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Multisource approaches in taxonomy gather different lines of evidence in order to draw strongly supported taxonomic conclusions and constitute the basis of integrative taxonomy. In the case of overlooked taxa with disjunct distributions for which sampling is more challenging, integrative approaches help to propose stable hypotheses at the species and subspecies levels. Here, based on genetic and semio-chemical traits, we performed an integrative taxonomic analysis to evaluate species delimitation hypotheses within a monophyletic group of bumblebees (Hymenoptera, Apidae, Bombus) including the formerly recognized subgenera Eversmannibombus, Laesobombus and Mucidobombus which are now included in the subgenus Thoracobombus. Our results demonstrate the conspecificity of several polytypic taxa, and we formally recognize the subspecies Bombus laesus aliceae comb. nov. Cockerell, 1931, endemic to North Africa, based on its allopatry, unique mitochondrial haplotype and divergent cephalic labial gland secretions. This highlights the need to maintain studying polytypic complexes of bumblebee taxa for which phylogenetic relationships could be still entangled and eventually implement conservation strategies for taxonomically differentiated lineages.
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Zoologica Scripta. 2021;00:1–17.
© 2021 Royal Swedish Academy of Sciences
Received: 18 November 2020
Revised: 6 February 2021
Accepted: 12 March 2021
DOI: 10.1111/zsc.12486
Resolving the species status of overlooked West- Palaearctic
Paul HughWilliams7
1Laboratory of Zoology, Research institute
for Biosciences, University of Mons, Mons,
2Université de Lorraine, INRAE, URAFPA,
Nancy, France
3Academy of Sciences of the Czech
Republic, Institute of Organic Chemistry
and Biochemistry, Prague, Czech Republic
4Faculty of Tropical AgriSciences, Czech
University of Life Sciences, Prague, Czech
5Department of Biotechnology and
Biosciences, University of Milano- Bicocca,
Milano, Italy
6Department of Plant Protection, Faculty of
Agriculture, Yasouj University, Yasouj, Iran
7Department of Life Sciences, The Natural
History Museum, London, UK
8Evolutionary Biology & Ecology,
Université Libre de Bruxelles, Bruxelles,
Baptiste Martinet, Evolutionary Biology &
Ecology, Université Libre de Bruxelles, C.P.
160/12, Avenue F.D. Roosevelt, 50, B- 1050
Brussels, Belgium.
Funding Information
The research has received funding from the
European Community's Seventh Framework
Program, STEP Project (Status and Trends
of European Pollinators, www.step- proje, grant agreement no 244090,
FP7/2007- 2013), by the Federal German
Ministry for the Environment, Nature
Conservation and Nuclear Safety (BMU)
through the International Climate Initiative
(IKI) and the “Fonds de la Recherche
Scientifique – FNRS” (Brussels, Belgium).
Multisource approaches in taxonomy gather different lines of evidence in order to draw
strongly supported taxonomic conclusions and constitute the basis of integrative tax-
onomy. In the case of overlooked taxa with disjunct distributions for which sampling
is more challenging, integrative approaches help to propose stable hypotheses at the
species and subspecies levels. Here, based on genetic and semio- chemical traits, we
performed an integrative taxonomic analysis to evaluate species delimitation hypoth-
eses within a monophyletic group of bumblebees (Hymenoptera, Apidae, Bombus)
including the formerly recognised subgenera Eversmannibombus, Laesobombus and
Mucidobombus which are now included in the subgenus Thoracobombus. Our results
demonstrate the conspecificity of several polytypic taxa, and we formally recog-
nise the subspecies Bombus laesus aliceae comb. nov. Cockerell, 1931, endemic to
North Africa, based on its allopatry, unique mitochondrial haplotype and divergent
cephalic labial gland secretions. This highlights the need to maintain studying poly-
typic complexes of bumblebee taxa for which phylogenetic relationships could be
still entangled and eventually implement conservation strategies for taxonomically
differentiated lineages.
