Seq' and ARMS shall find: DNA (meta)barcoding of Autonomous Reef Monitoring Structures across the tree of life uncovers hidden cryptobiome of tropical urban coral reefs.

Coral reefs are among the richest marine ecosystems on Earth, but there remains much diversity hidden within cavities of complex reef structures awaiting discovery. While the abundance of corals and other macroinvertebrates are known to influence the diversity of other reef-associated organisms, much remains unknown on the drivers of cryptobenthic diversity. A combination of standardized sampling with 12 units of the Autonomous Reef Monitoring Structure (ARMS) and high-throughput sequencing was utilized to uncover reef cryptobiome diversity across the equatorial reefs in Singapore. DNA barcoding and metabarcoding of mitochondrial cytochrome c oxidase subunit I, nuclear 18S and bacterial 16S rRNA genes revealed the taxonomic composition of the reef cryptobiome, comprising 15,356 microbial ASVs from over 50 bacterial phyla, and 971 MOTUs across 15 metazoan and 19 non-metazoan eukaryote phyla. Environmental factors across different sites were tested for relationships with ARMS diversity. Differences among reefs in diversity patterns of metazoans and other eukaryotes, but not microbial communities, were associated with biotic (coral cover) and abiotic (distance, temperature and sediment) environmental variables. In particular, ARMS deployed at reefs with higher coral cover had greater metazoan diversity and encrusting plate cover, with larger-sized non-coral invertebrates influencing spatial patterns among sites. Our study showed that DNA barcoding and metabarcoding of ARMS constitute a valuable tool for quantifying cryptobenthic diversity patterns and can provide critical information for the effective management of coral reef ecosystems.

Mitogenomes Reveal Alternative Initiation Codons and Lineage-Specific Gene Order Conservation in Echinoderms.

The mitochondrial genetic code is much more varied than the standard genetic code. The invertebrate mitochondrial code, for instance, comprises six initiation codons, including five alternative start codons. However, only two initiation codons are known in the echinoderm and flatworm mitochondrial code, the canonical ATG and alternative GTG. Here, we analyzed 23 Asteroidea mitogenomes, including ten newly sequenced species and unambiguously identified at least two other start codons, ATT and ATC, both of which also initiate translation of mitochondrial genes in other invertebrates. These findings underscore the diversity of the genetic code and expand upon the suite of initiation codons among echinoderms to avoid erroneous annotations. Our analyses have also uncovered the remarkable conservation of gene order among asteroids, echinoids, and holothuroids, with only an interchange between two gene positions in asteroids over ∼500 Ma of echinoderm evolution.

Effects of missing data and data type on phylotranscriptomic analysis of stony corals (Cnidaria: Anthozoa: Scleractinia).

Across the tree of life, phylogenetic analysis is increasingly being performed using transcriptome data. As a result of heterogeneous gene expression within individual organisms and unequal sequencing depth between samples, coverage of homologous loci in such datasets is typically inhomogeneous. Consequently, missing data are a common feature of phylotranscriptomic inference, but their impact on phylogenetic analysis remains poorly characterised empirically. Considering the complexity of the evolutionary history of stony corals (Cnidaria: Anthozoa: Scleractinia), transcriptome data hold great promise for resolving their phylogeny, particularly if there is a good understanding of missing data and data type (either amino acid or DNA) effects. Here, we reconstructed a broad phylogenetic tree of 39 scleractinian species with 3 corallimorpharians as outgroups, including 15 transcriptomes that were newly sequenced and assembled in this study. Between 63 and 505 loci were used to analyse the scleractinian phylogeny, and we quantified differences in tree topology, tree shape, bootstrap support and effects of conflicting gene trees among datasets of varying completeness for both amino acid and DNA sequences. Even with almost 70% missing data, tree topologies appear to be mostly unaffected, although there are higher incongruence levels in the less complete datasets. Furthermore, DNA trees outperform amino acid trees in bootstrap support and robustness against incongruent loci. Overall, our findings indicate that high levels of missing data can still produce expected tree topologies, but identifying and omitting incongruent loci can lead to more consistent branch length estimates.

Revisiting mitogenome evolution in Medusozoa with eight new mitochondrial genomes.

