Class Ophiuroidea Research Articles

Development of two new sets of PCR primers for eDNA metabarcoding of brittle stars (Echinodermata, Ophiuroidea)

Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2,100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required.

Shallow- and Deep-Water Ophiura Species Produce a Panel of Chlorin Compounds with Potent Photodynamic Anticancer Activities.

A Pacific brittle star Ophiura sarsii has previously been shown to produce a chlorin (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) (1) with potent phototoxic activities, making it applicable to photodynamic therapy. Using extensive LC-MS metabolite profiling, molecular network analysis, and targeted isolation with de novo NMR structure elucidation, we herein identify five additional chlorin compounds from O. sarsii and its deep-sea relative O. ooplax: 10S-Hydroxypheophorbide a (2), Pheophorbide a (3), Pyropheophorbide a (4), (3S,4S,21R)-14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (5), and (3S,4S,21R)-14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (6). Chlorins 5 and 6 have not been previously reported in natural sources. Interestingly, low amounts of chlorins 1-4 and 6 could also be identified in a distant species, the basket star Gorgonocephalus cf. eucnemis, demonstrating that chlorins are produced by a wide spectrum of marine invertebrates of the class Ophiuroidea. Following the purification of these major Ophiura chlorin metabolites, we discovered the significant singlet oxygen quantum yield upon their photoinduction and the resulting phototoxicity against triple-negative breast cancer BT-20 cells. These studies identify an arsenal of brittle star chlorins as natural photosensitizers with potential photodynamic therapy applications.

New species, redescriptions and new records of deep-sea brittle stars (Echinodermata: Ophiuroidea) from the South China Sea, an integrated morphological and molecular approach

Deep-sea ophiuroids were collected by the manned submersible ‘Shenhaiyongshi’ from the South China Sea at depths of 500–3550 m, in 2017 to 2020. A total of 18 species were identified, including three new species and eight new records, increasing the total number of species known from the South China Sea to 304. Most of the ophiuroids recorded from the South China Sea were found in shallow waters (77.9%) and a few of them occurred only in deep water (20.4%). Three new species are described as Ophiacantha aster sp. nov., Ophiomoeris petalis sp. nov. and Ophiopristis shenhaiyongshii sp. nov. We provide comprehensive descriptions of morphological features, including characteristics of the arm skeletons, and a phylogenetic analysis based on COI and 16S sequences. Overall intraspecific and interspecific genetic distance variations among the families found in this study were 0.5% to 2.47% and 1.16% to 44.16%, respectively, along the South Pacific region to the South China Sea. Our phylogenetic analysis suggested that COI partial genes resolved the interspecies complexity in the class Ophiuroidea better than 16S partial genes. The order Euryalida had low interspecies genetic distance variation within the class Ophiuroidea. The present study suggests a high probability that species of Asteroschema and Gorgonocephalus are more widely spread around the Indo-Pacific region than previously expected.

Order Euryalida (Echinodermata, Ophiuroidea), new species and new records from the South China Sea and the Northwest Pacific seamounts.

Ophiuroids were collected by the manned submersible ‘Shenhaiyongshi’ from the deep-sea seamounts in the South China Sea and Northwest Pacific regions at 602–1920 m depth, during 2018 to 2020. A total of nine species was identified, including two new species and seven new records from the South China Sea and one new record from the Northwest Pacific region. Two new species are described as Asteroschemashenhaiyongshiisp. nov. and Asteroschemadomogranulatumsp. nov. The seven new records included five species from the genus Asteroschema, and one species each from the genera Asterostegus and Astrodendrum. Comprehensive descriptions of morphological features are provided, including characteristics of the arm skeleton, as well as a phylogenetic analysis based on 16S and COI sequences. Intraspecific genetic distance ranges of Euryalida species from the present study were 0.34% to 1.38%, which was relatively low compared to other orders in the class Ophiuroidea. The present study suggests a high probability that species of the order Euryalida are more widely spread around the Indo-Pacific region than previously expected.

