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Targeted enrichment of genomic DNA can profoundly increase the phylogenetic resolution of clades and inform taxonomy. Here, we redesign a custom bait set previously developed for the cnidarian class Anthozoa to more efficiently target and capture ultraconserved elements (UCEs) and exonic loci within the subclass Hexacorallia. We test this enhanced bait set (targeting 2476 loci) on 99 specimens of scleractinian corals spanning both the "complex" (Acroporidae, Agariciidae) and "robust" (Fungiidae) clades. Focused sampling in the staghorn corals (genus Acropora) highlights the ability of sequence capture to inform the taxonomy of a clade previously deficient in molecular resolution. A mean of 1850 (±298) loci were captured per taxon (955 UCEs, 894 exons), and a 75% complete concatenated alignment of 96 samples included 1792 loci (991 UCE, 801 exons) and ~1.87 million base pairs. Maximum likelihood and Bayesian analyses recovered robust molecular relationships and revealed that species-level relationships within the Acropora are incongruent with traditional morphological groupings. Both UCE and exon datasets delineated six well-supported clades within Acropora. The enhanced bait set will facilitate investigations of the evolutionary history of many important groups of reef corals, particularly where previous molecular marker development has been unsuccessful., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Corallimorpharia is a small Order of skeleton-less animals that is closely related to the reef-building corals (Scleractinia) and of fundamental interest in the context of understanding the potential impacts of climate change in the future on coral reefs. The relationship between the nominal Orders Corallimorpharia and Scleractinia is controversial-the former is either the closest outgroup to the Scleractinia or alternatively is derived from corals via skeleton loss. This latter scenario, the "naked coral" hypothesis, is strongly supported by analyses based on mitochondrial (mt) protein sequences, whereas the former is equally strongly supported by analyses of mt nucleotide sequences. The "naked coral" hypothesis seeks to link skeleton loss in the putative ancestor of corallimorpharians with a period of elevated oceanic CO2 during the Cretaceous, leading to the idea that these skeleton-less animals may be harbingers for the fate of coral reefs under global climate change. In an attempt to better understand their evolutionary relationships, we examined mt genome organization in a representative range (12 species, representing 3 of the 4 extant families) of corallimorpharians and compared these patterns with other Hexacorallia. The most surprising finding was that mt genome organization in Corallimorphus profundus, a deep-water species that is the most scleractinian-like of all corallimorpharians on the basis of morphology, was much more similar to the common scleractinian pattern than to those of other corallimorpharians. This finding is consistent with the idea that C. profundus represents a key position in the coral <-> corallimorpharian transition.

The relationship between Scleractinia and Corallimorpharia, Orders within Anthozoa distinguished by the presence of an aragonite skeleton in the former, is controversial. Although classically considered distinct groups, some phylogenetic analyses have placed the Corallimorpharia within a larger Scleractinia/Corallimorpharia clade, leading to the suggestion that the Corallimorpharia are "naked corals" that arose via skeleton loss during the Cretaceous from a Scleractinian ancestor. Scleractinian paraphyly is, however, contradicted by a number of recent phylogenetic studies based on mt nucleotide (nt) sequence data. Whereas the "naked coral" hypothesis was based on analysis of the sequences of proteins encoded by a relatively small number of mt genomes, here a much-expanded dataset was used to reinvestigate hexacorallian phylogeny. The initial observation was that, whereas analyses based on nt data support scleractinian monophyly, those based on amino acid (aa) data support the "naked coral" hypothesis, irrespective of the method and with very strong support. To better understand the bases of these contrasting results, the effects of systematic errors were examined. Compared to other hexacorallians, the mt genomes of "Robust" corals have a higher (A+T) content, codon usage is far more constrained, and the proteins that they encode have a markedly higher phenylalanine content, leading us to suggest that mt DNA repair may be impaired in this lineage. Thus the "naked coral" topology could be caused by high levels of saturation in these mitochondrial sequences, long-branch effects or model violations. The equivocal results of these extensive analyses highlight the fundamental problems of basing coral phylogeny on mitochondrial sequence data.