Mitogenomics has improved our understanding of medusozoan phylogeny. However, sequenced medusozoan mitogenomes remain scarce, and Medusozoa phylogeny studies often analyze mitogenomic sequences without incorporating mitogenome rearrangements. To better understand medusozoan evolution, we analyzed Medusozoa mitogenome phylogeny by sequencing and assembling eight mitogenomes from three classes (Cubozoa, Hydrozoa, and Scyphozoa). We reconstructed the mitogenome phylogeny using these mitogenomes and 84 other existing cnidarian mitogenomes to study mitochondrial gene rearrangements. All reconstructed mitogenomes had 13 mitochondrial protein-coding genes and two ribosomal genes typical for Medusozoa. Non-cubozoan mitogenomes were all linear and had typical gene orders, while arrangement of genes in the fragmented Cubozoa (Morbakka sp.) mitogenome differed from other Cubozoa mitogenomes. Gene order comparisons and ancestral state reconstruction suggest minimal rearrangements within medusozoan classes except for Hydrozoa. Our findings support a staurozoan ancestral medusozoan gene order, expand the pool of available medusozoan mitogenomes, and enhance our understanding of medusozoan phylogenetic relationships.

Taxonomy and Molecular Phylogeny of the Sea Anemone Macrodactyla (Haddon, 1898) (Cnidaria, Actiniaria), with a Description of a New Species from Singapore.

Sea anemones (Cnidaria, Actiniaria) are a successful group of marine invertebrates found in a diverse range of environments globally. In spite of their ubiquity, identities for many sea anemones remain unverified, especially those from the Indo-West Pacific region. Here, we clarify the taxonomy of the poorly known Macrodactyla aspera, a shallow-water species first described from the Torres Straits in northern Australia. We re-describe M. aspera based on new morphological and molecular data gathered from the type specimen, other museum vouchers, and from fresh material collected from Singapore. We tested the monophyly of Macrodactyla using three mitochondrial (12S, 16S and cox3) and one nuclear (28S) marker based on three congeners, recovering this genus to be polyphyletic. As a consequence, we transferred M. doreensis to the genus Heteractis, and describe a new species, Macrodactyla fautinae sp. nov. While both M. aspera and M. fautinae sp. nov. share the same arrangement and number of complete mesenteries, a similar distribution of cnidae, and are not symbiotically associated with any other biota, M. fautinae sp. nov. has perforated, lobe-like verrucae on its column, and lacks nematocyst batteries on its tentacles, unlike M. aspera. These two species also occur in similar habitats in Singapore. Finally, because M. aspera strongly resembles Dofleinia armata, the latter species flagged as a danger to public health due to its ability to inflict painful stings, we tested the relationship between these species and found them not to be closely related. However, tentacles of M. aspera, like D. armata, are densely covered with nematocyst batteries and harbour large nematocysts; we infer that M. aspera may also be capable of delivering stings that endanger public health. This study builds upon a growing number of studies that aim to ascertain identities and systematics of sea anemones historically reported from the Indo-West Pacific. Our findings will facilitate accurate species identification, which is crucial for advancing research, formulating conservation measures, and protecting public health.

Spirobranchus bakau sp. nov. from Singapore: yet another species of S. kraussii-complex (Polychaeta: Serpulidae).

The intertidal serpulid polychaete Spirobranchus kraussii was originally described from South Africa and has since been reported in numerous sub (tropical) localities around the world. Recently, however, S. kraussii was uncovered as a complex of morphologically similar and geographically restricted species, raising the need to revise S. cf. kraussii populations. We formally describe S. cf. kraussii from Singapore mangroves as Spirobranchus bakau sp. nov. based on morphological and molecular data. Despite their morphological similarities, Maximum Likelihood and Bayesian Inference analyses of 18S and Cyt b DNA sequence data confirm that S. bakau sp. nov. is genetically distinct from S. kraussii and other known species in the complex. Both analyses recovered S. bakau sp. nov. as part of a strongly supported clade (96% bootstrap, 1 posterior probability), comprising S. sinuspersicus, S. kraussii and S. cf. kraussii from Australia and Hawaii. Additionally, paratypes of S. kraussii var. manilensis, described from Manila Bay in the Philippines, were examined and elevated to the full species S. manilensis. Finally, we tested the hypothesis that fertilisation and embryonic development of S. bakau sp. nov. can occur under the wide range of salinities (19.630.9 psu) and temperatures (2531C) reported in the Johor Strait. Fertilisation success of ≥70% was achieved across a temperature range of 2532C and a salinity range of 2032 psu. Embryonic development, however, had a narrower salinity tolerance range of 2732 psu. Clarifying the taxonomic status of S. cf. kraussii populations reported from localities elsewhere in Singapore and Southeast Asia will be useful in establishing the geographical distribution of S. bakau sp. nov. and other members of the S. kraussii-complex.