A New Species of Ophiura (Echinodermata, Ophiuroidea) from Miocene Deep-Sea Deposits in the Pohang Basin, Korea

Dense aggregations of articulated ophiuroids, in external mould preservation, were discovered in the middle Miocene Duho Formation (Yeonil Group) of Pohang City, near the southeast coast of Korea. These specimens are here described as a new species, Ophiura pohangensis, which differs from congeners in having long, slender oral papillae, and tiny quadrangular arm comb papillae. The new species constitutes the first well-identified example of the class Ophiuroidea in the fossil record of Korea. Aggregation density of the new species is similar to that of occurrences of both fossil and extant Ophiura sarsii sarsii across Japan.

Diversity of Echinoderms in Intertidal Zone of Sundak Beach, Gunung Kidul, Yogyakarta, Indonesia

Indonesia is a maritime country with high diversity of marine biota, which one is from Phylum Echinoderms. Echinoderms are known as biota of marine which is are living on corals, sandy, and on the intertidal zone and deep ocean. The intertidal characteristics of Sundak Beach has a suitable structure to echinodems life because there are many corals, but there is many species has not been identified before. The purpose of this research is to know the diversity of echinoderms phylum on the intertidal zone. Sampling was carried out on August, 24th 2019 with purposive random sampling method along the coastal-line in intertidal zone. The result of this research are identified and classified on the each class. Based on the results, the are found species from Echinodea, Holothuroidea, and Ophiuroidea. Species from Echinoidea are Tripneustes gratilla, Echinometra mathaei, Heterocentrotus trigonarius, Diadema antillarum., and Echinothrix calamaris. Holothuroidea namely Holothuria atra. Ophiuroidea namely Ophiotrix fragilis, Ophiocoma scolopendrina, and Ophiocoma erinaceus. The conclusion from this study is the discovery of five species from class Echinoidea, one species from class Holothuroidea and three species from class Ophiuroidea.

The diversity of echinoderms in the seagrass meadows of Penang Island, Malaysia

Many studies had been done on the diversity of echinoderms in Malaysia, especially on holothuroids, but only a handful was conducted on seagrass meadows. The lack of studies conducted on echinoderms in seagrass meadows coupled with the steadily declining area of seagrass meadows represents a serious threat to the documentation of Malaysia’s echinoderm biodiversity. This study was conducted to provide a checklist of echinoderms found in the seagrass meadows of Penang Island. Four sampling trips were conducted from October 2019 to February 2020 and specimens were collected by hand during low tide in two sites, Pulau Gazumbo and Middle Bank. Additional specimens in the collection of Centre for Marine and Coastal Studies, Universiti Sains Malaysia (CEMACS, USM) were also included in this study. Ossicles of holothuroids were extracted, observed under a light microscope, and illustrated. A total of 31 specimens were examined and identified, comprised of 15 species: one from the class Asteroidea; seven from the class Ophiuroidea; two from the class Echinoidea; and five from the class Holothuroidea. The results would be able to serve as a basis for future studies on the taxonomy, diversity, and distribution of echinoderms in the seagrass meadows of Malaysia.

Echinodermata from Barra de Mamanguape, Northeast of Brazil, Tropical Southwestern Atlantic

The Mamanguape River estuary represents an important environment for marine species, where there are the combination of soft-bottoms and reefs zones. The present study reports on the echinoderm species inhabiting Barra de Mamanguape, at Northeast coast of Brazil. A total of 83 specimens were analyzed, belonging to 13 species, 10 families and the classes Crinoidea, Ophiuroidea, Echinoidea and Holothuroidea. The Ophiuroidea was the most representative with seven species. The crinoid Tropiometra carinata (Lamarck, 1816) and the ophiuroid Ophiophragmus luetkeni (Ljungman, 1872) were new records for Paraiba coast. Concise taxonomic descriptions, figures and ecological notes for recorded species were provided. Comparisons between young and adult specimens of Ophioderma appressa (Say, 1825) were included. This brings information about the arrangement of species in the ecosystem, besides contributes to fill knowledge gaps about echinoderms from Northeast coast.

Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features

Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.

