DOI: 10.5061/dryad.vmcvdnd32
title: Resolving Acuticulata (Metridioidea: Enthemonae: Actiniaria), a clade containing many invasive species of sea anemones

description: 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 phylogenetic relationships and outdated taxonomic frameworks hinder a full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.
description: 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.
description: # Resolving Acuticulata (Metridioidea: Enthemonae: Actiniaria), a clade
 containing many invasive species of sea anemones
 [https://doi.org/10.5061/dryad.vmcvdnd32](https://doi.org/10.5061/dryad.vmcvdnd32) ## Description of the data and file structure 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. Data included are the resulting assembled contigs. ### Files The identification for the files is as follows: Acri_ADK_10.contigs.fasta – *Acricoactis brachyacontis* Actisp_PML.contigs.fasta – *Actinothoe* sp. Aipge_PJM_2.contigs.fasta – *Aiptasiogeton eruptaurantia* Aipgh_AD_21.contigs.fasta – *Aiptasiogeton hyalinus* Aipmt_FSBC.contigs.fasta – *Aiptasiomorpha texaensis* Aiptm_AD_16.contigs.fasta – *Aiptasia mutabilis* Alics_196882.contigs.fasta – *Alicia sansibarensis* Alicsan_MAB_41.contigs.fasta – *Alicia sansibarensis* Antha.contigs.fasta – *Antholoba achates* Antha_FI_64.contigs.fasta – *Antholoba achates* Anthc_FI_Unid_22.contigs.fasta – *Anthothoe chilensis* Barta_BH_39.contigs.fasta – *Bartholomea annulata* Bellsp_AD_28.contigs.fasta – *Bellactis lux* Bolocfm_CAL_23.contigs.fasta – *Boloceroides* sp. Bolom_198131.contigs.fasta – *Boloceroides* sp. Bolomc_CAL_15.contigs.fasta – *Boloceroides* sp. Bolomcm_CAL_16.contigs.fasta – *Bunodeopsis* sp. Bolomcmu_CAL_23.contigs.fasta – *Boloceroides* sp. Bolomcmur_MAB_48_2.contigs.fasta – *Boloceroides* sp. Bunosp_CAL_23.contigs.fasta – *Bunodeopsis* sp. Callp_AMNH.contigs.fasta – *Calliactis polypus* Callt_4.contigs.fasta – *Calliactis tricolor* Cereh.contigs.fasta – *Cereus herpetodes* Diadl_DART.contigs.fasta – *Diadumene lineata* Diadl_DL_CB.contigs.fasta – *Diadumene* sp. Diadn_N_91_1.contigs.fasta – *Diadumene neozelanica* Diadp_GAIL.contigs.fasta – *Diadumene paranaensis* Diadsp_FG_07.contigs.fasta – *Diadumene* sp. Diadspe_FG_20.contigs.fasta – *Aiptasiogeton* sp. Diadspec_MAND.contigs.fasta – *Diadumene* sp. Diadspeci_orange.contigs.fasta – *Diadumene* sp. Diadspecie_WS2.contigs.fasta – *Diadumene neozelanica* FI_36.contigs.fasta – *Isoparactis fionae* FI_37.contigs.fasta – *Isoparactis fionae* FI_38.contigs.fasta – *Isoparactis fionae* FI_40.contigs.fasta – *Anthothoe chilensis* FI_532_2b.contigs.fasta – *Anthothoe chilensis* FI_532_6.contigs.fasta – *Anthothoe chilensis* FI_533_3.contigs.fasta – *Antholoba achates* FI_73.contigs.fasta – *Anthothoe chilensis* Gonap_Norway.contigs.fasta – *Gonactinia prolifera* Isopf_FI_29.contigs.fasta – *Isoparactis fionae* Neoam_26.contigs.fasta – *Neoaiptasia morbilla* Paipr.contigs.fasta – *Paraiptasia radiata* Parasp._ONIO.contigs.fasta – Acuticulata indet. Phyls_CAL_06.contigs.fasta – *Phyllodiscus semoni* Prots_Norway.contigs.fasta – *Protanthea simplex* Protsp_Blane.contigs.fasta – *Protanthea* sp. RedBrooding_FI_39.contigs.fasta – Sagartiidae sp. Sagae_5_1.contigs.fasta – *Cylista elegans* Triap_MAB_36.contigs.fasta – *Triactis producta* Verip_39.contigs.fasta – *Verrillactis paguri* resolving_acuticulata_code.txt - Code used for generating phylogenies ## Code/software See resolving_acuticulata_code.txt for details on the software used.