DNA Amplification and Sequencing:
Forty one specimens of 33 species were used for phylogenetic analyses: 25 opheliids, four polygordiids, and 10 scalibregmatids. The two outgroup taxa, a capitellid Notomastus sp. and an arenicolid A. marina were chosen based on Struck et al.’s (2011) annelid phylogeny (Table 1) with Notomastus sp. being used as the root terminal. Newly collected specimens [from Beaufort, NC (Ophelina sp1.); Costa Rica (Ophelina sp3.); Friday Harbor, WA (Notomastus sp., O. acuminata, Polygordius sp., and Scalibregma inflatum); Greenland (O. acuminata, O. cylindricaudata, and O. limacina); La Jolla, CA (A. brevis, Polyophthalmus sp., and T. mucronata); Lizard Island, Australia (Armandia sp1.); and off the Oregon coast (Ophelina sp2.)] were relaxed in 7.5% MgCl2 and fixed in 95% ethyl alcohol. Sequences for the remaining 26 species were accessed through GenBank (Table 1).

A Qiagen DNeasy tissue kit was used to extract genomic DNA from specimens according to the manufacturer’s instructions. Approximately 500 base pairs of the mitochondrial small subunit ribosomal DNA (16S) were amplified using the primers 16SarL and 16SbrL (Palumbi, 1996) with temperature profiles of 95C for 3 min, followed by 40 cycles of 95C for 40 s, 48C for 40 s, 68C for 50 s, and final extension at 68C for 5 min (see Supporting Information, Table S1).

Three nuclear loci were also sequenced. The small subunit ribosomal DNA (18S) was amplified using three primer sets: 1) 1F and 5R; 2) 3F and bi; and 3) a2.0 and 9R (Giribet et al., 1996, 1999). Temperature profiles for the 1F/5R and a2.0/9R primer sets were 95C for 3 min, followed by 40 cycles of 95C for 30 s, 52C for 30 s, 72C for 90 s, and final extension at 72C for 8 min. The temperature profile for the 3F/bi primer set was 95C for 3 min, followed by 40 cycles of 95C for 30 s, 49C for 30 s, 72C for 90 s, and final extension at 72C for 8 min. Approximately 930 base pairs of the large subunit ribosomal DNA (28S) were amplified using the primers Po28F1 and Po28R4 (Struck et al., 2006), and ~360 base pairs of the nuclear protein coding gene Histone H3 were amplified using the primers H3aF and H3aR (Colgan et al., 1998). Both genes were amplified using the same temperature profiles of 94C for 2 min, followed by 35 cycles of 94C for 45 s, 48C for 60 s, 72C for 90 s, and final extension at 72C for 10 min.

Amplification reactions (25 ml) were conducted containing 2 ml of DNA template, 1 ml of forward and reverse primers, 12.5 ml GoTaq Green Master Mix (Promega), and 8.5 ml H2O. ExoSAP-IT (Affymetrix) was used to purify PCR products. Sequencing was done by either Retrogen (San Diego, CA) or Eurofins MWG Operon (Louisville, KY). Sequences were edited using Geneious 5.5.6 (www.geneious.com) and aligned with MAFFT 3.8 (Katoh and Kuma, 2002) under default settings with no manual alterations. The combined molecular dataset consisted of 3,955 total characters, 1,075 of which were parsimony informative and 436 were uninformative.

Phylogenetic Analysis:
Parsimony analyses on the combined genes (16S, 18S, 28S, and H3) were conducted in PAUP* 4.0b10 (Swofford, 2002) using a heuristic search with random stepwise addition of the terminals for 1,000 replicates, with tree bisection and reconnection. The character matrix was equally weighted, and gaps were treated as missing data. Clade support was assessed using jackknifing with 37% deletion of sites over 1,000 replicates with 10 random additions per iteration. Maximum likelihood analyses were performed in RAxML 7.2.8 (Stamatkis, 2006) as a four-gene partitioned dataset and under the General Time Reversible1Gamma (GTR1G) model. Bootstrap (thorough option) values were estimated using 100 pseudoreplicates under the same model.

BCO-DMO Processing:

- added conventional header with dataset name, PI name, version date, reference information
- renamed parameters to BCO-DMO standard
- replaced blanks with NA
- transformed gene columns to rows
- added html links to GenBank accession pages

polych_accnums.csv