Resolving Acuticulata (Metridioidea: Enthemonae: Actiniaria), a clade containing many invasive species of sea anemones

Acuticulata is a globally distributed group in the actiniarian superfamily Metridiodea comprised of taxa with ecological, economic, and scientific significance. Prominent members such as Exaiptasia diaphana and Diadumene lineata serve as model organisms for studying coral symbiosis, bleaching phenomena, and ecological invasions. Despite their importance, unresolved phylogenetic relationships and outdated taxonomic frameworks hinder a full understanding of the diversity and evolution of the taxa in this clade. In this study, we employ a targeted sequence-capture approach to construct a robust phylogeny for Acuticulata, addressing long-standing questions about familial monophyly and comparing the results to results from a more conventional, five-gene dataset. Specimens from previously underrepresented families and global regions, including the Falkland Islands, were included to elucidate evolutionary interrelationships and improve resolution. Our results support the monophyly of Aliciidae, Boloceroididae, Diadumenidae, Gonactiniidae, and Metridiidae. Our results highlight the need for taxonomic revision within the family Sagartiidae, as the specimens we included from this family were recovered in four distinct clades. Based on our results, we transfer Paraiptasia from Aiptasiidae to Sagartiidae.These findings emphasize the utility of genome-scale data for resolving phylogenetic ambiguities for morphologically problematic taxa and suggest a framework for future integrative taxonomic and ecological studies within Acuticulata.

Methods
Actiniarian specimens were collected either by hand, while SCUBA diving, or via trawls. Specimens were identified using external polyp anatomy and preserved in 95% ethanol to ensure the integrity of molecular data. Twelve specimens from the Falkland Islands were collected by hand while SCUBA diving (maximum depth of 20m) during the 2019 research cruise for the “Fine Scaling of the Marine Management Area of the Falkland Islands” project. Collection data for each specimen is contained in Appendix 1.

DNA extraction was performed using the E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s protocol, except with a reduced elution volume to increase concentration. The quality and quantity of extracted DNA were evaluated using a Qubit 2.0 fluorometer and a NanoDrop spectrophotometer, measuring DNA concentration (ng/μL). Specimens with remaining tissue have been deposited at the American Museum of Natural History (AMNH). Taxa sampled represent 75% of the acuticulate diversity at the family-level (9 of 12 valid families), and include several unidentified individuals collected from the Falkland Islands in 2019. No members of Haliactinidae Carlgren, 1949, Haliplanellidae Hand, 1956, nor Sagartiomorphidae Carlgren, 1934 were considered in the phylogenetic framework presented here. 

Up to 1000 ng of DNA per sample was used for library preparations, which were conducted using the Kapa HyperPrep Kit (Kapa Biosciences, Wilmington, MA, USA), optimized for target capture. Universal Y-yoke oligonucleotide adapters and iTru dual-indexed primers were used as described in Glenn et al. Eleven libraries were pooled in equimolar ratios, each contributing approximately 100 ng, for a total of 1.3–2.3 μg of DNA per pool for target enrichment. 

Target enrichment and sequencing were conducted at Arbor Biosciences (Ann Arbor, MI, USA). The hexacoral-v2 bait set  used contained a total of 25,288 baits targeting 2,476 (1,127 UCE and 1,349 exon) loci. Target enrichment was conducted according to the MyBaits v.IV protocol with bait concentrations of 500 ng per reaction. Libraries were sequenced using 150bp paired-end sequencing on an Illumina NovaSeq platform. 

Paired-end reads were trimmed to remove low-quality bases and adapters using TrimGalore. Trimmed reads were then assembled into contigs using SPAdes genome assembler v3.15.5.