A phylogenetic analysis of the Primnoidae (Anthozoa: Octocorallia: Calcaxonia) with analyses of character evolution and a key to the genera and subgenera.

BACKGROUND: Previous phylogenetic analyses of primnoid octocorals utilizing morphological or molecular data have each recovered evolutionary relationships among genera that are largely incongruent with each other, with some exceptions. In an effort to reconcile molecular-based phylogenies with morphological characters, phylogenetic reconstructions were performed with 33 of 43 primnoid genera using four loci (mtMutS, COI, 28S and 18S), and ancestral state reconstructions were performed using 9 taxonomically relevant characters. In addition, an updated illustrated key to the current 48 genus-level (43 genera, 5 subgenera) primnoids is presented. RESULTS: Ancestral state reconstruction recovered the ancestral colony shape of primnoids as dichotomous planar. Convergence was detected among all 9 characters, and reversals to the character state of the common ancestor occurred in 4 characters. However, some characters were found to be informative. For example, the weak ascus scale of Metafannyella is not likely homologous to the ascus scales of Onogorgia and Fannyella, and the monophyly of two subgenera within Thouarella, which contain polyps in either whorls or an isolated arrangement, was supported. Phylogenetic analyses were generally consistent with previous studies, and resulted in the synonymy of one genus and a subgenus, the elevation of two subgenera, and the transfer of two species back to an original genus. For example, body wall ornamentation of Fanellia was re-evaluated, indicating a synonymy with Callogorgia; the utility of polyp arrangement for the subgenus Plumarella (Dicholaphis) was not supported, and is synonymized with the nominate subgenus Plumarella (Plumarella); the subgenera Plumarella (Faxiella) and Plumarella (Verticillata) are raised to generic status; and the two Plumarella species (P. diadema and P. undulata) are transferred back to Thouarella based on the homology of their marginal scales. CONCLUSIONS: Altogether, and similar to other octocorallian groups, these results indicate that many of the morphological characters examined among primnoids, particularly colony morphology, are labile and exhibit complex evolutionary histories. However, some morphological characters such as coordination of polyps, presence of the ascus body wall scale, number of rows of body wall scales, and number of marginal scales help identify many clades, and are suitable for robust systematic assessments among primnoids.

Molecular evolution of calcification genes in morphologically similar but phylogenetically unrelated scleractinian corals.

Molecular phylogenies of scleractinian corals often fail to agree with traditional phylogenies derived from morphological characters. These discrepancies are generally attributed to non-homologous or morphologically plastic characters used in taxonomic descriptions. Consequently, morphological convergence of coral skeletons among phylogenetically unrelated groups is considered to be the major evolutionary process confounding molecular and morphological hypotheses. A strategy that may help identify cases of convergence and/or diversification in coral morphology is to compare phylogenies of existing "neutral" genetic markers used to estimate genealogic phylogenetic history with phylogenies generated from non-neutral genes involved in calcification (biomineralization). We tested the hypothesis that differences among calcification gene phylogenies with respect to the "neutral" trees may represent convergent or divergent functional strategies among calcification gene proteins that may correlate to aspects of coral skeletal morphology. Partial sequences of two nuclear genes previously determined to be involved in the calcification process in corals, "Cnidaria-III" membrane-bound/secreted α-carbonic anhydrase (CIII-MBSα-CA) and bone morphogenic protein (BMP) 2/4, were PCR-amplified, cloned and sequenced from 31 scleractinian coral species in 26 genera and 9 families. For comparison, "neutral" gene phylogenies were generated from sequences from two protein-coding "non-calcification" genes, one nuclear (ß-tubulin) and one mitochondrial (cytochrome b), from the same individuals. Cloned CIII-MBSα-CA sequences were found to be non-neutral, and phylogenetic analyses revealed CIII-MBSα-CAs to exhibit a complex evolutionary history with clones distributed between at least 2 putative gene copies. However, for several coral taxa only one gene copy was recovered. With CIII-MBSα-CA, several recovered clades grouped taxa that differed from the "non-calcification" loci. In some cases, these taxa shared aspects of their skeletal morphology (i.e., convergence or diversification relative to the "non-calcification" loci), but in other cases they did not. For example, the "non-calcification" loci recovered Atlantic and Pacific mussids as separate evolutionary lineages, whereas with CIII-MBSα-CA, clones of two species of Atlantic mussids (Isophyllia sinuosa and Mycetophyllia sp.) and two species of Pacific mussids (Acanthastrea echinata and Lobophyllia hemprichii) were united in a distinct clade (except for one individual of Mycetophyllia). However, this clade also contained other taxa which were not unambiguously correlated with morphological features. BMP2/4 also contained clones that likely represent different gene copies. However, many of the sequences showed no significant deviation from neutrality, and reconstructed phylogenies were similar to the "non-calcification" tree topologies with a few exceptions. Although individual calcification genes are unlikely to precisely explain the diverse morphological features exhibited by scleractinian corals, this study demonstrates an approach for identifying cases where morphological taxonomy may have been misled by convergent and/or divergent molecular evolutionary processes in corals. Studies such as this may help illuminate our understanding of the likely complex evolution of genes involved in the calcification process, and enhance our knowledge of the natural history and biodiversity within this central ecological group.

