Phylogenetic analysis
A 633-bp sequence was obtained from
the Cyt b gene and a 478-bp sequence was obtained from the CR,
which were integrated into one 1111-bp sequence. After excluding some
samples of poor quality, 78 individual sequences from 2015 and 173
individual sequences from 2016 were obtained. For all mtDNA sequences,
which combined the Cyt b and CR genes, coalescent sequences were
aligned by the algorithm Clustal W in Mega v7 (Kumar et al. 2016). Then,
DnaSP v5 and FaBox websites were used to generate haplotypes from the
original individual sequences (Villesen 2007, Librado and Rozas 2009).
In addition, 94 reported haplotypes were also downloaded from the
National Center for Biotechnology Information (NCBI)for further
alignment. The haplotypes were derived from 370 dunlin individuals
breeding in eastern Asia and Alaska, including
six
subspecies, C . a. actites , C . a.
kistchinski , C . a. sakhalina , C. a. hudsonia ,C. a. pacifica , and C. a. arcticola (GenBank
accessions for CR: KP205084–KP205177; GenBank accessions for Cytb : KP205178–KP205271) (Miller et al. 2014).
To infer to the phylogenetic relationships between dunlins from the
breeding areas and those sampled at Jiangsu, all sequences were analyzed
with Bayes inference (BI) and maximum-likelihood (ML) algorithms. The
program PhyML v3.1 was applied to infer phylogenetic relationships among
haplotypes under the ML criterion, with 1000 bootstrap replicates used
to evaluate clade support (Guindon et al. 2010). The best-fit nucleotide
substitution model for ML analysis was distinguished using Jmodeltest
v2.1.7
based on the Akaike information criterion (AIC) (Darriba et al. 2012).
MrBayes v3.2.2 was used to infer phylogenetic relationships by a
multigene Bayesian algorithm based on the model, as suggested by
Mrmodeltest v3.7 software (Ronquist et al. 2012). Four concurrent Markov
Chain Monte Carlo chains (one cold chain and three hot chains) were run
simultaneously for 5.0 × 107 generations. Trees were
sampled every 1000 generations, and the burn-in of the initial 25% of
samples was discarded.
Tracer
v1.5 was used to test and check the reliability of the results of
Bayesian analysis.