Diversity of Zoanthids (Anthozoa: Hexacorallia) on Hawaiian Seamounts: Description of the Hawaiian Gold Coral and Additional Zoanthids

The Hawaiian gold coral has a history of exploitation from the deep slopes and seamounts of the Hawaiian Islands as one of the precious corals commercialised in the jewellery industry. Due to its peculiar characteristic of building a scleroproteic skeleton, this zoanthid has been referred as Gerardia sp. (a junior synonym of Savalia Nardo, 1844) but never formally described or examined by taxonomists despite its commercial interest. While collection of Hawaiian gold coral is now regulated, globally seamounts habitats are increasingly threatened by a variety of anthropogenic impacts. However, impact assessment studies and conservation measures cannot be taken without consistent knowledge of the biodiversity of such environments. Recently, multiple samples of octocoral-associated zoanthids were collected from the deep slopes of the islands and seamounts of the Hawaiian Archipelago. The molecular and morphological examination of these zoanthids revealed the presence of at least five different species including the gold coral. Among these only the gold coral appeared to create its own skeleton, two other species are simply using the octocoral as substrate, and the situation is not clear for the final two species. Phylogenetically, all these species appear related to zoanthids of the genus Savalia as well as to the octocoral-associated zoanthid Corallizoanthus tsukaharai, suggesting a common ancestor to all octocoral-associated zoanthids. The diversity of zoanthids described or observed during this study is comparable to levels of diversity found in shallow water tropical coral reefs. Such unexpected species diversity is symptomatic of the lack of biological exploration and taxonomic studies of the diversity of seamount hexacorals

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ßtubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils

Octocoral Mitochondrial Genomes Provide Insights into the Phylogenetic History of Gene Order Rearrangements, Order Reversals, and Cnidarian Phylogenetics

We use full mitochondrial genomes to test the robustness of the phylogeny of the Octocorallia, to determine the evolutionary pathway for the five known mitochondrial gene rearrangements in octocorals, and to test the suitability of using mitochondrial genomes for higher taxonomic-level phylogenetic reconstructions. Our phylogeny supports three major divisions within the Octocorallia and show that Paragorgiidae is paraphyletic, with Sibogagorgia forming a sister branch to the Coralliidae. Furthermore, Sibogagorgia cauliflora has what is presumed to be the ancestral gene order in octocorals, but the presence of a pair of inverted repeat sequences suggest that this gene order was not conserved but rather evolved back to this apparent ancestral state. Based on this we recommend the resurrection of the family Sibogagorgiidae to fix the paraphyly of the Paragorgiidae. This is the first study to show that in the Octocorallia, mitochondrial gene orders have evolved back to an ancestral state after going through a gene rearrangement, with at least one of the gene orders evolving independently in different lineages. A number of studies have used gene boundaries to determine the type of mitochondrial gene arrangement present. However, our findings suggest that this method known as gene junction screening may miss evolutionary reversals. Additionally, substitution saturation analysis demonstrates that while whole mitochondrial genomes can be used effectively for phylogenetic analyses within Octocorallia, their utility at higher taxonomic levels within Cnidaria is inadequate. Therefore for phylogenetic reconstruction at taxonomic levels higher than subclass within the Cnidaria, nuclear genes will be required, even when whole mitochondrial genomes are available

Two Distinct, Geographically Overlapping Lineages of the Corallimorpharian Ricordea Florida (Cnidaria: Hexacorallia: Ricordeidae)

We examined the genetic variation of the corallimorpharian Ricordea florida; it is distributed throughout the Caribbean region and is heavily harvested for the marine aquarium trade. Eighty-four distinct individuals of R. florida were sequenced from four geographically distant Caribbean locations (Curaçao, Florida, Guadeloupe, and Puerto Rico). Analysis of the ribosomal nuclear region (ITS1, 5.8S, ITS2) uncovered two geographically partially overlapping genetic lineages in R. florida, probably representing two cryptic species. Lineage 1 was found in Florida and Puerto Rico, and Lineage 2 was found in Florida, Puerto Rico, Guadeloupe, and Curaçao. Because of the multi-allelic nature of the ITS region, four individuals from Lineage 1 and six from Lineage 2 were cloned to evaluate the levels of hidden intra-individual variability. Pairwise genetic comparisons indicated that the levels of intra-individual and intra-lineage variability (\u3c1%) were approximately an order of magnitude lower than the divergence (~9%) observed between the two lineages. The fishery regulations of the aquarium trade regard R. florida as one species. More refined regulations should take into account the presence of two genetic lineages, and they should be managed separately in order to preserve the long-term evolutionary potential of this corallimorpharian. The discovery of two distinct lineages in R. florida illustrates the importance of evaluating genetic variability in harvested species prior to the implementation of management policies

