MicroCT illuminates the unique morphology of Shiinoidae (Copepoda: Cyclopoida), an unusual group of fish parasites.

The copepod family Shiinoidae Cressey, 1975 currently comprises nine species of teleost parasites with unusual morphology and a unique attachment mechanism. Female shiinoids possess greatly enlarged antennae that oppose a rostrum, an elongate outgrowth of cuticle that originates between the antennules. The antennae form a moveable clasp against the rostrum which they use to attach to their host. In this study, we use micro-computed tomography (microCT) to examine specimens of Shiinoa inauris Cressey, 1975 in situ attached to host tissue in order to characterize the functional morphology and specific muscles involved in this novel mode of attachment and to resolve uncertainty regarding the segmental composition of the regions of the body. We review the host and locality data for all reports of shiinoids, revise the generic diagnoses for both constituent genera Shiinoa Kabata, 1968 and Parashiinoa West, 1986, transfer Shiinoa rostrata Balaraman, Prabha & Pillai, 1984 to Parashiinoa as Parashiinoa rostrata (Balaraman, Prabha & Pillai, 1984) n. comb., and present keys to the females and males of both genera.

Ultra-black Camouflage in Deep-Sea Fishes.

At oceanic depths >200 m, there is little ambient sunlight, but bioluminescent organisms provide another light source that can reveal animals to visual predators and prey [1-4]. Transparency and mirrored surfaces-common camouflage strategies under the diffuse solar illumination of shallower waters-are conspicuous when illuminated by directed bioluminescent sources due to reflection from the body surface [5, 6]. Pigmentation allows animals to absorb light from bioluminescent sources, rendering them visually undetectable against the dark background of the deep sea [5]. We present evidence suggesting pressure to reduce reflected bioluminescence led to the evolution of ultra-black skin (reflectance <0.5%) in 16 species of deep-sea fishes across seven distantly related orders. Histological data suggest this low reflectance is mediated by a continuous layer of densely packed melanosomes in the exterior-most layer of the dermis [7, 8] and that this layer lacks the unpigmented gaps between pigment cells found in other darkly colored fishes [9-13]. Using finite-difference, time-domain modeling and comparisons with melanosomes found in other ectothermic vertebrates [11, 13-21], we find the melanosomes making up the layer in these ultra-black species are optimized in size and shape to minimize reflectance. Low reflectance results from melanosomes scattering light within the layer, increasing the optical path length and therefore light absorption by the melanin. By reducing reflectance, ultra-black fish can reduce the sighting distance of visual predators more than 6-fold compared to fish with 2% reflectance. This biological example of efficient light absorption via a simple architecture of strongly absorbing and highly scattering particles may inspire new ultra-black materials.

Erratum: Author Correction: Biodiversity estimates and ecological interpretations of meiofaunal communities are biased by the taxonomic approach.

[This corrects the article DOI: 10.1038/s42003-018-0119-2.].

Biodiversity estimates and ecological interpretations of meiofaunal communities are biased by the taxonomic approach.

Accurate assessments of biodiversity are crucial to advising ecosystem-monitoring programs and understanding ecosystem function. Nevertheless, a standard operating procedure to assess biodiversity accurately and consistently has not been established. This is especially true for meiofauna, a diverse community (>20 phyla) of small benthic invertebrates that have fundamental ecological roles. Recent studies show that metabarcoding is a cost-effective and time-effective method to estimate meiofauna biodiversity, in contrast to morphological-based taxonomy. Here, we compare biodiversity assessments of a diverse meiofaunal community derived by applying multiple taxonomic methods based on comparative morphology, molecular phylogenetic analysis, DNA barcoding of individual specimens, and metabarcoding of environmental DNA. We show that biodiversity estimates are strongly biased across taxonomic methods and phyla. Such biases affect understanding of community structures and ecological interpretations. This study supports the urgency of improving aspects of environmental high-throughput sequencing and the value of taxonomists in correctly understanding biodiversity estimates.

Improved phylogenetic resolution within Siphonophora (Cnidaria) with implications for trait evolution.

Siphonophores are a diverse group of hydrozoans (Cnidaria) that are found at most depths of the ocean - from the surface, like the familiar Portuguese man of war, to the deep sea. They play important roles in ocean ecosystems, and are among the most abundant gelatinous predators. A previous phylogenetic study based on two ribosomal RNA genes provided insight into the internal relationships between major siphonophore groups. There was, however, little support for many deep relationships within the clade Codonophora. Here, we present a new siphonophore phylogeny based on new transcriptome data from 29 siphonophore species analyzed in combination with 14 publicly available genomic and transcriptomic datasets. We use this new phylogeny to reconstruct several traits that are central to siphonophore biology, including sexual system (monoecy vs. dioecy), gain and loss of zooid types, life history traits, and habitat. The phylogenetic relationships in this study are largely consistent with the previous phylogeny, but we find strong support for new clades within Codonophora that were previously unresolved. These results have important implications for trait evolution within Siphonophora, including favoring the hypothesis that monoecy arose at least twice.