Bombus, cryptic species, DNA sequences, integrative taxonomy, male marking secretion
The importance of the species status in biology makes its
accurate definition essential (Mayr,1969). However, criteria
for delimiting species are still controversial (Agapow,2005;
De Queiroz,2007). These disagreements are exemplified by
the numerous species delimitation approaches using alterna-
tive diagnostic criteria (De Queiroz,2007). While traditional
taxonomy is mainly based on discrete morphological traits
(Cipola etal.,2014; Ji & Du,2014; Rampini etal.,2012),
such traits can fail to detect species in taxon groups with low
or no morphological differentiation (i.e., cryptic species) or
in groups exhibiting large morphological variability at the
intraspecific level (e.g., in some bumblebee species com-
plexes: Carolan etal., 2012; Ghisbain, Lozier, etal.,2020;
Williams etal., 2012, 2020). Subsequently, many attempts
have been made to improve species delimitation by using al-
ternative features such as shapes (Aytekin etal.,2007; Gérard
etal.,2020), genetic markers (White etal.,2014), or semio-
chemical markers (Martin etal.,2008). Nevertheless, each of
these approaches presents its own limitations (for bumble-
bees see review in Lecocq, Dellicour, etal.,2015; Williams
etal., 2015). One solution is to use a multisource approach
to gather different lines of evidence of speciation to robustly
test taxonomic hypotheses (Arribas et al., 2013; Lecocq,
Dellicour, etal.,2015; Roe & Sperling,2007 a, b). The de-
velopment of integrative taxonomy based on the unified spe-
cies concept (USC) provides a methodological framework for
this taxonomic approach (De Queiroz,2007; Schlick- Steiner
etal.,2010). Many biologists now agree with the USC, rec-
ognising that species are evolving fragments of metapopu-
lation lineages where delimitation criteria do not evolve at
the same rate (De Queiroz,2007). Therefore, multiple oper-
ational criteria must be considered independently to evaluate
taxonomic status (Schlick- Steiner etal.,2010).
When using a set of multiple independent traits, Padial
etal.,(2010) discussed the limitations of two commonly used
frameworks called “integration by cumulation” and “integra-
tion by congruence”. The integration by cumulation assumes
that all taxonomic characters are contingent and even one sin-
gle character could be the basis for species delimitation Padial
etal., (2010). This approach might, however, overestimate
biodiversity by detecting intraspecific variation as species
status (Padial etal.,2010). On the contrary, the integration by
congruence is a strict approach in which two or more criteria
must be divergent across taxa, although this method might
underestimate biodiversity, being unable to detect recent
domestication processes or cryptic species. Assigning sub-
species taxonomic status to distinct allopatric lineages where
differentiation is highlighted in at least one (but not all) cri-
teria can be used as a rational alternative option to reduce the
underestimate risk of the congruence approach (Hawlitschek
etal.,2012; Lecocq, Brasero, etal.,2015).
Here, we revise the taxonomic status inside a monophyletic
group of bumblebees (Apidae, Bombus) including the formerly
recognised subgenera Eversmannibombus Skorikov 1938,
Laesobombus Krüger1920 and Mucidobombus Krüger1920,
now all included in the subgenus Thoracobombussensulato
(Cameron et al., 2007; Williams et al., 2008). The
Eversmannibombus group includes a single taxon: Bombus
persicus Radoszkowski1881. Two subspecies that are pheno-
typically diagnosable by the coat colour have been described:
(a) B. persicus eversmanniellus Skorikov1923 with tergites 1
to 4 white mixed with brown on tergite 2 (Figure1a) and (b)
B. persicus persicus Radoszkowski1881 with tergites 1 to 4
white fringed with yellow or completely yellow hairs. This
species is geographically restricted to mountainous steppes
in eastern Turkey, Northern Iran and the Caucasus. Most of
this range corresponds to the subspecies eversmanniellus
while the subspecies persicus is restricted to Northern Iran
(Rasmont, Franzen, etal.,2015; Rasmont & Iserbyt,2014).
