

Mitogenomics supports an unexpected taxonomic relationship for the extinct diving duck Chendytes lawi and definitively places the extinct Labrador Duck.

Chendytes lawi, an extinct flightless diving anseriform from coastal California, was traditionally classified as a sea duck, tribe Mergini, based on similarities in osteological characters. We recover and analyze mitochondrial genomes of C. lawi and five additional Mergini species, including the extinct Labrador Duck, Camptorhynchus labradorius. Despite its diving morphology, C. lawi is reconstructed as an ancient relictual lineage basal to the dabbling ducks (tribe Anatini), revealing an additional example of convergent evolution of characters related to feeding behavior among ducks. The Labrador Duck is sister to Steller's Eider which may provide insights into the evolution and ecology of this poorly known extinct species. Our results demonstrate that inclusion of full length mitogenomes, from taxonomically distributed ancient and modern sources can improve phylogeny reconstruction of groups previously assessed with shorter single-gene mitochondrial sequences.

Cell tracking supports secondary gastrulation in the moon jellyfish Aurelia.

The moon jellyfish Aurelia exhibits a dramatic reorganization of tissue during its metamorphosis from planula larva to polyp. There are currently two competing hypotheses regarding the fate of embryonic germ layers during this metamorphosis. In one scenario, the original endoderm undergoes apoptosis and is replaced by a secondary endoderm derived from ectodermal cells. In the second scenario, both ectoderm and endoderm remain intact through development. In this study, we performed a pulse-chase experiment to trace the fate of larval ectodermal cells. We observed that prior to metamorphosis, ectodermal cells that proliferated early in larval development concentrate at the future oral end of the polyp. During metamorphosis, these cells migrate into the endoderm, extending all the way to the aboral portion of the gut. We therefore reject the hypothesis that larval endoderm remains intact during metamorphosis and provide additional support for the "secondary gastrulation" hypothesis. Aurelia appears to offer the first and only described case where a cnidarian derives its endoderm twice during normal development, adding to a growing body of evidence that germ layers can be dramatically reorganized in cnidarian life cycles.

Sea-level driven glacial-age refugia and post-glacial mixing on subtropical coasts, a palaeohabitat and genetic study.

Using a novel combination of palaeohabitat modelling and genetic mixture analyses, we identify and assess a sea-level-driven recolonization process following the Last Glacial Maximum (LGM). Our palaeohabitat modelling reveals dramatic changes in estuarine habitat distribution along the coast of California (USA) and Baja California (Mexico). At the LGM (approx. 20 kya), when sea level was approximately 130 m lower, the palaeo-shoreline was too steep for tidal estuarine habitat formation, eliminating this habitat type from regions where it is currently most abundant, and limiting such estuaries to a northern and a southern refugium separated by 1000 km. We assess the recolonization of estuaries formed during post-LGM sea-level rise through examination of refugium-associated alleles and approximate Bayesian computation in three species of estuarine fishes. Results reveal sourcing of modern populations from both refugia, which admix in the newly formed habitat between the refuges. We infer a dramatic peak in habitat area between 15 and 10 kya with subsequent decline. Overall, this approach revealed a previously undocumented dynamic and integrated relationship between sea-level change, coastal processes and population genetics. These results extend glacial refugial dynamics to unglaciated subtropical coasts and have significant implications for biotic response to predicted sea-level rise.

Benefits of commercial weight-loss programs on blood pressure and lipids: a systematic review.

Our objective was to compare the effect of commercial weight-loss programs on blood pressure and lipids to control/education or counseling among individuals with overweight/obesity. We conducted a systematic review by searching MEDLINE and Cochrane Database of Systematic Reviews from inception to November 2014 and references identified by the programs. We included randomized, controlled trials ≥12weeks in duration. Two reviewers extracted information on study design, interventions, and mean change in systolic blood pressure (SBP), diastolic blood pressure (DBP), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), triglycerides, and total cholesterol and assessed risk of bias. We included 27 trials. Participants' blood pressure and lipids were normal at baseline in most trials. At 12months, Weight Watchers showed little change in blood pressure or lipid outcomes as compared to control/education (2 trials). At 12months, Atkins' participants had higher HDL-c and lower triglycerides than counseling (4 trials). Other programs had inconsistent effects or lacked long-term studies. Risk of bias was high for most trials of all programs. In conclusion, limited data exist regarding most commercial weight-loss programs' long-term effects on blood pressure and lipids. Clinicians should be aware that Weight Watchers has limited data that demonstrate CVD risk factor benefits relative to control/education. Atkins may be a reasonable option for patients with dyslipidemia. Additional well-designed, long-term trials are needed to confirm these conclusions and evaluate other commercial programs.

