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.