The Mucidobombus group includes a single taxon: Bombus
mucidus Gerstaecker 1869, located in the highest subal-
pine and alpine stage mountain ranges of Western Europe
(Cantabrian, Pyrenees, Alps, Apennines, Balkan Mountains,
and the Carpathians) (Rasmont, Franzen, etal.,2015). Three
subspecies, phenotypically diagnosable by coat colour,
are currently recognised: (a) B. mucidus mollis Pérez1879
(Figure1b) from the Cantabrian, Pyreneean and Western Alps
mountains; (b) B. mucidus Gerstaecker1869 from Central
and Eastern Alps, Apennines, and Balkan Mountains; and (c)
B. mucidus pittioniellus Tkalců 1969 from the mountains of
the Balkan Peninsula (Delmas,1976; Grandi,1957; Rasmont
& Iserbyt,2014; Tkalců, 1960) (Figure2).
Species status in Laesobombus have been the centre of a
major debate (refer Table1), although the common opinion
is to consider two distinct species inside this former subge-
nus: B. laesus Morawitz1875 (Figure1c,d) and B. mocsaryi
Kriechbaumer1877 (Figure1e,f), with a ginger and a black
spot on the mesosoma, respectively. However, many taxa
were previously described: B. laesus Morawitz 1875 from
Turkestan (Semiretschensk region), later described as B. lae-
sus mocsaryi from maculidorsis Skorikov1922; B. mocsaryi
Kriechbaumer 1877 from Hungary; and Bombus mocsaryi
aliceae Cockerell 1931 from Morocco. Currently, despite
their variation in coat colour, B. laesus Morawitz 1875
and B. mocsaryi Kriechbaumer 1877 are sometimes inter-
preted as a single species named B. laesus Morawitz,1875
(Rasmont, 1983; Reinig, 1971; Williams, 1998; Williams
etal.,2009) (Table1). These taxa are found in a large part of
the Palaearctic steppes and dry grasslands but have declined
recently and are becoming extremely rare and localised in
some areas (Rasmont, Franzen, etal.,2015) (Figure3).
In this study, we apply an integrative taxonomic approach
by congruence to the previously cited taxon by combining
three operational and independent criteria commonly used in
bumblebee taxonomy: a mitochondrial DNA marker (COI),
a nuclear DNA marker (PEPCK) and a semio- chemical trait
(cephalic labial gland secretions of males, herein referred to
as CLGS) and resolve the taxonomic affinities of these rare
and poorly known West- Palaearctic taxa.
We sampled male and female specimens of all clades of in-
terest across the West- Palaearctic region between 2001 and
2016 (Appendix S1, Table2) and determined them based on
their morphology and colour patterns (Pittioni, 1939). We
attributed a taxon name to the specimens without a priori
hypothesis as to their species status. The in- group com-
prises eversmanniellus from Turkey and Iran (n= 5); per-
sicus from Iran (n= 4); laesus from Iran, Kyrgyzstan and
Turkey (n=19); mocsaryi from France, Hungary, Mongolia
and Kyrgyzstan (n = 20); aliceae from Morocco (n = 5);
mollis from France, Andorra and Spain (n =8); mucidus
from Switzerland, Austria and Italy (n = 24); and pittion-
iellus from Montenegro, Albania and Macedonia (n=10).
We complemented the in- group with the six species from
the sylvarum- group of bumblebees (Brasero et al., 2020),
the sister group of our examined clades with the subgenus
Thoracobombus (Cameron et al.,2007). The outgroup in-
cludes the related species B. (Thoracobombus) mesomelas
(n=1) (formerly included in the Rhodobombus subgenus)
as well as the more distant species B. vestalis (n=1), which
belongs to the sister subgenus Psithyrus.
