Field

To complete the inventory of the island, samples will be taken from all elevation zones. The following four sites, Mouaputa (830 m), Tohiea (1207 m), Mouapu (762m), and Rotui (899 m) will be sampled from near sea level to the summit, where possible. Additional low and mid elevation sites will be sampled to fill in any elevations, geographical areas, or habitat types that were under sampled during the pilot study. In particular, we will target sites around Fairurani (741m) and Tearai (770m) to the northeast and Mateotea (714m) and Taufuapae (769m) to the northwest. Site selection will be done in consultation with the plant and fungal teams. Collections will be carried out using both quantitative and qualitative sampling methods. Insects, spiders, crustacea, mites, annelida, non-insect hexapods, and myriapods will be processed. Quantitative methods will provide statistically robust data (both numbers of species as well as abundance of individuals within species) for comparisons between sites and to provide a baseline for future surveys. Rare species and habitat types under-sampled quantitatively will be supplemented by standard qualitative sampling methods. CDC backpack aspirator (vegetation surface, aerial), Berlese extraction (forest litter/sedentary), and pitfall traps (forest litter/active) provide repeatable quantitative samples. Malaise trap (flying/climbing), flight intercept (flying/falling), blacklight (night flying), beating (vegetation), peeling barkexcavating wood (subcortical), sweep net (vegetation surface), and hand examination (vegetation) provide diverse nonquantitative samples. Teams of collectors will sample each site three times over the course of a 12-month period. Rainy season collections will be weather dependent, with heavy precipitation limiting high elevation sampling most. Each method will be evaluated to determine the number of samples per site and parameters of each sample. Depending on collections in year 1, selection of sites will be chosen for re-sampling in years 2 and 3. The quantitative sampling methods employed will allow for comparisons between sites and within sites over time. Additionally, environmental sequencing of mixed species samples could reduce processing effort.

Laboratory

Specimens will be sorted, databased, identified (to lowest taxonomic group possible), photographed, DNA-extracted, and mounted at the Gump Station immediately following each collection. Fragile specimens from each sample will be prepared for morphological identification on site (i.e., Lepidoptera and Miridae) with subsamples taken for DNA extraction. Specimens selected for barcoding will be prepared, photographed, and the whole abdomen will be removed and extracted nondestructively to allow for amplification of associated Wolbachia (endosymbiotic bacteria) from the gut. The abdomens will be removed from the lysis buffer, rinsed, and re-associated with the specimen. DNA extractions will be PCR amplified at either the Gump Station or Berkeley and up to 5 specimens of each morphospecies will be sequenced. Taxonomic workers (invertebrate coordinator, graduate students) working at Gump will identify specimens to species where possible. Where species or generic identification is not possible, the workers will distribute specimens to appropriate workers world-wide (see participants, http://polynesia.berkeley.edu) for identification and/or formal species identification. Voucher specimens and DNA extracts will be deposited in collections at the Essig Museum (Berkeley), Bishop Museum (Honolulu), the NHM Paris, with a diagnostic collection housed at Gump or elsewhere in French Polynesia. If and when long-term collection facilities are available in French Polynesia, specimens will be repatriated. LUCID keys will be prepared for several groups.

Timeline:

2008 – regular sampling of field sites, data processing, 1st cut taxonomic identification, COI barcoding; 2009, 2010 – additional sampling, continued specieslevel identification and formal description, COI barcoding.