To better understand the underlying causes of rarity and extinction risk in Acropora (staghorn coral), we contrast the minimum divergence ages and nucleotide diversity of an array of species with different range sizes and levels of threat. Time-calibrated Bayesian analyses based upon concatenated nuclear and mitochondrial sequence data implied contemporary range size and vulnerability are linked to species age. However, contrary to previous hypotheses that suggest geographically restricted Acropora species evolved in the Plio-Pleistocene, the molecular phylogeny depicts some Indo-Australian species have greater antiquity, diverging in the Miocene. Species age is not related to range size as a simple positive linear function and interpreting the precise tempo of evolution in this genus is greatly complicated by morphological homoplasy and a sparse fossil record. Our phylogenetic reconstructions provide new examples of how morphology conceals cryptic evolutionary relationships in this keystone genus, and offers limited support for the species groupings currently used in Acropora systematics. We hypothesize that in addition to age, other mechanisms (such as a reticulate ancestry) delimit the contemporary range of some Acropora species, as evidenced by the complex patterns of allele sharing and paraphyly we uncover. Overall, both new and ancient evolutionary information may be lost if geographically restricted and threatened Acropora species are forced to extinction. In order to protect coral biodiversity and resolve the evolutionary history of staghorn coral, further analyses based on comprehensive and heterogeneous morphological and molecular data utilizing reticulate models of evolution are needed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In corals, biocalcification is a major function that may be drastically affected by ocean acidification (OA). Scleractinian corals grow by building up aragonitic exoskeletons that provide support and protection for soft tissues. Although this process has been extensively studied, the molecular basis of biocalcification is poorly understood. Notably lacking is a comprehensive catalog of the skeleton-occluded proteins-the skeletal organic matrix proteins (SOMPs) that are thought to regulate the mineral deposition. Using a combination of proteomics and transcriptomics, we report the first survey of such proteins in the staghorn coral Acropora millepora. The organic matrix (OM) extracted from the coral skeleton was analyzed by mass spectrometry and bioinformatics, enabling the identification of 36 SOMPs. These results provide novel insights into the molecular basis of coral calcification and the macroevolution of metazoan calcifying systems, whereas establishing a platform for studying the impact of OA at molecular level. Besides secreted proteins, extracellular regions of transmembrane proteins are also present, suggesting a close control of aragonite deposition by the calicoblastic epithelium. In addition to the expected SOMPs (Asp/Glu-rich, galaxins), the skeletal repertoire included several proteins containing known extracellular matrix domains. From an evolutionary perspective, the number of coral-specific proteins is low, many SOMPs having counterparts in the noncalcifying cnidarians. Extending the comparison with the skeletal OM proteomes of other metazoans allowed the identification of a pool of functional domains shared between phyla. These data suggest that co-option and domain shuffling may be general mechanisms by which the trait of calcification has evolved.

Madrepora is one of the most ecologically important genera of reef-building scleractinians in the deep sea, occurring from tropical to high-latitude regions. Despite this, the taxonomic affinities and relationships within the genus Madrepora remain unclear. To clarify these issues, we sequenced the mitochondrial (mt) genome of the most widespread Madrepora species, M. oculata, and compared this with data for other scleractinians. The architecture of the M. oculata mt genome was very similar to that of other scleractinians, except for a novel gene rearrangement affecting only cox2 and cox3. This pattern of gene organization was common to four geographically distinct M. oculata individuals as well as the congeneric species M. minutiseptum, but was not shared by other genera that are closely related on the basis of cox1 sequence analysis nor other oculinids, suggesting that it might be unique to Madrepora., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Members of the universal stress protein (USP) family were originally identified in stressed bacteria on the basis of a shared domain, which has since been reported in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants. Although not previously characterized in metazoans, here we report that USP genes are distributed in animal genomes in a unique pattern that reflects frequent independent losses and independent expansions. Multiple USP loci are present in urochordates as well as all Cnidaria and Lophotrochozoa examined, but none were detected in any of the available ecdysozoan or non-urochordate deuterostome genome data. The vast majority of the metazoan USPs are short, single-domain proteins and are phylogenetically distinct from the prokaryotic, plant, protist, and fungal members of the protein family. Whereas most of the metazoan USP genes contain introns, with few exceptions those in the cnidarian Hydra are intronless and cluster together in phylogenetic analyses. Expression patterns were determined for several cnidarian USPs, including two genes belonging to the intronless clade, and these imply diverse functions. The apparent paradox of implied diversity of roles despite high overall levels of sequence (and implied structural) similarity parallels the situation in bacteria. The absence of USP genes in ecdysozoans and most deuterostomes may be a consequence of functional redundancy or specialization in taxon-specific roles.