Genetic trait analyses
We sequenced two genes commonly used in bumblebee
phylogeny (Cameron etal.,2007; Williams etal.,2019): the
barcode fragment of the fast- evolving cytochrome oxidase 1
(hereafter referred to as COI) mitochondrial gene (from 100
specimens), and the nuclear slow- evolving protein- coding
gene phosphoenolpyruvate carboxykinase (hereafter referred
to as PEPCK) (Appendix S1, Table2). We carried out poly-
merase chain reaction (PCR) amplifications with primer pair
LepF1/LepR1 (Hebert etal.,2003) for COI and FHv4- RHv4
(Cameron etal.,2007) for PEPCK. We performed sequenc-
ing procedures described in Lecocq etal.,(2013). Sequences
were aligned with BioEdit version 7.2.5 (Hall,1999). The
FIGURE 1 Pictures of some
bumblebee taxa examined as part of this
study: (a) Bombus persicus eversmanniellus
female from Turkey; (b) B. mucidus mollis
male from Pyrenees, France; (c) B. laesus
male from Turkey; (d) B. laesus female
from Kyrgyzstan; (e) B. mocsaryi female
from Kyrgyzstan; (f) B. mocsaryi female
from Morocco. All white lines correspond to
a scale of 1cm. Photo credit P. Rasmont
(a) (b)
(c) (d)
(e) (f)
final molecular datasets spanned 660 bp from COI and
889bp from PEPCK. Sequences are available on GenBank
(accession numbers in Appendix S1).
The phylogenetic analyses were performed for each gene
independently with a Bayesian method (MB) to explore the
genetic divergence as well as to define lineages within the
different groups. We partitioned each gene to explore the best
substitution model: (a) PEPCK into two exons and two in-
trons; (b) COI and PEPCK exons by base position (1st, 2nd
and 3rd). Each dataset was submitted to the Akaike infor-
mation criterion corrected for small sample sizes (Hurvich
& Tsai,1989) to choose the best- fitting substitution models
with JModelTest Server version 2.0 (Posada,2008). The cho-
sen models were: (a) For COI: GTR+G (1st), HKY (2nd) and
GTR+G (3rd); (b) for PEPCK, exon 1: F81 (1st), JC (2nd)
and K80+ I (3rd); PEPCK, exon 2: JC (1st), TrN (2nd), and
JC+I(3rd); PEPCK, intron 1: TPM uf+I; PEPCK, intron
2: TPM1 uf. For the MB method, we performed Bayesian
inference analyses with MrBayes version 3.1.2 (Ronquist &
Huelsenbeck,2003). Ten independent analyses were achieved
for each gene (100 million generations, four chains with
mixed models, default priors, saving trees every 1,000 gen-
erations). We assessed convergence by examining (a) like-
lihood plots (for stationarity) and convergence statistics in
FIGURE 2 West- Palaearctic distribution of Bombus persicus and Bombus mucidus including B. mucidus mucidus (white circles), B. mucidus
mollis (black diamonds) and B. mucidus pittioniellus (black squares). Adapted from Rasmont, Franzen, etal.(2015) with the taxonomic conclusions
of the present study
MrBayes version 3.1.2 and (b) ESS values in Tracer version
1.7.1 (Rambaut etal.,2018), which led us to conservatively
discard the first 10 million generations as a burn- in proce-
dure. A majority rule 50% consensus tree was constructed.
We only considered as statistically significant the clades
supported branch supports with high posterior probabilities
(≥0.95) (Wilcox etal.,2002).
We applied bGMYC methods to the COI dataset by using
R Package “bGMYC” (Reid & Carstens,2012). A range of
probabilities <0.05 was considered as strong evidence that
taxa were heterospecific while a range of probabilities 0.05– 1
suggested that taxa were conspecific (Reid & Carstens,2012).
BEAST version 1.7.4 (Drummond etal.,2012) was used to
generate ultrametric trees (required for bGMYC method)
with a phylogenetic clock model to calculate a posterior dis-
tribution of trees (length of the MCMC chain: 1 billion gener-
ations). The first million sampled trees were burned- in using
the maximum clade credibility method and setting the poste-
rior probability limit to 0. The bGMYC analysis was based
on 1,000 trees sampled every 10,000 generations. For each
of these 1,000 trees, the MCMC was made of 100,000 gen-
erations, discarding the first 90,000 as burn- in and sampling
every 100 generations. In order to provide a “heat map” of
species delineation probability, posterior probability distribu-
tion has been applied against the first sample tree.
