Frederic Sinniger

Potential of DNA Sequences to Identify Zoanthids (Cnidaria: Zoantharia)

Abstract The order Zoantharia is known for its chaotic taxonomy and difficult morphological identification. One method that potentially could help for examining such troublesome taxa is DNA barcoding, which identifies species using standard molecular markers. The mitochondrial cytochrome oxidase subunit I (COI) has been utilized to great success in groups such as birds and insects; however, its applicability in many other groups is controversial. Recently, some studies have suggested that barcoding is not applicable to anthozoans. Here, we examine the use of COI and mitochondrial 16S ribosomal DNA for zoanthid identification. Despite the absence of a clear barcoding gap, our results show that for most of 54 zoanthid samples, both markers could separate samples to the species, or species group, level, particularly when easily accessible ecological or distributional data were included. Additionally, we have used the short V5 region of mt 16S rDNA to identify eight old (13 to 50 years old) museum samples. We discuss advantages and disadvantages of COI and mt 16S rDNA as barcodes for Zoantharia, and recommend that either one or both of these markers be considered for zoanthid identification in the future.

The Parazoanthidae (Hexacorallia: Zoantharia) DNA taxonomy: description of two new genera

The taxonomy of the hexacorallian order Zoantharia is very problematic due to the lack of easily accessible and informative morphological taxonomic characters. This is particularly true in the widespread family Parazoanthidae, members of which use a wide variety of different organisms as substrates. Recently, DNA-based studies have proven to be of great use in clarifying relationships among Parazoanthidae. Here we reconsider Parazoanthidae taxonomy based on analyses of multiple molecular markers [mitochondrial cytochrome oxidase subunit 1 (COI), 16S ribosomal DNA (mt 16S rDNA), and the nuclear internal transcribed spacer region (ITS rDNA)], coupled with ecological and morphological characteristics. Two new genera are described in this study: Hydrozoanthus n. gen. within the new family Hydrozoanthidae, and Antipathozoanthus n. gen in the family Parazoanthidae. The genetic data further suggest that the revised genus Parazoanthus is still polyphyletic and is composed of three distinctive subclades. However, as currently these subclades can essentially be differentiated by genetic data, these subclades should remain within Parazoanthus until further molecular, ecological and morphological studies help to clarify their status and relationships to each other.

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 metazoans.

Worldwide Analysis of Sedimentary DNA Reveals Major Gaps in Taxonomic Knowledge of Deep-Sea Benthos

Deep-sea sediments represent the largest but least known ecosystem on earth. With increasing anthropogenic pressure, it is now a matter of urgency to improve our understanding of deep-sea biodiversity. Traditional morpho-taxonomic studies suggest that the ocean floor hosts extraordinarily diverse benthic communities. However, due to both its remoteness and a lack of expert taxonomists, assessing deep-sea diversity is a very challenging task. Environmental DNA (eDNA) metabarcoding offers a powerful tool to complement morpho-taxonomic studies. Here we use eDNA to assess benthic metazoan diversity in 39 deep-sea sediment samples from bathyal and abyssal depths worldwide. The eDNA dataset was dominated by meiobenthic taxa and we identified all animal phyla commonly found in the deep-sea benthos; yet, the diversity within these phyla remains largely unknown. The large numbers of taxonomically unassigned molecular operational taxonomic units (OTUs) were not equally distributed among phyla, with nematodes and platyhelminthes being the most poorly characterized from a taxonomic perspective. While the data obtained here reveal pronounced heterogeneity and vast amounts of unknown biodiversity in the deep sea, they also expose the difficulties in exploiting metabarcoding datasets resulting from the lack of taxonomic knowledge and appropriate reference databases. Overall, our study demonstrates the promising potential of eDNA metabarcoding to accelerate the assessment of deep-sea biodiversity for pure and applied deep-sea environmental research but also emphasises the necessity to integrate such new approaches with traditional morphology-based examination of deep-sea organisms.