Background: Classical morphological taxonomy places the approximately 1400 recognized species of Scleractinia (hard corals) into 27 families, but many aspects of coral evolution remain unclear despite the application of molecular phylogenetic methods. In part, this may be a consequence of such studies focusing on the reef-building (shallow water and zooxanthellate) Scleractinia, and largely ignoring the large number of deep-sea species. To better understand broad patterns of coral evolution, we generated molecular data for a broad and representative range of deep sea scleractinians collected off New Caledonia and Australia during the last decade, and conducted the most comprehensive molecular phylogenetic analysis to date of the order Scleractinia., Methodology: Partial (595 bp) sequences of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene were determined for 65 deep-sea (azooxanthellate) scleractinians and 11 shallow-water species. These new data were aligned with 158 published sequences, generating a 234 taxon dataset representing 25 of the 27 currently recognized scleractinian families., Principal Findings/conclusions: There was a striking discrepancy between the taxonomic validity of coral families consisting predominantly of deep-sea or shallow-water species. Most families composed predominantly of deep-sea azooxanthellate species were monophyletic in both maximum likelihood and Bayesian analyses but, by contrast (and consistent with previous studies), most families composed predominantly of shallow-water zooxanthellate taxa were polyphyletic, although Acroporidae, Poritidae, Pocilloporidae, and Fungiidae were exceptions to this general pattern. One factor contributing to this inconsistency may be the greater environmental stability of deep-sea environments, effectively removing taxonomic "noise" contributed by phenotypic plasticity. Our phylogenetic analyses imply that the most basal extant scleractinians are azooxanthellate solitary corals from deep-water, their divergence predating that of the robust and complex corals. Deep-sea corals are likely to be critical to understanding anthozoan evolution and the origins of the Scleractinia.

The phylogenetic conundrum posed by the Chaetognatha, a cryptic phylum consisting largely of planktonic predators, is the subject of two short papers in this issue of Current Biology. These analyses go some way towards defining the phylogenetic position of the chaetognaths, which possess features apparently spanning the protostome/deuterostome divide.

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

A significant proportion of mammalian genes are not represented in the genomes of Drosophila, Caenorhabditis or Saccharomyces, and many of these are assumed to have been vertebrate innovations. To test this assumption, we conducted a preliminary EST project on the anthozoan cnidarian, Acropora millepora, a basal metazoan. More than 10% of the Acropora ESTs with strong metazoan matches to the databases had clear human homologs but were not represented in the Drosophila or Caenorhabditis genomes; this category includes a surprising diversity of transcription factors and metabolic proteins that were previously assumed to be restricted to vertebrates. Consistent with higher rates of divergence in the model invertebrates, three-way comparisons show that most Acropora ESTs match human sequences much more strongly than they do any Drosophila or Caenorhabditis sequence. Gene loss has thus been much more extensive in the model invertebrate lineages than previously assumed and, as a consequence, some genes formerly thought to be vertebrate inventions must have been present in the common metazoan ancestor. The complexity of the Acropora genome is paradoxical, given that this organism contains apparently few tissue types and the simplest extant nervous system consisting of a morphologically homogeneous nerve net.

Hybridisation between coral species clearly occurs in vitro, but the evolutionary significance of this cross-fertility is still the subject of much debate. Compelling genetic and reproductive evidence support introgressive hybridization amongst Indo-Pacific members of the scleractinian genus Acropora. Although population genetic analyses indicate that interspecific hybridization events are relatively rare, they are likely be important on evolutionary time scales, creating the capacity for adaptive evolution by increasing genomic diversity and heterozygosity. However, in a recent paper based exclusively on the three endemic Caribbean Acropora species, Vollmer and Palumbi (2002) dispute the occurrence of reticulation in corals. Here we use data from both the Vollmer and Palumbi study and our earlier paper on the same species (van Oppen et al., 2000) to show that reticulation has occurred amongst the Caribbean Acropora species. Furthermore, conclusions based on the limited Caribbean Acropora fauna cannot simply be extrapolated to Indo-Pacific corals, and it is inappropriate to view some coral species as 'immortal mules'.