Skimming genomes for systematics and DNA barcodes of corals.

Numerous genomic methods developed over the past two decades have enabled the discovery and extraction of orthologous loci to help resolve phylogenetic relationships across various taxa and scales. Genome skimming (or low-coverage genome sequencing) is a promising method to not only extract high-copy loci but also 100s to 1000s of phylogenetically informative nuclear loci (e.g., ultraconserved elements [UCEs] and exons) from contemporary and museum samples. The subphylum Anthozoa, including important ecosystem engineers (e.g., stony corals, black corals, anemones, and octocorals) in the marine environment, is in critical need of phylogenetic resolution and thus might benefit from a genome-skimming approach. We conducted genome skimming on 242 anthozoan corals collected from 1886 to 2022. Using existing target-capture baitsets, we bioinformatically obtained UCEs and exons from the genome-skimming data and incorporated them with data from previously published target-capture studies. The mean number of UCE and exon loci extracted from the genome skimming data was 1837 ± 662 SD for octocorals and 1379 ± 476 SD loci for hexacorals. Phylogenetic relationships were well resolved within each class. A mean of 1422 ± 720 loci was obtained from the historical specimens, with 1253 loci recovered from the oldest specimen collected in 1886. We also obtained partial to whole mitogenomes and nuclear rRNA genes from >95% of samples. Bioinformatically pulling UCEs, exons, mitochondrial genomes, and nuclear rRNA genes from genome skimming data is a viable and low-cost option for phylogenetic studies. This approach can be used to review and support taxonomic revisions and reconstruct evolutionary histories, including historical museum and type specimens.

Vectored dispersal of Symbiodinium by larvae of a Caribbean gorgonian octocoral.

The ability of coral reefs to recover from natural and anthropogenic disturbance is difficult to predict, in part due to uncertainty regarding the dispersal capabilities and connectivity of their reef inhabitants. We developed microsatellite markers for the broadcast spawning gorgonian octocoral Eunicea (Plexaura) flexuosa (four markers) and its dinoflagellate symbiont, Symbiodinium B1 (five markers), and used them to assess genetic connectivity, specificity and directionality of gene flow among sites in Florida, Panama, Saba and the Dominican Republic. Bayesian analyses found that most E. flexuosa from the Florida reef tract, Saba and the Dominican Republic were strongly differentiated from many E. flexuosa in Panama, with the exception of five colonies from Key West that clustered with colonies from Panama. In contrast, Symbiodinium B1 was more highly structured. At least seven populations were detected that showed patterns of isolation by distance. The symbionts in the five unusual Key West colonies also clustered with symbionts from Panama, suggesting these colonies are the result of long-distance dispersal. Migration rate tests indicated a weak signal of northward immigration from the Panama population into the lower Florida Keys. As E. flexuosa clonemates only rarely associated with the same Symbiodinium B1 genotype (and vice versa), these data suggest a dynamic host-symbiont relationship in which E. flexuosa is relatively well dispersed but likely acquires Symbiodinium B1 from highly structured natal areas prior to dispersal. Once vectored by host larvae, these symbionts may then spread through the local population, and/or host colonies may acquire different local symbiont genotypes over time.