Direct comparisons of commercial weight-loss programs on weight, waist circumference, and blood pressure: a systematic review.

BACKGROUND: Obesity is common in the U.S. and many individuals turn to commercial programs to lose weight. Our objective was to directly compare weight loss, waist circumference, and systolic and diastolic blood pressure (SBP, DBP) outcomes between commercially available weight-loss programs. METHODS: We conducted a systematic review by searching MEDLINE and the Cochrane Database of Systematic Reviews from inception to November 2014 and by using references identified by commercial programs. We included randomized, controlled trials (RCTs) of at least 12 weeks duration that reported comparisons with other commercial weight-loss programs. Two reviewers extracted information on mean change in weight, waist circumference, SBP and DBP and assessed risk of bias. RESULTS: We included seven articles representing three RCTs. Curves participants lost 1.8 kg (95%CI: 0.1, 3.5 kg) more than Weight Watchers in one comparison. There was no statistically significant difference in waist circumference change among the included programs. The mean reduction in SBP for SlimFast participants was 4.5 mmHg (95%CI: 0.4, 8.6 mmHg) more than that of Atkins participants in one comparison. There was no significant difference in mean DBP changes among programs. CONCLUSIONS: There is limited evidence that any one of the commercial weight-loss programs has superior results for mean weight change, mean waist circumference change, or mean blood pressure change.

Efficacy of commercial weight-loss programs: an updated systematic review.

BACKGROUND: Commercial and proprietary weight-loss programs are popular obesity treatment options, but their efficacy is unclear. PURPOSE: To compare weight loss, adherence, and harms of commercial or proprietary weight-loss programs versus control/education (no intervention, printed materials only, health education curriculum, or <3 sessions with a provider) or behavioral counseling among overweight and obese adults. DATA SOURCES: MEDLINE and the Cochrane Database of Systematic Reviews from inception to November 2014; references identified by program staff. STUDY SELECTION: Randomized, controlled trials (RCTs) of at least 12 weeks' duration; prospective case series of at least 12 months' duration (harms only). DATA EXTRACTION: Two reviewers extracted information on study design, population characteristics, interventions, and mean percentage of weight change and assessed risk of bias. DATA SYNTHESIS: We included 45 studies, 39 of which were RCTs. At 12 months, Weight Watchers participants achieved at least 2.6% greater weight loss than those assigned to control/education. Jenny Craig resulted in at least 4.9% greater weight loss at 12 months than control/education and counseling. Nutrisystem resulted in at least 3.8% greater weight loss at 3 months than control/education and counseling. Very-low-calorie programs (Health Management Resources, Medifast, and OPTIFAST) resulted in at least 4.0% greater short-term weight loss than counseling, but some attenuation of effect occurred beyond 6 months when reported. Atkins resulted in 0.1% to 2.9% greater weight loss at 12 months than counseling. Results for SlimFast were mixed. We found limited evidence to evaluate adherence or harms for all programs and weight outcomes for other commercial programs. LIMITATION: Many trials were short (<12 months), had high attrition, and lacked blinding. CONCLUSION: Clinicians could consider referring overweight or obese patients to Weight Watchers or Jenny Craig. Other popular programs, such as Nutrisystem, show promising weight-loss results; however, additional studies evaluating long-term outcomes are needed. PRIMARY FUNDING SOURCE: None. ( PROSPERO: CRD4201-4007155).

The early expansion and evolutionary dynamics of POU class genes.

The POU genes represent a diverse class of animal-specific transcription factors that play important roles in neurogenesis, pluripotency, and cell-type specification. Although previous attempts have been made to reconstruct the evolution of the POU class, these studies have been limited by a small number of representative taxa, and a lack of sequences from basally branching organisms. In this study, we performed comparative analyses on available genomes and sequences recovered through "gene fishing" to better resolve the topology of the POU gene tree. We then used ancestral state reconstruction to map the most likely changes in amino acid evolution for the conserved domains. Our work suggests that four of the six POU families evolved before the last common ancestor of living animals-doubling previous estimates-and were followed by extensive clade-specific gene loss. Amino acid changes are distributed unequally across the gene tree, consistent with a neofunctionalization model of protein evolution. We consider our results in the context of early animal evolution, and the role of POU5 genes in maintaining stem cell pluripotency.