We also applied the Poisson Tree Processes (PTP) model,
complementary to the GMYC model to avoid potential biases
of the time- calibrating procedure and only relies on the num-
ber of DNA- nucleotide substitutions using the branch lengths
from a metric gene tree (Zhang etal.,2013). We used the
online implementation of the bPTP server (https://speci es.h- to recognise coalescent as evidence for candidate
species in our dataset applying default PTP options, as al-
ready performed in integrative taxonomic approaches applied
to bumblebee taxonomy in the past (Potapov et al., 2017;
Williams etal.,2019).
Eco- chemical trait analyses
We focused on the main reproductive trait involved in the bum-
blebee pre- mating recognition (Ayasse etal.,2001; Valterová
etal.,2019): the cephalic labial gland secretions (CLGS) of
males. The CLGS constitute a semio- chemical species- specific
trait (Calam,1969) providing efficient diagnostic characters
for species delimitation (Martinet etal.,2018, 2019). They are
complex mixtures of mainly aliphatic compounds synthesised
de novo (Žáček etal.,2013) in the head of bumblebee males.
By main compounds, we mean the compounds that have the
highest relative amounts (RA) among all compounds of CLGS
at least in one individual of the taxon. All specimens were
killed by freezing at −20°C and the CLGS were extracted
with 400µl of heptane (method described in De Meulemeester
TABLE 1 List of taxa of the Laesobombus from previous taxonomic revisions
Morawitz(1875) Kriechbaumer(1877) Radoszkowski(1888) Skorikov(1922) Cockerell(1931) Panfilov(1956) Williams(1998)
B. sidemii - - B. laesus
B. laesus B. laesus mocsaryi
Form maculidorsis
B. tianschanicus
B. laesus
B. mocsaryi B. mocsaryi aliceae B. mocsaryi
B. maculidorsis
etal.,2011). Samples were stored at −40°C prior to analy-
sis. In total, we were able to sample 35 specimens belonging
to the three groups Eversmannibombus, Laesobombus and
Mucidobombus (Appendix S1, Table2).
We qualified the CLGS composition by gas
chromatography– mass spectrometry (GC/MS) using a Focus
GC (Thermo Scientific) with a non- polar DB- 5ms capillary
column [5% phenyl (methyl) polysiloxane stationary phase;
column length 30m; inner diameter 0.25mm; film thickness
0.25μm] coupled to DSQ II quadrupol mass analyser (Thermo
Scientific) with 70eV electron impact ionisation. We used a
splitless injection mode (220°C) and helium as a carrier gas
(1ml/min). The temperature program of the oven was set to
70°C for 2min and then heated up at a rate of 10°C/min to
320°C. The temperature was then held at 320°C for 5min.
Compounds were identified in XcaliburTM using the reten-
tion times (tr) and mass spectra of each peak, in comparison
with those at National Institute of Standards and Technology
library (NIST, USA). Double- bond positions (C = C) were
determined by dimethyl disulfide (DMDS) derivatisation
(Cvacka etal.,2008). We quantified the CLGS of all samples
by a gas chromatograph– flame ionisation detector Shimadzu
GC- 2010 with a SLB- 5ms non- polar capillary column (5%
phenyl (methyl) polysiloxane stationary phase; 30- m column
length; 0.25- mm inner diameter; 0.25- µm film thickness) with
the same chromatographic conditions as in GC/MS. Peak
areas of compounds were detected in GCsolution Postrun
(Shimadzu Corporation) with automatic peak detection and
FIGURE 3 West- Palaearctic distribution of Bombus laesus including B. laesus laesus (white circles), B. laesus mocsaryi (black diamonds)
and B. laesus aliceae (black squares). Adapted from Rasmont, Franzen, etal.(2015) with the taxonomic conclusions of the present study
noise measurement. We calculated RA (in %) of compounds
in each sample by dividing the peak areas of compounds by
the total area of compounds. We discarded all compounds
for which RA was recorded as <0.1% for all specimens (De
Meulemeester etal.,2011). The data matrix (Appendix S2) for
each taxon was based on the alignment of each relative pro-
portion of the compound between all samples performed with
GCAligner version 1.0 (Dellicour & Lecocq,2013). Before
each sample injection, a standard (Kováts) containing a mix of
hydrocarbons (alkanes) from C10 (decane) to C40 (tetracon-
tane) was injected to facilitate the alignment and the identifi-
cation of compounds. Kováts indices were calculated with GC
Kováts version 1.0 (Dellicour & Lecocq, 2013). Clustering
method was performed using R version 3.3.2 (R Development
Core Team, 2020) to detect CLGS differentiation between
taxa. We transformed data (log (x+1)) to reduce the great dif-
ference of abundance between compounds (De Meulemeester
etal.,2011). A Pearson r correlation distance matrix based on
the CLGS data matrix (RA of each compound) was computed.
