DOI: 10.5061/dryad.gxd2547rw
title: A cryptic radiation of Caribbean sea slugs revealed by integrative analysis: Cyerce ‘antillensis’ (Sacoglossa: Caliphyllidae) is six distinct species

description: Integrative studies have revealed cryptic radiations in several Caribbean
 lineages of heterobranch sea slugs, raising questions about the
 evolutionary mechanisms that promote speciation within the tropical
 Western Atlantic. Cyerce Bergh 1871 is a genus comprising 12 named species
 in the family Caliphyllidae, lacking the photosynthetic ability of other
 sacoglossans but noted for vibrant colors on the large cerata (dorsal
 leaf-like appendages) that characterize many species. Two species are
 widely reported from the Caribbean: C. cristallina (Trinchese, 1881) and
 C. antillensis Engel, 1927. Here, we present an integrative assessment of
 diversity in Caribbean Cyerce. Four methods of molecular species
 delimitation supported seven species in samples from the Caribbean and
 adjacent subtropical Western Atlantic. Six delimited species formed a
 monophyletic lineage in phylogenetic analyses but were &gt;9%
 divergent at the barcoding COI locus and could be differentiated using
 ecological, reproductive and/or morphological traits. We redescribe C.
 antillensis Engel, 1927, a senior synonym for the poorly known C.
 habanensis Ortea & Templado 1989, and describe five new species.
 Evolutionary shifts in algal host use, penial armature and larval life
 history may have synergistically promoted the rapid divergence of endemic
 species with restricted distributions in this radiation, substantially
 increasing global diversity of the genus.
description: Specimen
 collections Samples comprising 92 specimens
 were collected from the Caribbean and adjacent subtropical western
 Atlantic waters. Most specimens were collected by the authors with
 permission from the State of Florida (FL) under Special Activity Licenses
 (07SR-1034, SAL-11-1034-SR, SAL-19-1034-SR), or from the host country as
 needed, including special permission granted to authors by the Autoridad
 de los Recursos Acuaticos de Panama (ARAP 27-2015 and ANAM SE/A-64-15),
 and from the Dept. of Fisheries of the Bahamas to Joseph R. Pawlik and
 participants of research cruises in 2003, 2004, 2007 and 2010. Exceptions
 were specimens collected from Lauderdale by the Sea, FL (by A. Dimitris);
 Summerland Key, FL (by Y. Gryzmbowski); and Dominica (by D. Willette).
 Some Caribbean specimens were identified as <em>C.
 </em>cf.<em> cristallina </em>based on their distinctive
 external morphology, but no Mediterranean samples were available for
 comparison; all other Caribbean samples were provisionally identified as
 <em>C. antillensis</em> in accordance with recent literature
 (Valdés et al., 2006; Goodheart et al., 2016). Specimens were preserved in 95–100%
 ethanol for molecular analysis. The type material of newly described
 species was deposited at the Natural History Museum of Los Angeles County
 (LACM) and the California Academy of Sciences (CASIZ). Non-type vouchers
 were deposited at the museums listed above as well as at the research
 collections of Cal State L.A. and the California State Polytechnic
 University Pomona (CPIC). Sequences from the National Center
 for Bioinformatics (NCBI) database were included for 13 specimens
 representing nine species of nominal <em>Costasiella</em>,
 <em>Sohgenia</em> and<em> Cyerce </em>spp., based
 on prior work analyzing relationships within Limapontioidea (Krug et al.,
 2015, 2018; Filho et al., 2019). Additional specimens of
 <em>Cyerce</em> were included in phylogenetic analyses to test
 whether Caribbean samples identified as <em>C.