Octocoral biodiversity in Portugal: a barcoding approach coupling long-range PCR and long-read sequencing to assemble mitochondrial genomes

As florestas de corais de zonas mais profundas ou de águas temperadas são caracterizadas por comunidades de elevada diversidade comummente denominadas por “florestas animais” pois são dominadas por espécies filtradoras não fotossintéticas que formam habitats tridimensionalmente complexos. Octocorallia é uma vasta e diversificada subclasse de antozoários que incluem os corais moles e gorgónias (Alcyonacea), os corais azuis (Helioporacea) e as penas marinhas (Pennatulacea). Estas espécies também apresentam um grande valor ecológico pois atuam como engenheiros do ecossistema com a capacidade de manter um habitat fornecendo abrigo e alimento para várias espécies de peixe, incluindo espécies com elevado interesse para a pesca comercial. Infelizmente os impactos antropogénicos persistem devido ao crescimento populacional exponencial e ao desenvolvimento da indústria global criando uma enorme pressão sobre estes ecossistemas marinhos. Felizmente, o nosso conhecimento tem crescido acerca da gravidade de como os impactos estão a afetar a integridade dos ecossistemas em zonas mais profundas. Tais impactos antropogénicos incluem poluição como derrames de petróleo, acidificação dos oceanos e atividade intensas de pesca. Corais, como as gorgónias, têm um crescimento lento e alta longevidade tornando-os mais vulneráveis a distúrbios no ecossistema pois apresentam uma baixa taxa de recuperação natural. Devido à sua importância ecológica, existe um grande interesse na comunidade científica de desenvolver estudos e implementar medidas de conservação e restauração destes habitats. A perda da biodiversidade, causada por impactos antropogénicos, tem sido um motivo preocupante globalmente. O desenvolvimento de ferramentas genéticas inovadores que facilitam na identificação de espécies, especialmente de espécies que são morfologicamente muito semelhantes, mas geneticamente diferentes, têm vindo a complementar os métodos de monitoração de biodiversidade existentes. A next-generation sequencing (NGS) éuma plataforma recente de sequenciação que fornece elevadas leituras de sequenciação numa única execução, pois fornecem informação rápida e massiva por sequenciação com esforços e custos mínimos em comparação com as técnicas tradicionais de sequenciação de Sanger. Esta técnica promove o aperfeiçoamento e complementação dos métodos tradicionais, mas apresenta algumas limitações. O primeiro indica que a região que é amplificada por PCR e sequenciada está limitada a um tamanho pequeno do genoma mitocondrial total como um fragmento do COI (marcador genético tradicional). O segundo indica que, para alguns grupos de invertebrados, estes marcadores genéticos mitocondriais não possuem polimorfismo suficiente para distinguir espécies próximas. Por fim, o barcoding de ADN requer uma biblioteca de referência completa e com qualidade. Estas bibliotecas contêm dados genéticos que são fundamentais para estudos ecológicos e também são ferramentas valiosas para a avaliação da biodiversidade como “eDNA metabarcoding” que posteriormente podem ser incorporados em estratégias de gestão e conservação. Alguns grupos de corais exigem um sistema de classificação baseado em informação genética que complemente a classificação através de morfológica. A utilização de genomas mitocondriais completos também tem sido uma ferramenta emergente e eficaz em taxonomia e estudos filogenéticos, pois são bastante úteis para avaliar relações filogenéticas ao nível de espécies porque têm uma taxa evolutiva muito mais rápida do que o genoma nuclear. Pois requer mais que um gene, ou preferencialmente, o mitogenoma completo para resolver relações evolutivas entre espécies. O genoma mitocondrial em antozoários, em particular na subclasse Octocorallia, exibe uma taxa de evolução molecular muito lenta em comparação com outros grupos taxonómicos. Como tal, o barcoding de ADN através da sequenciação de apenas um gene mitocondrial (normalmente o tradicional COI) é inadequado para distinguir muitas espécies de corais. A Oxford Nanopore Techniques (ONT) é uma técnica de sequenciação de terceira geração capaz de produzir longas fáceis e rápidas leituras de sequenciação. Uma tecnologia ideal para a construção de mitogenomas devido à sua capacidade de realizar leituras longas e repetitivas de sequenciação com uma contiguidade muito maior que garante a integridade da informação genética e construção completa do mitogenoma. Enquanto que os métodos tradicionais de sequenciação de mitogenomas completo, como o primer-walk (i.e., sequenciação de Sanger) sequenciam apenas um único fragmento de ADN de cada vez a um custo muito mais elevado. Para além disso, os octocorais exibem cinco ordens de genes diferentes em que podem ter arranjos de ordem genética diferentes ou existe um bloco de cinco ou mais genes que foram invertidos, ou seja, a codificação da cadeia é revertida. O objetivo geral desta tese foi desenvolver uma abordagem de sequenciação, construção e anotação de mitogenomas completos de diferentes grupos de corais com aplicação universal e que permita sequenciação em larga escala. O estudo é focado em espécies de octocorais maioritariamente presentes na costa portuguesa e alguns representantes de Scleractinians de Cabo Verde e de Espanha. Como tal, os objetivos específicos foram: 1) desenvolver primers específicos para corais para amplificar o mitogenoma completo usando dados de referências que estão disponíveis publicamente; 2) expandir as bibliotecas de referencia disponíveis através de sequenciação, assembly e anotação dos mitogenomas de espécies