DNA barcoding reveals long-term speciation processes in subspecies of the Melipona (Michmelia) seminigra complex (Hymenoptera: Apidae)

The stingless bee Melipona (Michmelia) seminigra Friese is a polytypic species widely distributed in Brazilian Amazon and Bolivia. Seven subspecies are recognized, four are described, which inhabit mutually exclusive areas in the Amazon basin, although zones of hybridization are recorded. The three other subspecies, despite being recognized by taxonomists are undescribed. Melipona seminigra is a good honey-producer and an important pollinator of native flora and crops. Partial DNA sequence of cytochrome c oxidase subunit 1 (COI) mitochondrial gene (526 bp) was used to identify the four described subspecies of M. seminigra (M. s. abunensis, M. s. merrillae, M. s. pernigra, M. s. seminigra) and two other possible subspecies (M. s. ssp1 and M. s. ssp2). We added public data (sequences of ten other Melipona species) of the same subgenera and carried out phylogenetic analyses. The aim was to evaluate if subspecies in the M. seminigra complex could be delimited using COI and measure the genetic distances between them. Our results revealed that the genetic distances between subspecies of M. seminigra ranged from 0.4 to 2.7% (average 1.80 ± 0.47) and among Melipona species from 0.2 to 2.9% (average 2.13 ± 0.5). The average haplotype diversity was 0.8770 ± 0.0140 and average nucleotide diversity 0.0166 ± 0.0004. Phylogenetic and clustering analysis revealed well delimited clusters for subspecies of M. seminigra and that the inter subspecies divergences are similar to inter species divergence. Our findings indicate that the COI gene can be used for delimiting subspecies of M. seminigra.