Ancestral state reconstruction of ontogeny supports a bilaterian affinity for Dickinsonia.

Despite numerous attempts, classification of the Precambrian fossil Dickinsonia has eluded scientific consensus. This is largely because Dickinsonia and its relatives are structurally simple, lacking morphological synapomorphies to clarify their relationship to modern taxa. However, there is increasing precedence for using ontogeny to constrain enigmatic fossils, and growth of the type species Dickinsonia costata is well understood. This study formalizes the connection between ontogeny in Dickinsonia-which grows by the addition of metameric units onto one end of its primary axis-with terminal addition, defined as growth and patterning from a posterior, subtermial growth zone. We employ ancestral state reconstruction and stochastic character mapping to conclude that terminal addition is a synapomorphy of bilaterian animals. Thus, terminal addition allies Dickinsonia with the bilaterians, providing evidence that large stem- or crown-group bilaterians made up a significant proportion of the Precambrian biota. This study also illustrates the potential for combining developmental and phylogenetic data in constraining the placement of ancient problematic fossil taxa on the evolutionary tree.

Temporal genetic analysis of the endangered tidewater goby: extinction-colonization dynamics or drift in isolation?

Extinction and colonization dynamics are critical to understanding the evolution and conservation of metapopulations. However, traditional field studies of extinction-colonization are potentially fraught with detection bias and have rarely been validated. Here, we provide a comparison of molecular and field-based approaches for assessment of the extinction-colonization dynamics of tidewater goby (Eucyclogobius newberryi) in northern California. Our analysis of temporal genetic variation across 14 northern California tidewater goby populations failed to recover genetic change expected with extinction-colonization cycles. Similarly, analysis of site occupancy data from field studies (94 sites) indicated that extinction and colonization are very infrequent for our study populations. Comparison of the approaches indicated field data were subject to imperfect detection, and falsely implied extinction-colonization cycles in several instances. For northern California populations of tidewater goby, we interpret the strong genetic differentiation between populations and high degree of within-site temporal stability as consistent with a model of drift in the absence of migration, at least over the past 20-30 years. Our findings show that tidewater goby exhibit different population structures across their geographic range (extinction-colonization dynamics in the south vs. drift in isolation in the north). For northern populations, natural dispersal is too infrequent to be considered a viable approach for recolonizing extirpated populations, suggesting that species recovery will likely depend on artificial translocation in this region. More broadly, this work illustrates that temporal genetic analysis can be used in combination with field data to strengthen inference of extinction-colonization dynamics or as a stand-alone tool when field data are lacking.

Convergent evolution of ecomorphological adaptations in geographically isolated Bay gobies (Teleostei: Gobionellidae) of the temperate North Pacific.

North Pacific Bay gobies (Teleostei: Gobioidei: Gobionellidae) inhabit bays, beaches, coastal lagoons, and estuaries of temperate Asia and North America, but are absent from the boreal northernmost Pacific. Previously, morphological characters conventionally subdivided the clade into two groups - an elongate-bodied, infaunal-inhabiting "Astrabe" group, and a deeper-bodied, non-infaunal "Chasmichthys" group - each with a disjunct East-West (amphi-) Pacific distribution. Here we use mitochondrial and multi-locus nuclear DNA sequence data to show that several morphological characters previously used to delimit these two groups have in fact arisen independently on both sides of the Pacific, revealing convergence of ecologically adaptive characters within a geographically divided clade. Basal divergence of the resultant tree coincides with a dramatic global cooling event at the Eocene/Oligocene transition, without evidence of subsequent trans-Pacific migration. A novel approach to partitioning sequence data by relative rate, as opposed to traditional gene/codon position partitioning, was used to help distinguish phylogenetic signal from noise on a per-site basis. Resulting improvements in topology and nodal support, along with decreased computational effort, suggest that this partitioning strategy may be useful for future studies in phylogenetics and phylogenomics.

Stem cell dynamics in Cnidaria: are there unifying principles?