que existem em Portugal; 3) confirmar as ordens de genes das espécies-alvo e ver se estão em conformidade com o que foi descrito na literatura; 4) Fazer uma reconstrução filogenética com base nos dados da sequenciação do mitogenoma para inferir a localização filogenética e a afinidade genética das espécies-alvo dentro de Octocorallia, incluindo espécies que não foram identificadas de forma conclusiva com base na morfologia. A abordagem desenvolvida consistiu em desenhar primers universais que permitiram a amplificação do mitogenoma por PCR de longo alcance (produtos de PCR > 8000 pb), seguido de sequenciação com tecnologia de terceira geração, ou seja, dados de “long reads” obtidos com Oxford Nanopore Technologies. Uma vez que o genoma mitocondrial é circular, procurou-se desenhar primers em duas regiões opostas e equidistantes de maneira a amplificar o mitogenoma em duas reações de PCR, uma para cada metade da molécula. Um total de 17 mitogenomas de octocorais foram construídos com sucesso através de de novo assembly. Os mitogenomas circulares codificam 14 genes codificadores de proteínas (Nad1-6, Nad4L, cox1-3, Cytb, mtMutS, Atp6 e Atp8), dois genes de RNA ribossómico (r12S e r16S) e um RNA de transferência (trnM). Das cinco ordens de genes existentes em octocorais, foram sequenciadas espécies que representavam três tipos de organização do mitogenoma: Isidella elongata exibindo a ordem de genes B, Corallium rubrum exibindo a ordem de genes C e as restantes espécies exibiram a ordem de genes A, embora não tenhamos conseguido reconstruir o mitogenoma da Isidella elongata devido a um erro na combinação de primers durante a amplificação. A reconstrução filogenética com base em 16 genes mitocondriais permitiu inferir a localização filogenética e a afinidade genética das espécies-alvo dentro de Octocorallia, incluindo espécies que não foram identificadas de forma conclusiva com base na morfologia. Posicionamos e identificamos, pelo menos ao nível de genérico, todos os 17 mitogenomas reconstruídos na árvore filogenética de Octocorallia e recuperamos três ramos principais que correspondem a estudos filogenéticos anteriores e um ramo que aparenta ser basal ao ramo Holaxonia-Alcyoniina. Esta abordagem com sequenciação de terceira geração com a Oxford Nanopore Technology permitiu a sequenciação de “long-read” fornecendo sequenciação em larga escala de muitas amostras numa única execução de sequenciação. Essa abordagem que inclui PCR de longo alcance e sequenciação de “long-read” poderá ter a desvantagem de introduzir erros durante a amplificação (PCR), mas com elevada cobertura de sequenciação é possível escapar a esta limitação. A amplificação por PCR aumenta o número de cópias do mitogenoma do extrato de ADN purificado em multiplex juntamente com várias amostras agrupadas com barcoding. Esse processo reduz a proporção de ADN genómico (não mitocondrial) nos extratos de ADN e permite sequenciar de uma maneira bastante mais direcionada no qual permite agrupar várias amostras na mesmo processo de sequenciação. Esta abordagem possibilitou construir bibliotecas de referência completas cobrindo todos os mitogenomas, no qual melhorou assim as bibliotecas de referência disponíveis que posteriormente poderão auxiliar na avaliação da biodiversidade de corais com base no metabarcoding de eDNA (ADN ambiental). Mas o mais importante foi que esta abordagem pode ser altamente aplicável para sequenciar o mitogenoma completo de amostras ambientais (eDNA) por meio de metabarcoding. Desta forma, esta abordagem, juntamente com o eDNA, é valiosa para catalogar a biodiversidade e auxiliar na avaliação da biodiversidade e possíveis medidas de conservação.Coral communities found either in tropical or deeper locations or in temperate waters can be classified as “marine animal forests” as they are dominated by habitat-forming suspension feeders, creating three-dimensional forest-like structures. Octocorallia is a wide and diverse subclass of Anthozoa that includes soft corals and gorgonians (Alcyonacea), blue corals (Helioporacea), and sea feathers (Pennatulacea). Their ecological importance, slow growth, and susceptibility to degradation caused by anthropogenic impacts make them vulnerable marine ecosystems. This leads to an interest of the scientific community to develop studies and implement conservation and restoration measures. Yet for many of these organisms, their identity is uncertain or debatable. The development of genetic tools that facilitate the identification of species, especially species that are morphologically identical but genetically different, has been complementing existing biodiversity monitoring methods. DNA barcoding allows high throughput multispecies identification but using sequencing of just one mitochondrial gene (typically the COI) is inadequate to distinguish many coral species. This study aims to develop an approach to barcode the mitochondrial genomes of octocorals by coupling long-range PCR with a 3rd generation sequencing platform (i.e., long-read sequencing) by designing coral-specific primers to amplify the mitogenomes, expanding the available reference library, confirming the gene orders arrangements of the target species and their placement in the Octocorallia phylogeny tree. We successfully identified and placed, at least at the genus level, all 17 reconstructed mitogenomes in the Octocorallia phylogenetic tree and we were able to identify three of the five existing gene orders within octocorals. This approach complemented and expanded the reference libraries that are applicable for eDNA metabarcoding to catalog biodiversity and assist in biodiversity assessment and possible conservation measures