“Locally extinct” coral species Seriatopora hystrix found at upper mesophotic depths in Okinawa

Following the bleaching events of 1998 and 2001, Seriatopora hystrix disappeared from shallow reefs around Okinawa Island, Japan (van Woesik et al. 2011). Here we report finding S. hystrix in a mesophotic coral ecosystem near Motobu Peninsula on Okinawa Island. This diverse coral community occurs on the reef downslope from ~35 m and extends to at least 47 m depth, with the lower boundary yet to be defined. Along with Acropora tenella (Fig. 1) and Pachyseris speciosa (Fig. 2), S. hystrix (Fig. 3) is one of the most abundant species in this community. Following the ‘‘extinction’’ of shallow S. hystrix, no new recruitment events were observed in nearby shallow reefs; thus, the presence of a deep population of this species likely does not originate in recent recruitment but would demonstrate that S. hystrix has not gone extinct in this location. Potentially, this deep population could contribute to the recolonization of S. hystrix at shallower depths, although that is strongly dependent on the level of genetic structuring over depth (Bongaerts et al. 2011; van Oppen et al. 2011). The importance of other ‘‘shallow’’ species in community composition is being further investigated to determine the possible role of this reef as refugium.

Metabolomic profiling reveals deep chemical divergence between two morphotypes of the zoanthid Parazoanthus axinellae

Metabolomics has recently proven its usefulness as complementary tool to traditional morphological and genetic analyses for the classification of marine invertebrates. Among the metabolite-rich cnidarian order Zoantharia, Parazoanthus is a polyphyletic genus whose systematics and phylogeny remain controversial. Within this genus, one of the most studied species, Parazoanthus axinellae is prominent in rocky shallow waters of the Mediterranean Sea and the NE Atlantic Ocean. Although different morphotypes can easily be distinguished, only one species is recognized to date. Here, a metabolomic profiling approach has been used to assess the chemical diversity of two main Mediterranean morphotypes, the “slender” and “stocky” forms of P. axinellae. Targeted profiling of their major secondary metabolites revealed a significant chemical divergence between the morphotypes. While zoanthoxanthin alkaloids and ecdysteroids are abundant in both morphs, the “slender” morphotype is characterized by the presence of additional and bioactive 3,5-disubstituted hydantoin derivatives named parazoanthines. The absence of these specific compounds in the “stocky” morphotype was confirmed by spatial and temporal monitoring over an annual cycle. Moreover, specimens of the “slender” morphotype are also the only ones found as epibionts of several sponge species, particularly Cymbaxinella damicornis thus suggesting a putative ecological link.

Cosmopolitanism of microbial eukaryotes in the global deep seas

Deep sea environments cover more than 65% of the earth’s surface and fulfil a range of ecosystem functions, yet they are also amongst the least known habitats on earth. Whilst the discovery of key geological processes, combined with technological developments, has focused interest onto geologically active areas such as hydrothermal vents, most abyssal biodiversity remains to be discovered ( Danovaro et al. 2010 ). However, as for terrestrial reservoirs of biodiversity, the world’s largest biome is under threat from anthropogenic activities ranging from environmental change to the exploitation of minerals and rare‐earth elements ( Kato et al. 2011 ). It is therefore important to understand the magnitude, nature and composition of deep sea biological communities to inform us of levels of local adaptation, functionality and resilience with respect to future environmental perturbation. In this issue of Molecular Ecology, Bik et al. utilize 454 Roche metagenetic environmental sequencing to assess microbial metazoan community composition and phylogenetic identity across deep sea depth gradients and between ocean basins. The analyses suggest that although the majority of microbial eukaryotic taxa are regionally restricted, a small percentage might maintain cosmopolitan deep sea distributions, and an even smaller fraction appear to be eurybathic (live across depth gradients).

Descriptions of two azooxanthellate Palythoa species (Subclass Hexacorallia, Order Zoantharia) from the Ryukyu Archipelago, southern Japan