The study of stem cells in cnidarians has a history spanning hundreds of years, but it has primarily focused on the hydrozoan genus Hydra. While Hydra has a number of self-renewing cell types that act much like stem cells--in particular the interstitial cell line--finding cellular homologues outside of the Hydrozoa has been complicated by the morphological simplicity of stem cells and inconclusive gene expression data. In non-hydrozoan cnidarians, an enigmatic cell type known as the amoebocyte might play a similar role to interstitial cells, but there is little evidence that I-cells and amoebocytes are homologous. Instead, self-renewal and transdifferentiation of epithelial cells was probably more important to ancestral cnidarian development than any undifferentiated cell lineage, and only later in evolution did one or more cell types come under the regulation of a "stem" cell line. Ultimately, this hypothesis and competing ones will need to be tested by expanding genetic and developmental studies on a variety of cnidarian model systems.

Flying shells: historical dispersal of marine snails across Central America.

The geological rise of the Central American Isthmus separated the Pacific and the Atlantic oceans about 3 Ma, creating a formidable barrier to dispersal for marine species. However, similar to Simpson's proposal that terrestrial species can 'win sweepstakes routes'-whereby highly improbable dispersal events result in colonization across geographical barriers-marine species may also breach land barriers given enough time. To test this hypothesis, we asked whether intertidal marine snails have crossed Central America to successfully establish in new ocean basins. We used a mitochondrial DNA genetic comparison of sister snails (Cerithideopsis spp.) separated by the rise of the Isthmus. Genetic variation in these snails revealed evidence of at least two successful dispersal events between the Pacific and the Atlantic after the final closure of the Isthmus. A combination of ancestral area analyses and molecular dating techniques indicated that dispersal from the Pacific to the Atlantic occurred about 750 000 years ago and that dispersal in the opposite direction occurred about 72 000 years ago. The geographical distribution of haplotypes and published field evidence further suggest that migratory shorebirds transported the snails across Central America at the Isthmus of Tehuantepec in southern Mexico. Migratory birds could disperse other intertidal invertebrates this way, suggesting the Central American Isthmus may not be as impassable for marine species as previously assumed.

Evolutionary origin of rhopalia: insights from cellular-level analyses of Otx and POU expression patterns in the developing rhopalial nervous system.

In Cnidaria, the medusae of Scyphozoa and its sister-group Cubozoa uniquely possess rhopalia at their bell margin. These sensory centers coordinate behavior and development. We used fluorescent in situ hybridization and confocal microscopy to examine mRNA expression patterns in Aurelia sp.1 (Cnidaria, Scyphozoa) during early medusa formation, while simultaneously visualizing the developing nervous system by immunofluorescence. The genes investigated include AurOtx1, and the POU genes, AurPit1, and AurBrn3, homologs of genes known to function in cephalar neural organization and sensory cell differentiation across Bilateria. Our results show that AurOtx1 expression defines the major part of the oral neuroectodermal domain of the rhopalium, within which distinct populations of AurBrn3- and AurPit1-expressing sensory cells develop. Thus, despite the unique attributes of rhopalial evolution, we suggest that the rhopalial nervous system of scyphozoan medusae involves similar patterns of differential expression of genes that function in bilaterian cephalic structure and neuroendocrine system development. We propose that rhopalia evolved from preexisting sensory structures that developed distinct populations of sensory cells differentially expressing POU genes within Otx oral-neuroectodermal domains. This implies some commonality of developmental genetic functions involving these genes in the still poorly constrained common ancestor of bilaterians and cnidarians.

Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria.

The similarity in the genetic regulation of arthropod and vertebrate appendage formation has been interpreted as the product of a plesiomorphic gene network that was primitively involved in bilaterian appendage development and co-opted to build appendages (in modern phyla) that are not historically related as structures. Data from lophotrochozoans are needed to clarify the pervasiveness of plesiomorphic appendage-forming mechanisms. We assayed the expression of three arthropod and vertebrate limb gene orthologs, Distal-less (Dll), dachshund (dac), and optomotor blind (omb), in direct-developing juveniles of the polychaete Neanthes arenaceodentata. Parapodial Dll expression marks pre-morphogenetic notopodia and neuropodia, becoming restricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appendages, Dll activity is primarily restricted to proximal domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a pair of segmental nerves, and in newly differentiating mesoderm. Domains of omb expression include the brain, nerve cord ganglia, one pair of anterior cirri, presumed precursors of dorsal musculature, and the same pharyngeal ganglia and presumed interneurons that express dac. Contrary to their roles in outgrowing arthropod and vertebrate appendages, Dll, dac, and omb lack comparable expression in Neanthes appendages, implying independent evolution of annelid appendage development. We infer that parapodia and arthropodia are not structurally or mechanistically homologous (but their primordia might be), that Dll's ancestral bilaterian function was in sensory and central nervous system differentiation, and that locomotory appendages possibly evolved from sensory outgrowths.

