Gavia Species Identification and Diet Composition from Feces, Central North Slope, Alaska, 2012-2014

Fecal samples were collected opportunistically from known Yellow-billed Loon latrine sites on the shorelines of breeding territories. Observed fecal depositions or wet feces at the sites were stored in 95% ethanol at ambient temperature in the field then transferred to -20°C until lab processing when, for each fecal sample, approximately 1 g of fecal material was transferred to a 15 mL conical tube containing 5 mL of Longmire Buffer (LMB) (Longmire et al. 1997).

This amplicon-based environmental DNA (eDNA) study was designed with a targeted list of species of interest present in Genbank at the time of development and was not intended to identify all species present within each sample. Given design parameters and method limitations, it is possible unassigned DNA sequences represent previously undocumented variants of expected/detected species, represent non-target species, or represent sequencing errors intrinsic in the laboratory method. It is expected that not all species present in the eDNA sample were detected. Salvelinus fontinalis and Thymallus arcticus baicalensis are not expected in waters of the North Slope of Alaska, but reference sequences in NCBI Genbank were used to design primers to exclude these species. Each sample was vortexed gently until homogeneous. DNA was extracted from a 400 µL subsample of the homogenized LMB preserved fecal sample using a Qiagen (Germantown, MD) DNeasy Blood and Tissue kit following manufacturer’s suggested protocols, with the exception that volumes were doubled. Polymerase chain reaction (PCR) primers were designed following Menning and Talbot (2018) to target multiple loci of multiple fish species found in Alaska as well as all five loon species (Menning et al ****). eDNA libraries were prepared using a two-step PCR protocol. In the first step, 2 mixtures of equimolar concentrations of all fish primers and Gavia sp. primers, respectively were used in 2 separate PCR reactions for each DNA. These reactions consisted of 12.5 µL 2X Type-it Multiplex PCR Master Mix (Qiagen), 2.5 µL 10X primer mix (2 µM of each primer mix), and 10 µL of template DNA. Reactions were thermocycled 95°C for 5 minutes, followed by 32 cycles of 95°C for 30 seconds, 60°C for 3 minutes, and 72°C for 30 seconds, with a final extension step at 60°C for 30 minutes. Excess primers and dNTPs were removed from products using ExoSap (Affymetrix, Santa Clara, CA), quantified by fluorometry using a Quant-IT dsDNA Broad Range Assay Kit (Thermo Fisher Scientific, Waltham, MA), normalized to equal concentrations, and pooled by sample. In the second step PCR to produce barcoded amplicons, 1.0 µL pooled PCR product, 2.0 µL molecular grade water (Thermo Fisher Scientific), 5.0 µL Phusion High-Fidelity PCR Master Mix with HF Buffer (New England BioLabs, Ipswich, MA), and 1.0 µL of each I5 and I7 Nextera XT (Illumina, San Diego, CA) index primer were thermocycled under the following conditions: 98°C for 30 seconds, followed by 20 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 40 seconds, with a final 5-minute elongation step at 72°C. Second-step PCR products were quantified by fluorometry using a Quant-IT dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific), normalized to equal concentrations, and pooled. Quantified, pooled PCR products were electrophoresed on a 1% polyacrylamide gel to visualize PCR fragment sizes and gel-purified using QIAquick Gel Extraction Kit (Qiagen). Gel-purified PCR products were quantified by fluorometry using a Quant-IT dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific), diluted to 4 nM concentrations following Illumina guidelines (Illumina Document # 15039740 v01), re-quantified by fluorometry using a Quant-IT dsDNA High-Sensitivity Assay Kit, and further diluted to 20 pM following the Illumina NextSeq Protocol A (Illumina Document #15048776 v02) for library dilution. All remaining steps for library preparation followed Illumina MiSeq protocols (Illumina Part #15034097 Rev. B). The final DNA library and PhiX were diluted to 15 pM. Sequencing (2 X 151 paired-end cycle runs) was performed with a 10% PhiX spike using a MiSeq Reagent Kit v2 (300 cycle) (Illumina Part #MS-102-2002) on an Illumina MiSeq.