<strong>Ophiuroid locomotion from fundamental structures to integrated systems</strong>

The phylum Echinodermata includes an estimated 7,000 extant and 13,000 extinct species. Each living class represents a separate body plan with a unique mode of movement and locomotion. Brittle stars (Class Ophiuroidea) utilize complex arm musculoskeletal physiology for rapid locomotion, in contrast to the other echinoderm classes, which are typically slow moving. Ophiuroid locomotion and its origins are poorly understood. This paper is a review of the current state of ophiuroid research, with a focus on topics relevant to ophiuroid movement and locomotion, including anatomy, physiology, functional morphology, disparity, ecology, and evolutionary history.

A farewell to arms: using X-ray synchrotron imaging to investigate autotomy in brittle stars

Autotomy, the self-induced loss of a body structure, occurs in every living class of echinoderms and is related to the remarkable regeneration capabilities of the group. It is particularly prevalent in brittle stars (Class Ophiuroidea). Autotomy is facilitated by mutable collagenous tissue, which undergoes nervous system-mediated changes in tensile stiffness, tensile strength, and viscosity. The previous investigations of autotomy have been based on observations of the external surface, surgical manipulation of internal structures, or data on the morphology of structures post-autotomy. We used fast phase-contrast X-ray synchrotron imaging to visualize full autotomy events in vivo in the arms of specimens of the brittle star Ophioderma brevispina. This method requires no chemical or surgical manipulation and enabled us to identify several key stages in the autotomy process. We used this methodology to observe critical changes within the internal structure of the arm as it transitions from a functional mechanical apparatus to a dysfunctional disarticulated state. This method is the first in which the full intersegmental plane of the arm can be observed during autotomy. It can be applied to visualize autotomy and motion in vivo in other brittle star taxa, as well as in other groups such as asteroids and arthropods.

Global biogeographic structuring of tropical shallow‐water brittle stars

AbstractAimBiogeographic barriers emerged in the tropical oceans as continental masses moved with plate tectonics, and as the tropics contracted to lower latitudes from the late Eocene. These barriers have shaped tropical marine biodiversity. We characterize large‐scale diversity patterns for tropical brittle stars and investigate the effect of biogeographic barriers on these in space and time.LocationShallow‐water (<200 m) tropical oceans.TaxonTropical shallow‐water brittle stars (class Ophiuroidea).MethodsWe integrate phylogenetic and biogeographic modelling to test and quantify the biogeographic structuring across the major ocean basins for five families of brittle stars. These are well‐sampled in our phylogenies (173 species) and represent an important component of the brittle star fauna of tropical shallow waters. We define major bioregions based on patterns of compositional and phylogenetic beta diversity.ResultsWe find congruence between patterns of shared ancestry of regions and inferred biogeographic histories. Biogeographic reconstructions show that faunal patterns reflect the emergence of biogeographic barriers in the tropical world, with evidence of vicariant events driven by the opening of the Atlantic Ocean, the narrowing of the Tethyan Seaway and the rise of the Isthmus of Panama.Main conclusionsBiogeographic barriers almost completely isolated regional faunas. However, divergence age estimates predate the onset of the different barriers, suggesting that changes associated with the gradual emergence of the barriers had a strong effect on the evolutionary history of tropical shallow‐water brittle stars. Limited, very recent, bi‐directional dispersal was detected across the East Pacific Barrier, which is otherwise an important barrier for dispersal of brittle stars.

Dark offshoot: Phylogenomic data sheds light on the evolutionary history of a new species of cave brittle star

Caves are a useful system for testing evolutionary and biogeographic hypotheses, as they are isolated, and their environmental conditions have resulted in adaptive selection across different taxa. Although in recent years many more cave species have been discovered, cave-dwelling members of the class Ophiuroidea (brittle stars) remain scarce. Out of the more than two thousand species of brittle stars described to date, only three are regarded as true cave-dwellers. These occurrences represent rare colonising events, compared to other groups that are known to have successfully diversified in these systems. A third species from an anchihaline cave system in the Yucatan Peninsula, Mexico, has been previously identified from cytochrome oxidase I (COI) barcodes. In this study, we reassess the species boundaries of this putative cave species using a phylogenomic dataset (20 specimens in 13 species, 100 exons, 18.7 kbp). We perform species delimitation analyses using robust full-coalescent methods for discovery and validation of hypotheses on species boundaries, as well as infer its phylogenetic relationships with species distributed in adjacent marine regions, in order to investigate the origin of this cave-adapted species. We assess which hypotheses on the origin of subterranean taxa can be applied to this species by taking into account its placement within the genus Ophionereis and its demographic history. We provide a detailed description of Ophionereis commutabilis n. sp., and evaluate its morphological characters in the light of its successful adaptation to life in caves.