GIS based mapping of zoanthids along Saurashtra coast, Gujarat, India

Zooxanthellate zoanthids (or. Zoantharia) are the third largest order of Hexacorallia and are an integral part of the coral reef ecosystem. Worldwide coral reefs will continue to suffer under the synergistic effect of anthropogenic agent and climate change, thereby shifting towards more adaptive and resilient species. Zoanthids are looked upon as adaptive species under the current dynamics of climate change. Zoanthids are also studied for their biochemical properties like extraction of zoanthamine, Oxytoxic agent, Green Fluorensce Proteins (GFP). Hence understanding the ecology and spatial distribution patterns of zoanthids is important in formulating conservational and management policies pertaining to marine ecosystems. The present study encompasses the spatial distribution pattern of zoanthids along the Saurashtra coast of Gujarat, India. Nineteen stations have been selected from Okha to Bhavnagar and spatial distribution patterns of eight zoanthid species have been studied using modified belt transact method, GIS and IDW interpolation technique. The results indicated Palythoa mutuki as the most common and abundant species along the Saurashtra coast of Gujarat followed by Zoanthus sansibaricus and Palythoa tuberculosa. While species such as Zoanthus gigantus and Palythoa heliodiscus, been the rarest species along this coast. The study is first of its kind and attempt has been made to incorporate the modern tools which overcome the constraints of spatial variation in the distribution over traditional methods of biodiversity studies. The study also forms baseline study to monitor zoanthid progression in the future and developing georeferenced database along the Saurashtra coast of India for long term permanent transect monitoring and policy framework development

