Recently, it was suggested that North America has lost nearly 30% of its avifauna since the 1970s.  To mitigate such avian population declines, many birds in California are protected under the Migratory Bird Treaty Act and California Fish and Game Code. Avian presence, therefore, at Caltrans infrastructure projects, especially bridge construction, has caused costly delays.  To avoid this conflict and understand population-specific migration and nesting patterns, we used Anna’s hummingbird, Calypte anna, (a species whose ecology has led to conflicts with construction in California) as a case study to assess population-level genetic variation across California, USA.  We sequenced whole genomes at low coverage from 40 individuals across 9 California counties and assessed population differentiation.  This project initiates a general framework for assessing population-specific impacts of Caltrans projects on avian populations with the future goal of providing specific tools to avoid impacts with hummingbirds nesting on or around bridges. 

Sample collection

Calypte anna hummingbirds were trapped using previously published methods (Bandivadekar et al., 2018) by master hummingbird banders.

DNA extraction

Whole genomic DNA was extracted from 100-150ml of blood stored in lysis buffer using the DNeasy Blood & Tissue Kit (Qiagen).  The following modifications to the extraction protocol were used: samples were incubated overnight at 56°C, 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 200ml AE buffer heated to 56°C.  Whole genomic DNA was quantified using a Qubit Fluorometer and the quality of DNA was assessed using a 2% agarose gel.

Library preparation and sequencing

We used a modified library preparation based on Illumina’s Nextera protocol (Baym et al., 2015; Overgaard Therkildsen and Palumbi, 2017).  To start, genomic DNA was standardized to 3ng/ml then underwent a tagmentation step using TDE1 enzyme and buffer (Illumina).  Dual combination Nextera indexes (Illumina) were then added to tagged DNA fragments followed by a reconditioning step using the Kapa HiFi Kit (Kapa Biosystems).  Libraries were then double size selected using AMPure XP Beads (Beckman Coulter) and quantified using a Qubit Fluorometer (Thermo Fisher Scientific).  Sixteen libraries only went through a left side selection.

All libraries were pooled equimolarly then visualized with a Bioanalyzer (Agilent).  The pooled libraries were further size selected to 320-600bp fragments using a Blue Pippin (Sage Science) at the University of California, Davis Genome Center.  The final library was sequenced on an Illumina HiSeq with PE150 and the resulting sequences were demultiplexed by Novogene (Sacramento, CA, USA).