Integrating morphology and invivo skeletal mobility with digital models to infer function in brittle star arms.

Brittle stars (Phylum Echinodermata, Class Ophiuroidea) have evolved rapid locomotion employing muscle and skeletal elements within their (usually) five arms to apply forces in a manner analogous to that of vertebrates. Inferring the inner workings of the arm has been difficult as the skeleton is internal and many of the ossicles are sub-millimeter in size. Advances in 3D visualization and technology have made the study of movement in ophiuroids possible. We developed six virtual 3D skeletal models to demonstrate the potential range of motion of the main arm ossicles, known as vertebrae, and six virtual 3D skeletal models of non-vertebral ossicles. These models revealed the joint center and relative position of the arm ossicles during near-maximal range of motion. The models also provide a platform for the comparative evaluation of functional capabilities between disparate ophiuroid arm morphologies. We made observations on specimens of Ophioderma brevispina and Ophiothrix angulata. As these two taxa exemplify two major morphological categories of ophiuroid vertebrae, they provide a basis for an initial assessment of the functional consequences of these disparate vertebral morphologies. These models suggest potential differences in the structure of the intervertebral articulations in these two species, implying disparities in arm flexion mechanics. We also evaluated the differences in the range of motion between segments in the proximal and distal halves of the arm length in a specimen of O.brevispina, and found that the morphology of vertebrae in the distal portion of the arm allows for higher mobility than in the proximal portion. Our models of non-vertebral ossicles show that they rotate further in the direction of movement than the vertebrae themselves in order to accommodate arm flexion. These findings raise doubts over previous hypotheses regarding the functional consequences of ophiuroid arm disparity. Our study demonstrates the value of integrating experimental data and visualization of articulated structures when making functional interpretations instead of relying on observations of vertebral or segmental morphology alone. This methodological framework can be applied to other ophiuroid taxa to enable comparative functional analyses. It will also facilitate biomechanical analyses of other invertebrate groups to illuminate how appendage or locomotor function evolved.

Developmental transcriptomics of the brittle star Amphiura filiformis reveals gene regulatory network rewiring in echinoderm larval skeleton evolution

BackgroundAmongst the echinoderms the class Ophiuroidea is of particular interest for its phylogenetic position, ecological importance and developmental and regenerative biology. However, compared to other echinoderms, notably echinoids (sea urchins), relatively little is known about developmental changes in gene expression in ophiuroids. To address this issue, we have generated and assembled a large RNAseq data set of four key stages of development in the brittle star Amphiura filiformis and a de novo reference transcriptome of comparable quality to that of a model echinoderm—the sea urchin Strongylocentrotus purpuratus. Furthermore, we provide access to the new data via a web interface: http://www.echinonet.eu/shiny/Amphiura_filiformis/.ResultsWe have identified highly conserved genes associated with the development of a biomineralised skeleton. We also identify important class-specific characters, including the independent duplication of the msp130 class of genes in different echinoderm classes and the unique occurrence of spicule matrix (sm) genes in echinoids. Using a new quantification pipeline for our de novo transcriptome, validated with other methodologies, we find major differences between brittle stars and sea urchins in the temporal expression of many transcription factor genes. This divergence in developmental regulatory states is more evident in early stages of development when cell specification begins, rather than when cells initiate differentiation.ConclusionsOur findings indicate that there has been a high degree of gene regulatory network rewiring and clade-specific gene duplication, supporting the hypothesis of a convergent evolution of larval skeleton development in echinoderms.

Mg and Sr in Arctic echinoderm calcite: Nature or nurture?