Mito-nuclear discordance within Anthozoa, with notes on unique properties of their mitochondrial genomes.

Whole mitochondrial genomes are often used in phylogenetic reconstruction. However, discordant patterns in species relationships between mitochondrial and nuclear phylogenies are commonly observed. Within Anthozoa (Phylum Cnidaria), mitochondrial (mt)-nuclear discordance has not yet been examined using a large and comparable dataset. Here, we used data obtained from target-capture enrichment sequencing to assemble and annotate mt genomes and reconstruct phylogenies for comparisons to phylogenies inferred from hundreds of nuclear loci obtained from the same samples. The datasets comprised 108 hexacorals and 94 octocorals representing all orders and > 50% of extant families. Results indicated rampant discordance between datasets at every taxonomic level. This discordance is not attributable to substitution saturation, but rather likely caused by introgressive hybridization and unique properties of mt genomes, including slow rates of evolution driven by strong purifying selection and substitution rate variation. Strong purifying selection across the mt genomes caution their use in analyses that rely on assumptions of neutrality. Furthermore, unique properties of the mt genomes were noted, including genome rearrangements and the presence of nad5 introns. Specifically, we note the presence of the homing endonuclease in ceriantharians. This large dataset of mitochondrial genomes further demonstrates the utility of off-target reads generated from target-capture data for mt genome assembly and adds to the growing knowledge of anthozoan evolution.

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

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

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

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

An updated assessment of Symbiodinium spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B.

The adaptative bleaching hypothesis (ABH) states that, depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to "switch" or "shuffle" their algal symbionts), coral bleaching can lead to a change in the composition of their associated Symbiodinium community and, thus, contribute to the coral's overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations and to detect low levels of coral-associated Symbiodinium. Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional molecular methods, to assess the presence of low-abundance (background) Symbiodinium spp. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional Symbiodinium clade, among clades A-D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral-Symbiodinium associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or "shuffle" different Symbiodinium clades. Altogether our data suggest that corals from French Polynesia may favor a trade-off between optimizing symbioses with a specific Symbiodinium clade(s), maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change.

Molecular and Morphological Species Boundaries in the Gorgonian Octocoral Genus Pterogorgia (Octocorallia: Gorgoniidae).

Most gorgonian octocoral species are described using diagnostic characteristics of their sclerites (microscopic skeletal components). Species in the genus Pterogorgia, however, are separated primarily by differences in their calyx and branch morphology. Specimens of a morphologically unusual Pterogorgia collected from Saba Bank in the NE Caribbean Sea were found with calyx morphology similar to P. citrina and branch morphology similar to P. guadalupensis. In order to test morphological species boundaries, and the validity of calyx and branch morphology as systematic characters, a phylogenetic analysis was undertaken utilizing partial gene fragments of three mitochondrial (mtMutS, cytochrome b, and igr4; 726bp total) and two nuclear (ITS2, 166bp; and SRP54 intron, 143bp) loci. The datasets for nuclear and mitochondrial loci contained few phylogenetically informative sites, and tree topologies did not resolve any of the morphological species as monophyletic groups. Instead, the mitochondrial loci and SRP54 each recovered two clades but were slightly incongruent, with a few individuals of P. guadalupensis represented in both clades with SRP54. A concatenated dataset of these loci grouped all P. anceps and P. guadalupensis in a clade, and P. citrina and the Pterogorgia sp. from Saba Bank in a sister clade, but with minimal variation/resolution within each clade. However, in common with other octocoral taxa, the limited genetic variation may not have been able to resolve whether branch variation represents intraspecific variation or separate species. Therefore, these results suggest that there are at least two phylogenetic lineages of Pterogorgia at the species level, and the atypical Pterogorgia sp. may represent an unusual morphotype of P. citrina, possibly endemic to Saba Bank. Branch morphology does not appear to be a reliable morphological character to differentiate Pterogorgia species (e.g., branches "flat" or "3-4 edges" in P. guadalupensis and P. anceps, respectively), and a re-evaluation of species-level characters (e.g., sclerites) is needed.

