Computational protocol: Examination of Clock and Adcyap1 gene variation in a neotropical migratory passerine

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Protocol publication

[…] For Clock and Adcyap1 analysis, we collected a total of 60 blood and feather samples from adult male Painted Buntings [] trapped with mist-nets at three geographically distant breeding sites (). The westernmost sampling site was the Wichita Mountains National Wildlife Refuge, Oklahoma (OK; N34.4 –W98.4), where we collected blood samples from 19 adult (after second year) males that were also equipped with geolocators [see for complete methodology].The next sampling site was Johnson Bayou, Louisiana (LA; N30.0 –W89.1), which is near the eastern extent of the larger western population. These birds represent, at least geographically and in some aspects of their morphology, an intermediate population between Oklahoma and the Atlantic coast. At this site, we obtained 21 primary or secondary feathers plucked from migrants arriving at the breeding grounds early in spring migration during the years 2011, 2012, and 2013. We measured body mass (g) and wing chord (mm) for each individual. In Louisiana, at a second field site (LA; N31.3 –W91.3), we also collected 16 blood samples which were used for neutral loci analysis (see next section).We collected blood samples from 20 adult male Painted Buntings at Bald Head Island, North Carolina (NC; N33.5 –W78.0), which is a representative site of the allopatric eastern breeding population. In North Carolina, as in Oklahoma and in the second field site in Louisiana, we collected ~40 ul whole blood from the brachial vein of each individual and stored these samples in 0.4 ml of Queen’s lysis buffer at 4°C for later genetic analyses.All work with animals was performed with relevant state and federal banding permits (Permit #23215) and was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma (Protocol #R12-019). [...] We used the program ARLEQUIN v.3.5 [] to test for deviation from Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD) and estimated the p-values through a Markov chain Monte Carlo (MCMC) algorithm of one million iterations and significance level α < 0.05 [, ]. We estimated population differentiation at each gene, Clock and Adcyap1, and at the neutral loci through FST and RST statistics and significance level α < 0.05 [] and calculated the genetic distances between all pairs of populations (δμ2) based on a microsatellite stepwise mutation model []. We then used δμ2 to build a tree diagram with the Neighbor-joining method to illustrate genetic distances at Clock and Adcyap1 [, ].We implemented a Bayesian population clustering algorithm in the program STRUCTURE [] and analyzed Clock and Adcyap1 separately to determine the contribution of each locus to the observed population differentiation. The parameters for our Bayesian population clustering model included assumptions of correlated allele frequencies, admixed populations, and incorporated the sample locations of origin (LOCPRIOR) to assist the clustering algorithm []. For each value of K ranging from 1 to 3, we ran 10 simulations with 106 MCMC repetitions each, which included an initial burn-in of 105 iterations. We then plotted the log probability [L(K)] of the data over multiple runs in STRUCTURE HARVESTER [] to determine the most likely number of clusters among the samples []. We used the software CLUMPP to merge the models with the highest likelihood assignment values and used the software DISTRUCT to visualize the results [, ]. We tested for allele size differences across Oklahoma, Louisiana, and North Carolina populations by implementing unpaired Student’s t-tests.We also implemented a spatially explicit clustering algorithm in the R program TESS3 [] and computed ancestry coefficient estimations across populations based on Clock, Adcyap1, and four putative neutral genotypes. TESS3 implements least-squares and matrix factorization [, ] providing a population Q-matrix clustering output similar to STRUCTURE and therefore suited for result comparisons and cross-validations. […]

Pipeline specifications

Software tools bayou, Arlequin, CLUMPP, DISTRUCT, TESS
Applications Phylogenetics, Population genetic analysis
Organisms Homo sapiens