DNA extraction and species identification
Whole genomic DNA was extracted from muscle tissue (for museum collection birds or carcasses from rehabilitation centers), 100-150µl of blood stored in lysis buffer, or 2 to 3 blood spots from a blood collection card using the DNeasy Blood & Tissue Kit (Qiagen). The following modifications to the extraction protocol were used: samples were incubated overnight at 56ºC, the sample was passed over the spin column twice prior to washing, an extra column drying step was taken (14000rpm for 3min), and DNA was eluted in 200µl AE buffer heated to 56ºC. Whole genomic DNA was quantified using a Qubit Fluorometer (Thermo Fisher Scientific) and the quality of DNA was assessed using a 2% agarose gel.
Because nestling and fledgling hummingbirds are difficult to identify to species, we used molecular methods to determine which of the juvenile samples represented C. anna and thus could be used in our study. To identify nestlings and fledglings from the wildlife centers asC. anna we used Sanger sequencing to sequence 32 unknown individuals and 30 known samples from Anna’s (C. anna ) and other hummingbird species likely to be collected in the region: Costa’s (C. costae ), Allen’s (Selasphorus sasin ), Calliope (S. calliope ), and Rufous (S. rufus ) Hummingbirds. We amplified part of the NADH dehydrogenase subunit 2 (ND2) gene using H6313 and L5219 primers , cleaned the products using an ExoSAP protocol, then sequenced them at UCDNA Sequencing Facility at the University of California, Davis. We trimmed and aligned the resulting sequences and used the Neighbor-joining method to build the tree in Geneious v. 9.1.7 (http://www.geneious.com/). We visualized the phylogenetic tree with FigTree (http://tree.bio.ed.ac.uk/software/figtree/) using a Black-chinned Hummingbird sample to root the tree. Black-chinned Hummingbird was used because it was outside of the Selasphorus and Calyptesubclades between which we were identifying unknown samples. ND2 is a marker often used to create hummingbird phylogenies , and in our work has consistently separated individuals that are C. anna from other hummingbird species found in California.