Microsatellite and mtDNA data analysis
Allele size range and private alleles were determined using CONVERT
version 1.31 (Glaubitz, 2004). Genetic diversity parameters such as
number of alleles per locus (NA), number of private alleles (PA),
observed (HO ) and expected
(HE ) heterozygosities were calculated by using
POPGENE version 1.31 (Yeh, Yang, Boyle, Ye, & Mao, 1997). Polymorphic
information content (PIC) and inbreeding coefficient
(FIS) were calculated with Microsatellite Tollkit
software (Park, 2001) and FSTAT version 2.9.3.2 (Goudet, 1995). Genetic
differentiation tests between populations (pairwise FST)
and molecular variance analyses (AMOVA) were conducted using ARLEQUIN
(Excoffier, Laval, & Schneider, 2005). The genetic structures among
populations and individuals were investigated using STRUCTURE version
2.3.1 (Pritchard, Matthew, & Donnelly, 2000).We estimated the optimalK value using the ΔK statistic as described by
Evanno,
Regnaut,
&
Goudet
(2005) in STRUCTURE HARVESTER (Earl & vonHoldt, 2012). In addition,
GENETIX v 4.05 (Belkhir, Borsa, Chikhi, Raufaste, & Bonhomme, 2004)
program was applied for Factorial Correspondence Analysis.
Sequences of mtDNA gene regions were aligned with CLUSTAL W2 (Larkin et
al., 2007). mtDNA sequences were checked with reference sequences (Gen
Bank accession number for COI and cyt b , respectively :
GU085204.1, JQ820820.1) by using BLAST 2.2.20 (Zhang, Schwartz, Wagner,
& Miller, 2000). DNA sequence alignment was performed by uploading all
sequence information to the MEGA6 program (Tamura, Stecher, Peterson,
Filipski, & Kumar, 2013) for both gene regions. Sequences for detection
of SNP regions were uploaded to DnaSP v.5 (Librado & Rozas, 2009)
software and each bee was then assigned to a mitochondrial haplotype. We
compared mitochondrial haplotype frequencies in each population using
ARLEQUIN 2.00 (Schneider, Roessli, & Excoffier, 2000). As a result of
statistical analyzes intra and inter-group genetic variations (F
statistics) and genetic distances of 14 B. terrestris populations
were calculated. The genetic distance information of the populations was
uploaded to SplitsTree 4.0 (Huson & Bryant, 2006) software, to conduct
phylogenetic analysis and to construct Neigbour-Joining (NJ) tree. In
order to determine the evolutionary ancestral origin of each individual
in B. terrestris populations, the Maximum Likelihood (ML) method
based on the Tamura-Nei model was used (Tamura & Nei, 1993). Trees were
created in MEGA X program using 633 bp nucleotide sequences for 66
samples for COI gene region and 428 bp nucleotide sequences for
68 samples for cyt b region (Kumar, Stecher, Li, Knyaz, &
Tamura, 2018). B. lucorum , used as an out group (OG) in the
present study, were retrieved from Gen- Bank (Accession number:
JQ843492.1) to construct both NJ trees.