The complete plastomes of two H. flavescens individ-uals and two H. nujiangense individuals were aligned in Geneious with the MAFFT algorithm, and differences were identified by using the “Find Variations/SNPs” Two indices of genetic variation, haplotype diversity (Hd) and nucleotide diversity (π), were computed using DnaSP v6 (Rozas et al., 2017). analyse nucleotide diversity (Pi) using DnaSP v6.10.04 [39]. The parameters of sliding window analysis were set as: window length of 600 bp, the step size of 200 bp. DnaSP v6.11 (Rozas et al., 2017) was used to identify the number of haplotypes per location. Evolutionary divergence (nucleotide differences and p-distances) among the 32 accessions were evaluated using MEGA X . Rarefaction analyses were conducted via Analytic Rarefaction v1.3 (UGA Stratigraphy Lab, 2013) to check whether sample sizes from the various sampling locations analysed in … Bees (Hymenoptera, Apoidea and Anthophila) are distributed worldwide and considered the primary pollinators of angiosperm. Nucleotide diversity (average number of nucleotide differences per site between two sequences, π), number of variable sites, and PI sites were obtained using DnaSP v6 . For mtDNA we used DNAsp v6 (Librado and Rozas 2009) to calculate haplotype diversity. We calculated microsatellite allelic richness in MSA v4.05 (Dieringer and Schlötterer 2003) and expected heterozygosity in Arlequin v3.5 (Excoffier and Lischer 2010). was calculated using DnaSP v6. Regions with higher variability were selected as candidate markers. Haplotype networks In order to understand the relationships of the different haplotypes a haplotype network (of confirmed haplotypes) was constructed using Network v5.0.0.3 (www.fluxus-engineering.com) with median-joining calculation method (Bandelt et … Sliding window analysis (DnaSP v6 ) was conducted to generate Pi values of the plastid genomes. Megachilidae is one of the largest families of Anthophila. 388 Conservation Genetics (2020) 21:387–404 1 3 2010;Funketal.2012;Shaferetal.2015).Genomicmeth-ods,includingrestrictionenzymeassociatedDNAsequenc- Mol Ecol 24:1447–1466 Mariani S, Piscitelli MP, Uriz MJ (2001) Temporal and spatial co- Clarke KR, Gorley RN (2006) PRIMER v6: User manual/Tutorial. We estimated the genetic diversity and geographical range of each mOTU. Tamura 2015). Kimura 2 parameters (K2P) distance was calculated using MEGA-X. Hyper-variable regions were defined as a region with relatively high nucleotide diversity (Pi) and high species resolution. Thereafter we used DnaSP v6.12.01 to estimate genetic diversity indices and to generate haplotype data file by creating: (i) a haplotype list for Arlequin; and (ii) Roehl data file for Network software. occurrence in spawning and larval release of Cliona viridis PRIMER-E, Plymouth (Porifera: Hadromerida). What is more, the nucleotide polymorphism of the chloroplast genomes was estimated by DnaSP v6.11.01. DnaSP was also used to estimate levels of polymorphism within populations. All published plastomes were taken from NCBI (Table S1), rechecked by SnapGene 3.2.1 and manual aligned across the LSC-IRB-SSC-IRA regions for comparative analyses. We also conducted a sliding window analysis (window length: 500 bp, step size: 500 bp) by using DnaSP v6.0 [32] to calculate the nucleotide polymorphism (Pi) among the 4 species. The step size was set to 200bp, with a 500bp win-dow length.