Rampant drift in artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi).

Habitat fragmentation and its genetic consequences are a critically important issue in evaluating the evolutionary penalties of human habitat modification. Here, we examine the genetic structure and diversity in naturally subdivided and artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi), a small fish restricted to discrete coastal lagoons and estuaries in California, USA. We use five naturally fragmented coastal populations from a 300- km spatial scale as a standard to assess migration and drift relative to eight artificially fragmented bay populations from a 30- km spatial scale. Using nine microsatellite loci in 621 individuals, and a 522-base fragment of mitochondrial DNA control region from 103 individuals, we found striking differences in the relative influences of migration and drift on genetic variation at these two scales. Overall, the artificially fragmented populations exhibited a consistent pattern of higher genetic differentiation and significantly lower genetic diversity relative to the naturally fragmented populations. Thus, even in a species characterized by habitat isolation and subdivision, further artificial fragmentation appears to result in substantial population genetic consequences and may not be sustainable.

Confocal analysis of nervous system architecture in direct-developing juveniles of Neanthes arenaceodentata (Annelida, Nereididae).

BACKGROUND: Members of Family Nereididae have complex neural morphology exemplary of errant polychaetes and are leading research models in the investigation of annelid nervous systems. However, few studies focus on the development of their nervous system morphology. Such data are particularly relevant today, as nereidids are the subjects of a growing body of "evo-devo" work concerning bilaterian nervous systems, and detailed knowledge of their developing neuroanatomy facilitates the interpretation of gene expression analyses. In addition, new data are needed to resolve discrepancies between classic studies of nereidid neuroanatomy. We present a neuroanatomical overview based on acetylated alpha-tubulin labeling and confocal microscopy for post-embryonic stages of Neanthes arenaceodentata, a direct-developing nereidid. RESULTS: At hatching (2-3 chaetigers), the nervous system has developed much of the complexity of the adult (large brain, circumesophageal connectives, nerve cords, segmental nerves), and the stomatogastric nervous system is partially formed. By the 5-chaetiger stage, the cephalic appendages and anal cirri are well innervated and have clear connections to the central nervous system. Within one week of hatching (9-chaetigers), cephalic sensory structures (e.g., nuchal organs, Langdon's organs) and brain substructures (e.g., corpora pedunculata, stomatogastric ganglia) are clearly differentiated. Additionally, the segmental-nerve architecture (including interconnections) matches descriptions of other, adult nereidids, and the pharynx has developed longitudinal nerves, nerve rings, and ganglia. All central roots of the stomatogastric nervous system are distinguishable in 12-chaetiger juveniles. Evidence was also found for two previously undescribed peripheral nerve interconnections and aspects of parapodial muscle innervation. CONCLUSIONS: N. arenaceodentata has apparently lost all essential trochophore characteristics typical of nereidids. Relative to the polychaete Capitella, brain separation from a distinct epidermis occurs later in N. arenaceodentata, indicating different mechanisms of prostomial development. Our observations of parapodial innervation and the absence of lateral nerves in N. arenaceodentata are similar to a 19th century study of Alitta virens (formerly Nereis/Neanthes virens) but contrast with a more recent study that describes a single parapodial nerve pattern and lateral nerve presence in A. virens and two other genera. The latter study apparently does not account for among-nereidid variation in these major neural features.

First sequencing of ancient coral skeletal proteins.

Here we report the first recovery, sequencing, and identification of fossil biomineral proteins from a Pleistocene fossil invertebrate, the stony coral Orbicella annularis. This fossil retains total hydrolysable amino acids of a roughly similar composition to extracts from modern O. annularis skeletons, with the amino acid data rich in Asx (Asp + Asn) and Glx (Glu + Gln) typical of invertebrate skeletal proteins. It also retains several proteins, including a highly acidic protein, also known from modern coral skeletal proteomes that we sequenced by LC-MS/MS over multiple trials in the best-preserved fossil coral specimen. A combination of degradation or amino acid racemization inhibition of trypsin digestion appears to limit greater recovery. Nevertheless, our workflow determines optimal samples for effective sequencing of fossil coral proteins, allowing comparison of modern and fossil invertebrate protein sequences, and will likely lead to further improvements of the methods. Sequencing of endogenous organic molecules in fossil invertebrate biominerals provides an ancient record of composition, potentially clarifying evolutionary changes and biotic responses to paleoenvironments.