Demospongic Acids Revisited

The well-known fatty acids with a Δ5,9 unsaturation system were designated for a long period as demospongic acids, taking into account that they originally occurred in marine Demospongia sponges. However, such acids have also been observed in various marine sources with a large range of chain-lengths (C16–C32) and from some terrestrial plants with short acyl chains (C18–C19). Finally, the Δ5,9 fatty acids appear to be a particular type of non-methylene-interrupted fatty acids (NMA FAs). This article reviews the occurrence of these particular fatty acids in marine and terrestrial organisms and shows the biosynthetic connections between Δ5,9 fatty acids and other NMI FAs

Preliminary survey of zooxanthellate zoanthids (Cnidaria : Hexacorallia) of the Galapagos, and associated symbiotic dinoflagellates (Symbiodinium spp.)

Despite their presence in almost all marine ecosystems, the zoanthids (Cnidaria: Hexacorallia: Zoantharia) are poorly studied, in large part due to a lack of useful morphological identification characters. Recent research combining morphology with DNA markers has begun to shed new light on diversity and distribution of the order Zoantharia. Here, preliminary findings on the diversity and distribution of zooxanthellate zoanthid species from the genera Zoanthus and Palythoa are presented, documenting these genera in the Galapagos for the first time. A brief description of the species found is provided. Zoanthus and Palythoa appear to be limited in the Galapagos to rocky shores in warm shallow sublittoral and infralittoral waters (minimum temperature >18°C), isolated from the colder water that dominates much of the archipelago. Preliminary results from the internal transcribed spacer region of ribosomal DNA sequences of symbiotic dinoflagellates suggest that both Zoanthus and Palythoa spp. in the Galapagos possess only Symbiodinium clade C. Brief descriptions of the zooxanthellate zoanthid species found in the Galapagos are provided

Mitochondrial Genome of Savalia savaglia (Cnidaria, Hexacorallia) and Early Metazoan Phylogeny

Mitochondrial genomes have recently become widely used in animal phylogeny, mainly to infer the relationships between vertebrates and other bilaterians. However, only 11 of 723 complete mitochondrial genomes available in the public databases are of early metazoans, including cnidarians (Anthozoa, mainly Scleractinia) and sponges. Although some cnidarians (Medusozoa) are known to possess atypical linear mitochondrial DNA, the anthozoan mitochondrial genome is circular and its organization is similar to that of other metazoans. Because the phylogenetic relationships among Anthozoa as well as their relation to other early metazoans still need to be clarified, we tested whether sequencing the complete mitochondrial genome of Savalia savaglia, an anthozoan belonging to the order Zoantharia (=Zoanthidea), could be useful to infer such relationships. Compared to other anthozoans, S. savaglia's genome is unusually long (20,766 bp) due to the presence of several noncoding intergenic regions (3691 bp). The genome contains all 13 protein coding genes commonly found in metazoans, but like other Anthozoa it lacks most of the tRNAs. Phylogenetic analyses of S. savaglia mitochondrial sequences show Zoantharia branching closely to other Hexacorallia, either as a sister group to Actiniaria or as a sister group to Actiniaria and Scleractinia. The close relationships suggested between Zoantharia and Actiniaria are reinforced by strong similarities in their gene order and the presence of similar introns in the COI and ND5 genes. Our study suggests that mitochondrial genomes can be a source of potentially valuable information on the phylogeny of Hexacorallia and may provide new insights into the evolution of early metazoan

Complete mitochondrial genomes of two species of Stichopathes Brook, 1889 (Hexacorallia: Antipatharia: Antipathidae) from Rapa Nui (Easter Island)

We report the complete mitochondrial genomes of two antipatharian species, Stichopathes sp. SCBUCN-8849 and Stichopathes sp. SCBUCN-8850, collected between 120 and 180 m depth off Rapa Nui (∼ −27.1°, −109.4°). The size of the two mitogenomes are 20,389 bp (29.0% A, 15.2% C, 19.9% G, and 35.9% T) and 20,463 bp (29.0% A, 15.3% C, 19.9% G, and 35.8% T), respectively. Both mitogenomes have the classic Hexacorallia gene content of 13 protein-coding, two rRNA, and two tRNA genes plus a COX1 intron with embedded HEG as found in the Antipathidae and other antipatharian families