Abstract The Mg/Ca and Sr/Ca ratios in echinoderm skeletal calcite are used as a proxy for Phanerozoic seawater changes, since the skeletal concentrations are, to some extent, controlled by environmental factors. However, it remains unclear how the influence of environmental factors is modified by vital effects, especially in polar waters. Therefore, the goal of this study was to compare the ratios of Mg/Ca and Sr/Ca among the skeletal parts of 10 common Arctic echinoderm species belonging to three classes Echinoidea, Asteroidea and Ophiuroidea that contribute substantially to the carbon cycle in the Arctic benthic system. Significant differences were recorded in echinoid skeletal element concentrations among specific skeletal parts. The lowest Mg/Ca and Sr/Ca ratios were detected in the spines (mean Mg/Ca 37.5 ± 8.8 SD; Sr/Ca 1.8 ± 0.1). The components of the Aristotle's lantern (epiphyses, pyramids and rotulas) were characterised by the highest Mg levels (Mg/Ca 79.9 ± 6.0; 75.2 ± 9.1; 60.1 ± 3.8, respectively). It is likely that mouth parts experience greater mechanical pressure compared to other body parts, and the higher content of Mg in the Aristotle's lantern contributes to its robustness. We did not find any distinctive trends in the distribution of skeletal elements in the asteroid and ophiuroid skeletal parts. The heterogeneous concentrations of Mg and Sr in different skeleton parts of the echinoids suggest possible physiological regulation of the chemical composition rather than the composition only being influenced by the environment. We cannot recommend echinoderm skeletons as reliable indicators in palaeoenvironmental reconstructions due to the possible biological control of skeletal chemistry, which may interfere with the effect of environmental variables.

Diversity of Echinoderms in Balekambang Beach, Malang District, East Java

Echinoderms is a component of biodiversity in the coral reefs. Echinoderms play an important role in ecosystem of coral reefs especially in food web, generally as predators and detritivors. This report aims to understand composition and density relative of fauna echinoderms on the Balekambang beach. The Transect line method were used and data retrieval was conducted at three stations. The result showed that echinoderms found in Balekambang beach consists of 7 species that are included in three classes, they were <em>Phyllachantus imperialis</em>, <em>Tripneustes gratilla</em>, <em>Diadema setosum</em>, <em>Clypeaster</em> <em>reticulatus</em> (Class Echinoidea), <em>Archaster typicus</em> (Class Asteroidea), <em>Ophiotrix fragilis</em> and <em>Macrophiothrix belii</em> (Class Ophiuroidea). The highest abundance of echinoderms on the station where located in the mangrove area which was dominated by <em>Archaster typicus</em>. The diversity of echinoderms on Balekambang beach were moderate category. The observations of chemical and physics parameters of the aquatic environment and the substrate obtained quite optimal for life of Echinoderms

Checklist of the echinoderm fauna of the Adriatic Sea.

This paper presents a checklist of echinoderm species in the Adriatic Sea. The checklist is based on the review of the available literature data, with temporal coverage from the end of the 18th century to the present day, including the most recent investigations of benthic communities. A total of 108 species have been recorded: 2 species from class Crinoidea, 23 species from class Asteroidea, 22 species from class Ophiuroidea, 22 species from class Echinoidea and 39 species from class Holothuroidea. Non-indigenous echinoderm species have not been observed.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms.

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales.

Finding genes and lineages under selection in speciation.

What are the genes and traits that respond to selection and cause prezygotic reproductive isolation between species? This question has been hard to answer because genomes are large, the targets of selection may be scattered across the genome (Sabeti etal., ) and different genes may respond to the same selective pressure in different populations (Scheinfeldt etal., ). In this issue of Molecular Ecology, Weber etal. () use a clever comparative approach and leading-edge transcriptomic methods to identify the species and genes under positive selection for divergence between brittle stars (the echinoderm class Ophiuroidea) in the Ophioderma longicauda species complex. They found convincing evidence of positive or diversifying selection acting on two genes encoding ion channels that form part of the signal transduction cascade within the sperm in response to pheromones. Evidence for selection was concentrated in genes from one species (called C5, with internal fertilization and female parental care of brooded juveniles and not in the other species (called C3, with more conventional broadcast spawning and planktonic development of embryos and larvae). That analysis greatly extends the range of taxa, life history traits and molecules that are associated with positive selection in speciation. It also illustrates some of the current limitations on the application of RNAseq methods in the search for the targets of selection in nonmodel organisms like brittle stars. From both points of view, the new work by Weber etal. () has important implications for our understanding of speciation in the ocean.