<p>Plakortis2015_ESM 3</p><p>Molecular differences between Plakortis angulospiculatus and P. halichondrioides. Maximum likelihood phylogeny of Homoscleromorpha based on DNA sequencing of a 400 bp fragment at the 5’ end of the gene encoding the large (28S) subunit of nuclear ribosomal RNA. To confirm species identifications for this study, genomic DNA was extracted from specimens identified as Plakortis angulospiculatus and P. halichondrioides, using the Wizard Genomic DNA Purification Kit (Promega), following the manufacturer’s protocol for animal tissue. PCR amplification and sequencing followed the methods described by Thacker and Starnes (2003). Sequences obtained (labeled in bold font) were deposited in GenBank under accession numbers KP698747-KP698749. Sequences were aligned to other 28S sequences from representatives of Haploscleromorpha deposited in GenBank using the MAFFT alignment algorithm with its default parameters (Katoh and Standley 2013). This maximum likelihood phylogeny was constructed using RAxML software (Stamatakis et al. 2008). Scale bar indicates 0.2 substitutions per site. Numbers at nodes indicate percentage bootstrap support; values less than 50% are not shown. These sequences clearly distinguished between P. angulospiculatus and P. halichondrioides. Although this short fragment of DNA can be used to identify species, it may not accurately reconstruct relationships among species (Thacker et al. 2013).</p><p>Supplemental References</p><p>Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772-780</p><p>Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web- servers. Systematic Biol 75:758-771</p><p>Thacker RW, Hill AL, Hill MS, Redmond NE, Collins AG, Morrow CC, Spicer L, Carmack CA, Zappe ME, Pohlmann D, Hall C, Diaz MC, Bangalore PV (2013) Nearly complete 28S rRNA gene sequences confirm new hypotheses of sponge evolution. Integrative Comp Biol 53:373-387</p><p>Thacker RW, Starnes S (2003) Host specificity of the symbiotic cyanobacterium Oscillatoria spongeliae in marine sponges, Dysidea spp. Mar Biol 142:643-648</p>