Phylogenomic Analyses Support Traditional Relationships within Cnidaria.

Cnidaria, the sister group to Bilateria, is a highly diverse group of animals in terms of morphology, lifecycles, ecology, and development. How this diversity originated and evolved is not well understood because phylogenetic relationships among major cnidarian lineages are unclear, and recent studies present contrasting phylogenetic hypotheses. Here, we use transcriptome data from 15 newly-sequenced species in combination with 26 publicly available genomes and transcriptomes to assess phylogenetic relationships among major cnidarian lineages. Phylogenetic analyses using different partition schemes and models of molecular evolution, as well as topology tests for alternative phylogenetic relationships, support the monophyly of Medusozoa, Anthozoa, Octocorallia, Hydrozoa, and a clade consisting of Staurozoa, Cubozoa, and Scyphozoa. Support for the monophyly of Hexacorallia is weak due to the equivocal position of Ceriantharia. Taken together, these results further resolve deep cnidarian relationships, largely support traditional phylogenetic views on relationships, and provide a historical framework for studying the evolutionary processes involved in one of the most ancient animal radiations.

Spiralian phylogeny informs the evolution of microscopic lineages.

Despite rapid advances in the study of metazoan evolutionary history [1], phylogenomic analyses have so far neglected a number of microscopic lineages that possess a unique combination of characters and are thus informative for our understanding of morphological evolution. Chief among these lineages are the recently described animal groups Micrognathozoa and Loricifera, as well as the two interstitial "Problematica" Diurodrilus and Lobatocerebrum [2]. These genera show a certain resemblance to Annelida in their cuticle and gut [3, 4]; however, both lack primary annelid characters such as segmentation and chaetae [5]. Moreover, they show unique features such as an inverted body-wall musculature or a novel pharyngeal organ. This and their ciliated epidermis have led some to propose relationships with other microscopic spiralians, namely Platyhelminthes, Gastrotricha, and in the case of Diurodrilus, with Micrognathozoa [6, 7]-lineages that are grouped by some analyses into "Platyzoa," a clade whose status remains uncertain [1, 8-11]. Here, we assess the interrelationships among the meiofaunal and macrofaunal members of Spiralia using 402 orthologs mined from genome and transcriptome assemblies of 90 taxa. Lobatocerebrum and Diurodrilus are found to be deeply nested members of Annelida, and unequivocal support is found for Micrognathozoa as the sister group of Rotifera. Analyses using site-heterogeneous substitution models further recover a lophophorate clade and position Loricifera + Priapulida as sister group to the remaining Ecdysozoa. Finally, with several meiofaunal lineages branching off early in the diversification of Spiralia, the emerging concept of a microscopic, acoelomate, direct-developing ancestor of Spiralia is reviewed.

Stem cells in Nanomia bijuga (Siphonophora), a colonial animal with localized growth zones.

BACKGROUND: Siphonophores (Hydrozoa) have unparalleled colony-level complexity, precision of colony organization, and functional specialization between zooids (i.e., the units that make up colonies). Previous work has shown that, unlike other colonial animals, most growth in siphonophores is restricted to one or two well-defined growth zones that are the sites of both elongation and zooid budding. It remained unknown, however, how this unique colony growth and development is realized at the cellular level. RESULTS: To understand the colony-level growth and development of siphonophores at the cellular level, we characterize the distribution of proliferating cells and interstitial stem cells (i-cells) in the siphonophore Nanomia bijuga. Within the colony, we find evidence that i-cells are present at the tip of the horn, the structure within the growth zone that gives rise to new zooids. Co-localized gene expression of vasa-1, pl10, piwi, nanos-1, and nanos-2 suggests that i-cells persist in the youngest zooid buds and that i-cells become progressively restricted to specific regions within the zooids until they are mostly absent from the oldest zooids. The examined genes remain expressed in gametogenic regions. No evidence for i-cells is found in the stem between maturing zooids. Domains of high cell proliferation include regions where the examined genes are expressed, but also include some areas in which the examined genes were not expressed such as the stem within the growth zones. Cell proliferation in regions devoid of vasa-1, pl10, piwi, nanos-1, and nanos-2 expression indicates the presence of mitotically active epithelial cell lineages and, potentially, progenitor cell populations. CONCLUSIONS: We provide the first evidence for i-cells in a siphonophore. Our findings suggest maintenance of i-cell populations at the sites of growth zones and that these sites are the main source of i-cells. This restriction of stem cells to particular regions in the colony, in combination with localized budding and spatial patterning during pro-bud subdivision, may play a major role in facilitating the precision of siphonophore growth. Spatially restricted maintenance of i-cells in mature zooids and absence of i-cells along the stem may explain the reduced developmental plasticity in older parts of the colony.