 antillensis</em> formed a monophyletic group consistent with an
 endemic radiation. Samples of two species of the related genus
 <em>Sohgenia </em> were also included, given prior studies
 that recovered <em>Sohgenia </em> nested within
 <em>Cyerce</em> (Krug et al., 2015). Molecular studies have
 consistently recovered <em>Cyerce</em> outside family
 Caliphyllidae contrary to traditional morphological classification
 schemes, including phylogenetic studies using a few markers and many taxa
 (Krug et al., 2015; Christa et al., 2015), and phylogenomic analyses of
 1,160 genes for representative taxa (Krug et al., 2022). Given that no
 sister group has been identified for <em>Cyerce</em> using
 molecular data, we included<em> </em>an exemplar of
 <em>Costasiella</em> as the outgroup in all phylogenetic
 analyses based on phylogenetic results that recovered both genera as early
 splitting lineages at the base of Limapontioidea (Krug et al.,
 2022). ecological and life-history
 data collection Specimens of
 <em>Cyerce</em> spp. were collected from four sites within FL
 and a further 14 Caribbean sites. Each potential udotacean algal host was
 separately collected (0.5˗1 kg wet weight) at every sampling site; algal
 stipes were removed from the substrate, placed in a bag underwater and
 finally transferred to an aerated bin of seawater, where slugs were
 removed. Specimens were provisionally identified under a stereomicroscope
 and where possible, isolated and allowed to lay eggs. To determine if
 slugs could successfully change host algae, specimens from Tarpon Springs,
 FL and St Petersburg, FL were collected from <em>Penicillus
 lamourouxii </em>or <em>Udotea looensis</em> and either
 maintained on the field host (<em>N</em>=6 slugs per alga) or
 switched to the alternative host (<em>N</em>=6 slugs per
 treatment). Slugs were monitored daily for feeding and survival over 4
 weeks. Specimen
 collections DNA extraction, amplification and
 sequencing Specimen
 collections EZNA Mollusc DNA kits (Omega
 Bio-Tek) or a hot Chelex<sup>®</sup> protocol were used to
 extract DNA from one ceras or ~2 mg foot tissue, following described
 protocols (Krug et al., 2015; Medrano et al., 2019). Portions of two
 mitochondrial loci were then amplied by PCR: the cytochrome
 <em>c</em> oxidase I (COI) gene, using primers LCO1490 and
 HCO2198 (Folmer et al., 1994) with custom modifications as described
 (Ellingson & Krug, 2016); and the large ribosomal subunit rRNA
 (16S) gene, using primers 16Sar-L and 16Sbr-H (Palumbi, 1996). The nuclear
 Histone H3 gene was also amplified using universal primers (Colgan et al.,
 1998). Purified products were cycle-sequenced using PCR primers at
 Retrogen (San Diego, CA) or Source BioScience Sanger Sequencing Laboratory
 (Santa Fe Springs, CA). Chromatograms were edited in Geneious Pro R8
 (Kearse et al., 2012) and alignments performed using default parameters
 for Multiple Sequence Comparison by Log-Expectation (MUSCLE) (Edgar 2004).
 Published secondary structure models guided refinements of rRNA gene
 alignments (Krug et al., 2015). Regions of ambiguous alignment in
 hypervariable loop regions of rRNA genes were identified using the least
 stringent criteria in GBLOCKS (Castresana, 2000) and removed prior to
 analyses.
description: All data files can be viewed with a standard text
 editor. "DNA alignments" are text files in
 Phylip format used as inputs by the program RaxML, as specified in the
 README file. see: https://doi.org/10.1093/bioinformatics/btu033 "Arlequin input files" are input files for the program Arlequin, as specified in the README file. see: http://cmpg.unibe.ch/software/arlequin35/ "Popart input files" are input files for the program Popart, as specified in the README file. see: http://popart.otago.ac.nz
description: # Data files supporting "A cryptic radiation of Caribbean sea slugs:
 integrative analysis indicates Cyerce ‘antillensis’ (Sacoglossa:
 Caliphyllidae) is six species." Moreno, K., Rico, D.M., Middlebrooks,
 M., Medrano, S., Valdés, Á.A., and P.J. Krug. Zoological Journal of the
 Linnean Society, in press (2022). ## Description of the data and file
 structure DNA alignments A) Phylip file used for RaxML analysis of
 concatenated three-gene dataset (Cyerce_3gene_alignment_12_12_21.nex) B)
 Partitioning scheme used in RaxML analysis of concatenated three-gene
 dataset C) Phylip file used for analyses of COI dataset (Cyerce_COI.nex)
 D) Partitioning scheme used in RaxML analysis of COI dataset Arlequin
 input files E) Arlequin input file for population genetic analyses of COI
 dataset for Cyerce antillensis, using 655-bp haplotypes (Arlequin input
 2021 C_antillensis.ARP) F) Arlequin input file for population genetic
 analyses of COI dataset for Cyerce nicholasi, using 658-bp haplotypes
 edited to allow inclusion of shorter public sequences for two additional
 Florida Keys sites (Arlequin input 2021 C_nicholasi.ARP Popart input files
 G) Popart input file for network analyses of COI dataset for Cyerce
 antillensis, using 655-bp haplotypes for six populations (COI
 network_antillensis_network_formatted.nex) H) Popart input file for
 network analyses of COI dataset for Cyerce nicholasi, using 656-bp
 haplotypes for six populations (COI
 network_nicholasi_formatted_network.nex) I) Popart input file for network
 analyses of Histone 3 alleles for Cyerce antillensis complex, using 328-bp
 phased allelic haplotypes from seven species (H3 alleles for
 network_species names coded.nex) ## Sharing/Access information All raw
 sequences also archived in the National Center for Bioinformatics (NCBI)
 database; see associated publication for accession numbers. Voucher
 specimens deposited in collections; see associated publication for
 accession numbers.