Abstract Two new species of zoantharians (Hexacorallia, Zoantharia, Sphenopidae), Palythoa mizigama sp. n. and Palythoa umbrosa sp. n., are described from the Ryukyu Archipelago, southern Japan. Unlike almost all other known Palythoa spp., both species are azooxanthellate and inhabit low-light environments such as floors or sides of caves, crevasses, or hollows of shallow coral reefs. The two species were initially considered to be the same species from their similar habitat environments and highly similar morphological features. However, phylogenetic analyses of nuclear internal transcribed spacer (ITS) ribosomal DNA, mitochondrial 16S ribosomal DNA, and cytochrome oxidase subunit I (COI) sequences revealed that these two species have a genetically distant relationship within the genus Palythoa. Morphological characteristics, including polyp size, tentacle number, external/internal coloration, and types and sizes of cnidae were examined in this study. As a result, only tentacle coloration was found to be useful for the morphological distinction between the two species. Palythoa mizigama possesses white tentacles with black horizontal stripes while Palythoa umbrosa possesses white tentacles without any stripe patterns. Considering their distant phylogenetic relationship, it can be assumed that their unique yet similar morphological and ecological characteristics developed independently in each species as an example of parallel evolution.

Preliminary analyses of cultured Symbiodinium isolated from sand in the oceanic Ogasawara Islands, Japan

The dinoflagellate genus Symbiodinium is generally found in many tropical and subtropical marine invertebrates. Recently, reports have focused on free-living types. We examined free-living Symbiodinium from the Ogasawara (Bonin) Islands, a group of oceanic islands south of Japan. Examining sand samples, seven of eight initial isolates were successfully cultured. Genetic analyses of 18S, 28S and internal transcribed spacer (ITS) ribosomal DNA regions reveal that one isolate cultured with only IMK was identical to clade A isolated from coral reef sand in Okinawa, and four additional isolates cultured with only IMK comprised a new clade A lineage. Additionally, two isolates cultured with IMK and soil extract were closely related to a little-known divergent lineage within clade D. Our results demonstrate some free-living Symbiodinium types may have very wide distributions, and that utilizing different culturing techniques will further discovery of unique Symbiodinium lineages from environmental samples.

Unexpected diversity and new species in the sponge-Parazoanthidae association in southern Japan

Currently the genera Parazoanthus (family Parazoanthidae) and Epizoanthus (family Epizoanthidae) are the only sponge-associated zoantharians (Cnidaria, Anthozoa). The Parazoanthidae-sponge associations are widely distributed in tropical and subtropical waters from the intertidal to the deep sea in the Atlantic and Indo-Pacific Oceans. However, the taxonomic identification of both parties is often confused due to variable morphology and wide ecological ranges. In particular, Parazoanthidae species diversity remains poorly understood in the Indo-Pacific. In the present study, the diversity of the sponge-zoanthid association in the Indo-Pacific was investigated with 71 Parazoanthidae specimens collected from 29 different locations in Japan (n=22), Australia (n=6) and Florida, USA (n=1). For all specimens morphological analyses were performed and total DNA was extracted and amplified for four DNA markers (COI-mtDNA, mt 16S-rDNA, ITS-rDNA and ALG11-nuDNA). The combined data demonstrate that the specimens of this study are clearly different from those of all described Parazoanthus species, and lead us to erect Umimayanthus gen. n., within family Parazoanthidae, containing the three newly described species U. chanpuru sp. n., U. miyabi sp. n., U. nakama sp. n. The new genus also includes the previously described species U. parasiticus (Duchassaing and Michelotti, 1860; comb. nov.), previously belonging to the genus Parazoanthus. Neighbor joining, maximum likelihood and Bayesian posterior probability phylogenetic trees clearly demonstrate the monophyly of Umimayanthus gen. n. to the exclusion of all outgroup sequences. The phylogenetic results were also compared to morphological features, and polyp sizes, amount of sand content in tissues, types of connections between polyps, and cnidae data, in particular holotrichs-1, were useful in distinguishing the different species within this new genus. This new genus can be distinguished from all other Zoantharia by a unique and conserved 9 bp insertion and a 14 bp deletion in the mt 16S-rDNA region. Additionally, compared to Parazoanthus sensu stricto (i.e. P. axinellae [Schmidt, 1862]), Umimayanthus spp. are only found associated to sponges, and have a coenenchyme much less developed than Parazoanthus sensu stricto. Each new species can be distinguished from other congeners by a unique DNA sequence, numbers of tentacle, maximum sizes of holotrichs, associated sponge morphology, and colony morphology. The identification of the host sponge species is the next logical step in this research as this may also aid in the distinction of Umimayanthus species.