Phylogenomic analyses of deep gastropod relationships reject Orthogastropoda.

Gastropods are a highly diverse clade of molluscs that includes many familiar animals, such as limpets, snails, slugs and sea slugs. It is one of the most abundant groups of animals in the sea and the only molluscan lineage that has successfully colonized land. Yet the relationships among and within its constituent clades have remained in flux for over a century of morphological, anatomical and molecular study. Here, we re-evaluate gastropod phylogenetic relationships by collecting new transcriptome data for 40 species and analysing them in combination with publicly available genomes and transcriptomes. Our datasets include all five main gastropod clades: Patellogastropoda, Vetigastropoda, Neritimorpha, Caenogastropoda and Heterobranchia. We use two different methods to assign orthology, subsample each of these matrices into three increasingly dense subsets, and analyse all six of these supermatrices with two different models of molecular evolution. All 12 analyses yield the same unrooted network connecting the five major gastropod lineages. This reduces deep gastropod phylogeny to three alternative rooting hypotheses. These results reject the prevalent hypothesis of gastropod phylogeny, Orthogastropoda. Our dated tree is congruent with a possible end-Permian recovery of some gastropod clades, namely Caenogastropoda and some Heterobranchia subclades.

Resolving the evolutionary relationships of molluscs with phylogenomic tools.

Molluscs (snails, octopuses, clams and their relatives) have a great disparity of body plans and, among the animals, only arthropods surpass them in species number. This diversity has made Mollusca one of the best-studied groups of animals, yet their evolutionary relationships remain poorly resolved. Open questions have important implications for the origin of Mollusca and for morphological evolution within the group. These questions include whether the shell-less, vermiform aplacophoran molluscs diverged before the origin of the shelled molluscs (Conchifera) or lost their shells secondarily. Monoplacophorans were not included in molecular studies until recently, when it was proposed that they constitute a clade named Serialia together with Polyplacophora (chitons), reflecting the serial repetition of body organs in both groups. Attempts to understand the early evolution of molluscs become even more complex when considering the large diversity of Cambrian fossils. These can have multiple dorsal shell plates and sclerites or can be shell-less but with a typical molluscan radula and serially repeated gills. To better resolve the relationships among molluscs, we generated transcriptome data for 15 species that, in combination with existing data, represent for the first time all major molluscan groups. We analysed multiple data sets containing up to 216,402 sites and 1,185 gene regions using multiple models and methods. Our results support the clade Aculifera, containing the three molluscan groups with spicules but without true shells, and they support the monophyly of Conchifera. Monoplacophora is not the sister group to other Conchifera but to Cephalopoda. Strong support is found for a clade that comprises Scaphopoda (tusk shells), Gastropoda and Bivalvia, with most analyses placing Scaphopoda and Gastropoda as sister groups. This well-resolved tree will constitute a framework for further studies of mollusc evolution, development and anatomy.

Differential gene expression in the siphonophore Nanomia bijuga (Cnidaria) assessed with multiple next-generation sequencing workflows.

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Bioluminescent organs of two deep-sea arrow worms, Eukrohnia fowleri and Caecosagitta macrocephala, with further observations on Bioluminescence in chaetognaths.

Bioluminescence in the deep-sea chaetognath Eukrohnia fowleri is reported for the first time, and behavioral, morphological, and chemical characteristics of bioluminescence in chaetognaths are examined. Until this study, the only known species of bioluminescent chaetognath was Caecosagitta macrocephala. The luminescent organ of that species is located on the ventral edge of each anterior lateral fin, whereas that of E. fowleri runs across the center of the tail fin on both dorsal and ventral sides. Scanning electron microscopy showed that the bioluminescent organs of both species consist of hexagonal chambers containing elongate ovoid particles-the organelles holding bioluminescent materials. No other luminous organism is known to use hexagonal packing to hold bioluminescent materials. Transmission electron microscopy of particles from C. macrocephala revealed a densely packed paracrystalline matrix punctuated by globular inclusions, which likely correspond to luciferin and luciferase, respectively. Both species use unique luciferases in conjunction with coelenterazine for light emission. Luciferase of C. macrocephala becomes inactive after 30 min, but luciferase of E. fowleri is highly stable. Although C. macrocephala has about 90 times fewer particles than E. fowleri, it has a similar bioluminescent capacity (total particle volume) due to its larger particle size. In situ observations of C. macrocephala from a remotely operated vehicle revealed that the luminous particles are released to form a cloud. The discovery of bioluminescence in a second chaetognath phylogenetically distant from the first highlights the importance of bioluminescence among deep-sea organisms.