Distinct fungal communities associated with different organs of the mangrove Sonneratia alba in the Malay Peninsula.

Mangrove forests are key tropical marine ecosystems that are rich in fungi, but our understanding of fungal communities associated with mangrove trees and their various organs remains limited because much of the diversity lies within the microbiome. In this study, we investigated the fungal communities associated with the mangrove tree Sonneratia alba throughout Peninsular Malaysia and Singapore. At each sampling location, we collected leaves, fruits, pneumatophores and sediment samples and performed amplicon sequencing of the ribosomal internal transcribed spacer 1 to characterise the associated communities. Results show distinct fungal communities at each sampled location with further differentiation according to the plant part. We find a significant distance decay of similarity, particularly for sediment samples due to the greater variability of sediment environments relative to the more stable fungal habitats provided by living plant organs. We are able to assign taxonomy to the majority of sequences from leaves and fruits, but a much larger portion of the sequences recovered from pneumatophores and sediment samples could not be identified. This pattern underscores the limited mycological research performed in marine environments and demonstrates the need for a concerted research effort on multiple species to fully characterise the coastal microbiome and its role in the functioning of marine ecosystems.

The earliest diverging extant scleractinian corals recovered by mitochondrial genomes.

Evolutionary reconstructions of scleractinian corals have a discrepant proportion of zooxanthellate reef-building species in relation to their azooxanthellate deep-sea counterparts. In particular, the earliest diverging "Basal" lineage remains poorly studied compared to "Robust" and "Complex" corals. The lack of data from corals other than reef-building species impairs a broader understanding of scleractinian evolution. Here, based on complete mitogenomes, the early onset of azooxanthellate corals is explored focusing on one of the most morphologically distinct families, Micrabaciidae. Sequenced on both Illumina and Sanger platforms, mitogenomes of four micrabaciids range from 19,048 to 19,542 bp and have gene content and order similar to the majority of scleractinians. Phylogenies containing all mitochondrial genes confirm the monophyly of Micrabaciidae as a sister group to the rest of Scleractinia. This topology not only corroborates the hypothesis of a solitary and azooxanthellate ancestor for the order, but also agrees with the unique skeletal microstructure previously found in the family. Moreover, the early-diverging position of micrabaciids followed by gardineriids reinforces the previously observed macromorphological similarities between micrabaciids and Corallimorpharia as well as its microstructural differences with Gardineriidae. The fact that both families share features with family Kilbuchophylliidae ultimately points towards a Middle Ordovician origin for Scleractinia.

Complete mitochondrial genome of the sea star Archaster typicus (Asteroidea: Archasteridae).

The complete mitochondrial genome of the widespread and common Indo-Pacific sea star Archaster typicus has been sequenced in this study. The mitogenome is 16,230 base pairs (bp) in length, with 13 protein coding genes (PCGs), 22 tRNAs and 2 rRNAs. Gene order of its PCGs and rRNAs matches those of nine other asteroid taxa included for comparison in this study, and it has a similar nucleotide composition of 33.08% A, 26.38% T, 25.53% C and 15.01% G nucleotides. Phylogenetic analyses place A. typicus as the sister group to Acanthaster spp., consistent with previous inferences.

Mangrove-Associated Fungal Communities Are Differentiated by Geographic Location and Host Structure.

Marine fungi on the whole remain understudied, especially in the highly diverse Southeast Asian region. We investigated the fungal communities associated with the mangrove tree Avicennia alba throughout Singapore and Peninsular Malaysia. At each sampling location, we examined ten individual trees, collecting leaves, fruits, pneumatophores, and an adjacent sediment sample from each plant. Amplicon sequencing of the fungal internal transcribed spacer 1 and subsequent analyses reveal significant differences in fungal communities collected from different locations and host structures. Mantel tests and multiple regression on distance matrices show a significant pattern of distance decay with samples collected close to one another having more similar fungal communities than those farther away. Submergence appears to drive part of the variation as host structures that are never submerged (leaves and fruits) have more similar fungal communities relative to those that are covered by water during high tide (pneumatophores and sediment). We suggest that fungi of terrestrial origins dominate structures that are not inundated by tidal regimes, while marine fungi dominate mangrove parts and sediments that are submerged by the incoming tide. Given the critical functions fungi play in all plants, and the important role they can have in determining the success of restoration schemes, we advocate that fungal community composition should be a key consideration in any mangrove restoration or rehabilitation project.