The Genus Cerion (Gastropoda: Cerionidae) in the Florida Keys.

The systematic relationships and phylogeography of Cerion incanum, the only species of Cerion native to the Florida Keys, are reviewed based on partial sequences of the mitochondrial COI and 16S genes derived from 18 populations spanning the range of this species and including the type localities of all four described subspecies. Our samples included specimens of Cerion casablancae, a species introduced to Indian Key in 1912, and a population of C. incanum x C. casablancae hybrids descended from a population of C. casablancae introduced onto Bahia Honda Key in the same year. Molecular data did not support the partition of C. incanum into subspecies, nor could populations be apportioned reliably into subspecies based on morphological features used to define the subspecies. Phylogenetic analyses affirmed the derived relationship of C. incanum relative to other cerionids, and indicated a Bahamian origin for the Cerion fauna of southern Florida. Relationships among the populations throughout the Keys indicate that the northernmost populations, closest to the Tomeu paleoislands that had been inhabited by Cerion petuchi during the Calabrian Pleistocene, are the oldest. The range of Cerion incanum expanded as the archipelago that is the Florida Keys was formed since the lower Tarantian Pleistocene by extension from the northeast to the southwest, with new islands populated as they were formed. The faunas of the High Coral Keys in the northeast and the Oölite Keys in the southwest, both with large islands that host multiple discontinuous populations of Cerion, are each composed of well supported clades that are characterized by distinctive haplotypes. In contrast, the fauna of the intervening Low Coral Keys consist of a heterogeneous series of populations, some with haplotypes derived from the High Coral Keys, others from the Oölite Keys. Individuals from the C. incanum x C. casablancae hybrid population inhabiting the southeastern coast of Bahia Honda Key were readily segregated based on their mitogenome lineage, grouping either with C. incanum or with C. casablancae from Indian Key. Hybrids with C. casablancae mitogenomes had haplotypes that were more divergent from their parent mitogenome than were hybrids with C. incanum mitogenomes.

Rapid assessment of octocoral diversity and habitat on Saba Bank, Netherlands Antilles.

Saba Bank is a large submerged platform (approximately 2200 km(2)), average depth 30 m, located 4 km southwest of Saba Island in Netherlands Antilles, Caribbean Sea. Ships traveling to and from oil terminals on nearby St. Eustatius routinely anchor on the Bank, damaging benthic megafauna. Gorgonian octocorals are vulnerable to anchor damage, and they are common and conspicuous in shallow water (15-50 m) around the banks. This prompted a rapid assessment of octocoral habitat and diversity. The primary objectives were to estimate total species richness and to characterize habitats vis a vis gorgonians. Landsat imagery and multibeam bathymetry were employed to identify random sites for quantitative transects. A Seabotix LBV200L remotely operated vehicle (ROV) and SCUBA were used to collect and survey to 130 m. A total of 14 scuba dives and 3 ROV dives were completed in 10 days. During that time, 48 octocoral species were collected, including two likely undescribed species in the genera Pterogorgia and Lytreia. Gorgonian richness was exceptional, but not all species were collected, because the species accumulation curve remained steeply inclined after all surveys. Two shallow-water gorgonian habitat types were identified using multidimensional scaling and hierarchical cluster analyses: 1) a high diversity, high density fore-reef environment characterized by Eunicea spp., Gorgonia spp., and Pseudopterogorgia spp. and 2) a low diversity, low density plateau environment characterized by Pseudopterogorgia acerosa, Pterogorgia guadalupensis, and Gorgonia mariae. The analyses support hypotheses of broad (approximately 15 km) habitat homogeneity (ANOSIM, P>0.05), but a significant difference between fore-reef and plateau environments (ANOSIM, P<0.05). However, there was some indication of habitat heterogeneity along the 15 km study section of the 50 km platform edge along the southeast rim. Our results highlight the complexity and biodiversity of the Saba Bank, and emphasize the need for more scientific exploration.