An unweighted pair group method with arithmetic mean
(UPGMA) was used as a clustering method (R- package ape,
Suzuki & Shimodaira,2011). We assessed the uncertainty in
hierarchical cluster analysis using p- values calculated via mul-
tiscale bootstrap resampling with 10.000 bootstrap replica-
tions (significant branch support>0.85) (R- package pvclust,
Suzuki & Shimodaira,2011). We assessed CLGS differentia-
tions between taxa by performing a permutation multivariate
analysis of variance using distance matrix (PerMANOVA)
(R package vegan; Oksanen et al., 2011). When a significant
difference was detected, we performed a pairwise multiple
comparison with an adjustment of P- values (Bonferroni cor-
rection) to avoid the type I errors.
To determine the indicator compounds (IC) of each
taxon, we used the indicator value (IndVal) method (Claudet
etal.,2006; Dufrêne & Legendre,1997). The value given is
the product of relative abundance and relative frequency of
occurrence of a compound within a group. We evaluated the
statistical significance of a compound as an indicator at the
0.01 level with a randomisation procedure.
Decision framework of
taxonomic status
We followed the decision framework proposed by Lecocq,
Brasero, etal.(2015). This approach follows the USC (De
Quieroz, 2007) and corresponds to the strictest commonly
used framework called “integration by congruence” by
Padial etal.,(2010). We thus assigned a specific status to
a taxon (a) which is genetically differentiated in all genetic
markers (i.e., unique haplotypes); (b) which constitutes a
reciprocally monophyletic group with a highly supported
branch support; and (c) with a significant differentiation in
CLGS composition (including IndVal IC, PerMANOVA
TABLE 2 Number of Bombus females (F) and males (M) sampled for each operational criterion
Sampling country
nuDNA marker
mtDNA marker
(COI) CLGSGroup Taxon
Eversmannibombus persicus Iran 3F 4F -
eversmanniellus Iran, Turkey 2M, 1F 2M, 2F 3M
Laesobombus laesus Iran, Kyrgyzstan, Turkey 5M, 13F 6M, 13F 6M
mocsaryi France, Hungary,
Kyrgyzstan, Mongolia
3M, 12F 5M, 15F 2M
aliceae Morocco 5M 5M 5M
Mucidobombus mollis Andorra, France and
2M, 4F 3M, 4F 4M
mucidus Austria, France, Italy and
17M, 7F 14M, 4F 15M
pittioniellus Albania, Macedonia and
10F 5F -
Outgroups mlokosievitzi Brasero etal.(2020) 3F 3F -
sylvarum Brasero etal.(2020) 3M 3M -
veteranus Brasero etal.(2020) 3M 3M -
inexspectatus Brasero etal.(2020) 3M 3M -
ruderarius Brasero etal.(2020) 3F 3F -
velox Brasero etal.(2020) 1F 1F -
mesomelas Brasero etal.(2020 1F 1F -
vestalis Data from Genbank and
1F 1F -
and high bootstrap values>0.85). We assigned a subspecies
status to those allopatric populations which were not diverg-
ing in all lines of evidence but exhibiting original pheno-
typic features (Hawlitschek etal.,2012) such as a divergent
morphology or a derived CLGS signal (Lecocq, Dellicour,
etal.,2015; Martinet etal.,2019). Hair colour was not used
as an operational criterion for species delineation as col-
our patterns can be shared by long- separated heterospecific
taxa (Ghisbain, Lozier, etal.,2020; Williams etal.,2012);
this character is a strong variable at the intraspecific level
(Martinet et al., 2018; Williams et al.,2020) and can be
strongly influenced by evaluative pressures from Müllerian
mimicry at a regional level (Ezray etal., 2019; Ghisbain,
Lozier, etal.,2020).
