Samples for DNA analysis were preserved in nondenatured 95% ethanol for long-term storage after DNA extraction. Most samples were extracted within a few days of sorting and placement in 95% ethanol. For species smaller than ~25mm3, at least one intact individual was retained from at least one collection as a physical voucher. Up to three individuals from the remaining collections were removed and the entire organism extracted. For species larger than ~25mm3, an intact individual from one collection was retained where possible, as long as three other individuals were present from which to remove a small portion for extraction (i.e., at least 4 total individuals). If fewer than four individuals were collected, the smallest portion allowable for DNA extraction was removed from each from a non-taxonomically important region of the specimen.

DNA extraction was performed with the Qiagen DNeasy Kit, using standard protocols. Tissues were dissected under sterile conditions, and digested with proteinase K until no solid pieces of tissue were visible. Purified genomic DNA was eluted in Buffer AE, supplied in the DNeasy Kit. Elution volumes varied to reflect original individual size, but in general were 100-200 L.

An approximately 650 base pair region of the cytochrome oxidase C subunit I (COI) gene was amplified using standard primers and thermal cycler profiles, performed in a Perkin-
Elmer 480 thermal cycler. PCR products were electrophoresed through 1% agarose/TBE gels, stained with ethidium bromide, and visualized under UV light. Target bands were purifed from the PCR reactions using the Qiagen PCR Cleanup Kit, following manufacturer's protocols. Purified COI PCR products were eluted in Buffer EB (10mM Tris) in preparation for sequencing.

Sequencing reactions consisted of purified COI products, one of the PCR primers as sequencing primer, and the BigDye 3.1 Terminator sequencing chemistry (ABI). The reactions were one-quarter standard volume (1/4X) and were performed in an ABI 9600 thermal cycler according to manufacturer's protocols. The sequences were then purified by ethanol/EDTA/sodium chloride precipitation, followed by centrifugation to pellet the sequenced DNA. Sequences were washed with 80% ethanol and re-centrifuged, then dissolved in 10 L high-quality deionized formamide (HiDi, ABI) in preparation for sequence determination. This was conducted on a 4-capillary 3130 DNA Sequencer (ABI) using a 50cm capillary array and standard operating conditions. A one-hour electrophoresis time on the 3130 produced approximately 500-700 base pair reads in one direction, providing complete or almost complete bi-directional coverage of the COI gene fragment.

Both DNA strands were aligned for each single individual sequenced, and checked against each other for discrepancies. All discrepancies were corrected by eye (or, if discrepancies were not easily resolvable, the sequences were deemed unreadable and the PCR repeated) before producing edited, finalized COI barcodes.