DOI: 10.5061/dryad.cvdncjt3t
title: Integrative taxonomic analysis to reveal the species status of Bombus flavidus, combining COI and nuclear sequencing, wing morphometrics and secretions used for mate attraction as well as patterns of color polymorphism

description: Bumble bees, due to their morphological monotony and color diversity, have
 presented difficulties with species delimitation. Recent bumble bee
 declines have made it ever more imperative to resolve the status of
 species to address conservation concerns. Some of the taxa found to be
 most threatened are the often-rare socially parasitic bumble bees, which
 have additional trophic requirements. Among the socially parasitic bumble
 bees, Bombus flavidus Eversmann has contentious species status. While
 multiple separate species allied with Bombus flavidus have been suggested,
 until recently, recognition of two species, a Nearctic Bombus fernaldae
 (Franklin) and Palearctic B. flavidus, was favoured. Limited genetic data,
 however, suggested that even these could be a single widespread species,
 B. flavidus. We addressed the species status of this lineage using an
 integrative taxonomic approach, combining COI and nuclear sequencing, wing
 morphometrics and secretions used for mate attraction. We also explore
 patterns of color polymorphism that have previously confounded taxonomy in
 this lineage. Our results support the conspecific status of Bombus
 fernaldae and Bombus flavidus, however, sampling specimens from across the
 range of these two taxa revealed a distinct population within this broader
 species confined to eastern North America. This makes the distribution of
 the social parasite B. flavidus the broadest of any bumble bee, broader
 than the known distribution of any non-parasitic bumble bee species.
 Analysis of color phenotypes revealed that color polymorphisms are
 retained across the range of the species, but may be influenced by local
 mimicry complexes. Following these results, Bombus flavidus Eversmann,
 1852 is synonymized with Bombus fernaldae (Franklin, 1911) syn. nov. and a
 subspecific status, Bombus flavidus appalachiensis ssp. nov., is assigned
 to the distinct lineage ranging from the Appalachians to the eastern
 boreal regions of the United States and far southeastern Canada.
description: The full dataset includes 9 files : 1- file
 "COI_phylogeny_flavidus.nex" : We constructed a <i>COI
 </i>Bayesian phylogeny of 62 specimens of both the Nearctic and
 Palearctic lineages of Bombus flavidus, including 59 newly sequenced
 individuals spanning its range, and 3 sequences from North America
 obtained from GenBank. We included exemplar <i>COI
 </i>sequences of <i>B. barbutellus </i>(n = 4),
 <i>B. bohemicus </i>(n = 4), <i>B. norvegicus
 </i>(n = 7), <i>B. quadricolor </i>(n =
 3)<i>,</i> <i>B. skorikovi </i>(n = 1),
 <i>B. sylvestris</i> (n = 6), <i>B. vestalis</i>
 (n = 4) and <i>Bombus citrinus</i> (n = 1) as an outgroup,
 using both newly sequenced individuals and sequences extracted from
 GenBank. 2- file "Haplotypes_flavidus.nex" :
 TCS haplotype network using inferred haplotypes from a subset of the
 <i>COI</i> sequences (n=65 individuals, including 61
 <i>B. f</i><i>lavidus s.l.</i> and 4 <i>B.
 norvegicus</i>). 3- file
 "ITS_flavidus.nex" : Nuclear Internal Transcribed Spacer (ITS)
 gene sequenced on 10 individuals of Bombus flavidus, and 2 outgroups B.
 skorikovi and B. norvegicus. 4- file
 "PEPCK_flavidus.nex" : Sequence data for the PEPCK gene
 including 13 individuals of Bombus flavidus, and the two closest relatives
 as outgroups, <i>B. skorikovi</i> and <i>B.
 norvegicus</i>, using sequences for these available on
 Genbank. 5- file "CLGS_matrix_flavidus.xslx"
 : Matrix showing chemical composition of the cephalic labial gland
 secretions (CLGS) of males of Bombus flavidus. We extracted CLGS of 23
 specimens of the Nearctic lineage from Alaska (n = 4), Yukon (n = 5),
 Wyoming (n = 1) and Pennsylvania (n = 13) and 11 specimens of the
 Palearctic lineage<i> </i>from Siberia (n = 8) and Sweden (n =
 3). 6- file "wing_morpho_file1_flavidus.TPS"
 : Digitization of the 18 homologous landmarks on the veins and cells of
 the bumble bee wings if Bombus flavidus males with 2D Cartesian
 coordinates. Geometric morphometric analysis of wing venation includes 103
 total male specimens, 94 males of the Nearctic lineage from Oregon (n =
 78), Alaska (n = 2), Pennsylvania (n = 12) and Maine (n = 2) and 9
 specimens of the Palearctic lineage from Scandinavia (n = 5) and Russia (n
 = 4). 7- file
 "wing_morpho_file2_list_name_specimens.txt" : List of the
 species names of the 103 individuals used in the geometric morphometric
 analysis. 8 - file
 "wing_morpho_file3_list_location_specimen.txt" : List of the
 location of the 103 individuals used in the geometric morphometric
 analysis. 9- file
 "Color_pattern_oregon_flavidus.xslx" : Quantification of color
 pattern differences of Bombus flavidus across Oregon (77 males collected
 from 16 different localities across Oregon). Each specimen had their color
 pattern recorded for the head, dorsal thorax, pleuron (lateral thorax),
 and abdomen. The color patterns were partitioned into 68 total squares for
 each individual. Each square was given a score representing the color
 percentage of the pile (black, yellow or orange). The percentages of each
 color were summed, and each individual was given a total body pile percent
 black, yellow and orange.