As the closest outgroup to the Bilateria, the Phylum Cnidaria is likely to be critical to understanding the origins and evolution of body axes. Proteins of the decapentaplegic (DPP)/bone morphogenetic protein (BMP) 2/4 subfamily are central to the specification of the dorsoventral (D/V) axis in bilateral animals, albeit with an axis inversion between arthropods and chordates. We show that a dpp/BMP2/4 ortholog (bmp2/4-Am) is present in the reef-building scleractinian coral, Acropora millepora (Class Anthozoa) and that it is capable of causing phenotypic effects in Drosophila that mimic those of the endogenous dpp gene. We also show that, during coral embryonic development, bmp2/4-Am expression is localized in an ectodermal region adjacent to the blastopore. Thus, a representative of the DPP/BMP2/4 subfamily of ligands was present in the common ancestor of diploblastic and triploblastic animals where it was probably expressed in a localized fashion during development. A localized source of DPP/BMP2/4 may have already been used in axis formation in this ancestor, or it may have provided a means by which an axis could evolve in triploblastic animals.

Pax genes encode a family of transcription factors, many of which play key roles in animal embryonic development but whose evolutionary relationships and ancestral functions are unclear. To address these issues, we are characterizing the Pax gene complement of the coral Acropora millepora, an anthozoan cnidarian. As the simplest animals at the tissue level of organization, cnidarians occupy a key position in animal evolution, and the Anthozoa are the basal class within this diverse phylum. We have identified four Pax genes in Acropora: two (Pax-Aam and Pax-Bam) are orthologs of genes identified in other cnidarians; the others (Pax-Cam and Pax-Dam) are unique to Acropora. Pax-Aam may be orthologous with Drosophila Pox neuro, and Pax-Bam clearly belongs to the Pax-2/5/8 class. The Pax-Bam Paired domain binds specifically and preferentially to Pax-2/5/8 binding sites. The recently identified Acropora gene Pax-Dam belongs to the Pax-3/7 class. Clearly, substantial diversification of the Pax family occurred before the Cnidaria/higher Metazoa split. The fourth Acropora Pax gene, Pax-Cam, may correspond to the ancestral vertebrate Pax gene and most closely resembles Pax-6. The expression pattern of Pax-Cam, in putative neurons, is consistent with an ancestral role of the Pax family in neural differentiation and patterning. We have determined the genomic structure of each Acropora Pax gene and show that some splice sites are shared both between the coral genes and between these and Pax genes in triploblastic metazoans. Together, these data support the monophyly of the Pax family and indicate ancient origins of several introns.

A protein encoded by cDNAs from the human parasite Onchocerca volvulus and its homologs from Caenorhabditis elegans and Ancyclostoma caninum define a family of aspartic proteases that are most closely related to cathepsins D, but differ from them in lacking the N-glycosylation site known to be required for lysosomal targeting. The nematode proteins have a potential N-glycosylation site at the same position as mammalian cathepsins E and in common with these have atypically long N-terminal extensions. The literature implies that cathepsins E may be secreted, and adult female O. volvulus are known to secrete a specific inhibitor of aspartic proteases; we therefore predict that the protease is secreted as an enzyme-inhibitor complex.

Systematic relationships among the subclasses of Anthozoa, and especially among the orders Scleractinia, Actiniaria, and Corallimorpharia of subclass Zoantharia, were investigated by applying parsimony and distance methods of analysis to nucleotide sequence data obtained for the 5' end of the 28S rDNA. Exhaustive parsimony analysis indicates that the Ceriantipatharia are most representative of the ancestral Anthozoa. When applied to a wide range of scleractinians (nine taxa), actiniarians (seven taxa), and corallimorpharians (six taxa), both parsimony and distance analyses resolve three groups, one being the Scleractinia and the others containing both actiniarians and corallimorpharians. This indicates an unclear relationship between Actiniaria and Corallimorpharia and gives no support for Hand's hypothesis of scleractinian ancestry of actiniarians and corallimorphians; the monophyly of the Scleractinia, which is strongly supported by our analyses, is evidence to the contrary.