Based on our taxonomic conclusions, we proposed an updated
conservation status for Bombus laesus following the stand-
ardised protocol implemented by the International Union
for Conservation of Nature (IUCN) (e.g., Nieto etal.,2014).
Occurrence data used in the analyses are those published in
Rasmont, Franzen, etal.(2015) and Polce et al.(2018). We
evaluated the conservation status following Nieto etal.(2014),
measuring the area of occupancy (AOO) and extent of occur-
rence (EOO) of B. laesus. The AOO is a measure of the area
in which the species occurs and corresponds to the sum of
the area of grids the species occupies. For this purpose, we
defined square grids of 5×5km, as previously suggested in
FIGURE 4 Genetic differentiation within the three former Bombus subgenera Laesobombus, Mucidobonus and Eversmannibombus, now
included in the broader subgenus Thoracobombus. Majority rule (50%) consensus tree based on Bayesian analyses of COI (cytochrome oxidase
1). Values above tree branches are Bayesian posterior probabilities/bPTP (Poisson Tree Process) values. The tree is rooted with the most distant
outgroup Bombus (Psithyrus) vestalis
bumblebees (Drossart etal.,2019). The EOO is a measure of
the geographic range size of a species and is calculated by
drawing a convex hull which is defined as the smallest poly-
gon containing all the sites of occurrence. The final conserva-
tion status was proposed following the criteria used in Nieto
Genetic trait analyses
The phylogenetic analyses showed the expected differentia-
tion between outgroup and in- group and highlighted similar
relationship between taxa. Both COI and PEPCK phylogenetic
analyses detected three monophyletic bootstrap- supported
groups inside our ingroups (Figures4 and 5): (a) a first clade
comprising the taxa laesus+mocsaryi +aliceae; (b) a second
clade comprising mollis+mucidus +pittioniellus and (c) a
third clade comprising eversmanniellus+persicus.
The tree generated by bGMYC analyses on COI sequences
split off the tree in several groups with low probabilities
(<0.05) to be conspecific with the other ones (Figure6). The
tree generated by the bGMYC analysis showed the delimita-
tion of three prospective species within our in- group (p<.05).
Three groups were highlighted using this threshold: (a)
mocsaryi+laesus + aliceae (bGMYC conspecificity proba-
bilities between individuals included in the group, p>.09– 1);
(b) mucidus+mollis + pittioniellus (p>.38– 1) and (c) persi-
cus+eversmanniellus (p>.08– 1). The complementary PTP
FIGURE 5 Majority rule (50%) consensus tree based on Bayesian analyses of PEPCK (phosphoenolpyruvate carboxykinase). Values above
tree branches are Bayesian posterior probabilities/bPTP (Poisson Tree Process) values. The tree is rooted with the most distant outgroup Bombus
(Psithyrus) vestalis
analysis with the most support and performed on the same
dataset corroborated the results of the bGMYC by recognis-
ing the same candidate species with high support (refer the
grey values accompanying the posterior probabilities in the
Figures4 and 5).
Eco- chemical trait analyses
The cluster analysis revealed three strongly supported groups
(bootstrap>85%) (Figure7): eversmanniellus (Turkey); mu-
cidus (Alps, Apennines) + mollis (Pyrenees); and mocsaryi
(Hungary), aliceae (Morocco) + laesus (Turkey). In total,
113 compounds were detected by chemical analyses (37 from
eversmanniellus, 53 from mucidus+mollis and 93 from moc-
saryi, aliceae+laesus, respectively; see Appendix S2). Main
compounds were detected for each group: octadec- 9- en- 1- yl
acetate (38%– 51%) for eversmanniellus group; octadec- 9- en-
1- ol (7%– 61%) and octadec- 9- en- 1- yl acetate (23%– 68%) for
mucidus group; and tetradecyl acetate (0%– 47%), octadec- 9-
enyl acetate (4%– 64%) and octadec- 9- enoic acid (2%– 27%)
for laesus group (Figure 7). Despite the separation we ob-
served between mocsaryi from Hungary, laesus from Turkey
and aliceae from Morocco, our statistical analysis did not
significantly support this differentiation (bootstrap <85%).
Moreover, the IndVal method revealed several significant indi-
cator compounds (IC) (IndVal value>70) for the eversmann-
iellus group (IC=9), mucidus group (IC=2) and laesus group
(IC=24) (Appendix S2). PerMANOVA test confirmed the
differentiation between (a) eversmanniellus and laesus+moc-
saryi +aliceae group (F=11.8; p<.05); (b) eversmanniellus
and mucidus (F=14.007; p<.05); and (c) B. mucidus and B.
laesus+B. mocsaryi group (F=49.458; p<.05).
Decision framework of
taxonomic status
Based on the genetic traits and supported by CLGS (for the
sampled taxa), three species are supported in the studied in-
group following the methodological framework of Lecocq,
Brasero, etal.,(2015): B. laesus, B. mucidus and B. persicus
(Table3). The genetic traits COI/PEPCK show no significative
difference among the subspecies of Bombus persicus (evers-
manniellus and persicus) and of Bombus mucidus (mollis,
mucidus and pittioniellus). These results also confirm that the
lineage mocsaryi is subspecific to Bombus laesus. Moreover,
based on the combination of its private COI haplotype, its al-
lopatry and tenuous divergence in CLGS, we formally recog-
nise a subspecies status for the North African lineage aliceae
Cockerell1931 within Bombus laesus. We also provide the
first description of the males of this subspecies. We designated
a lectotype and a series of paralectotypes that are deposited in
the collection of the Laboratory of Zoology of the University
of Mons (Belgium). We also provide the first description of
the males of this subspecies. We designated a lectotype and a
series of paralectotypes that are hosted in the collection of the
Laboratory of Zoology of the University of Mons (Belgium).
First description of the males: Body length measured
in lateral aspect from the base of the antenna to the poste-
rior edge of tergite 7:12.4mm±0.3mm (SE), head width:
FIGURE 6 bGMYC results based on the COI (cytochrome oxidase 1) phylogenetic tree. The vertical and horizontal lines correspond to the
three different groups shown on the tree. The scale corresponds to the probability to be conspecific
Coloration: (a) Head: large patch of yellow hairs on the
clypeus reaching the inner border of compound eyes. The an-
tennal socket is covered with yellow hairs intermixed with
some black hairs. The inner border of compound eyes has
some long black setae. The vertex is covered with a large
patch of yellow hairs. (b) Mesosoma: yellow- haired collare
on the whole pronotum, extending to the pleurae and reaching
the scutellum under the tegulae. Wide and yellowish collare
on the pronotum and the mesonotum reaching the middle of
the tegulae. Black hairs under the tegulae on the mesonotum.
The scutellum is yellow haired. (c) Metasoma: Terga 1– 7 are
yellow- haired; the tergum 7 has black- haired intermixed with
some long yellow hairs at the lateral extremities. (d) Legs:
coxa, trochanter and femur yellow haired intermixed with
black hairs. Tibia bordered with yellowish setae.
Conservation status
Our spatial analysis showed an EOO of 7,654,628.492 km2
and an AOO of 10,675.000 km2 at the European scale. Based
on these values only, B. laesus could be considered as “Least
FIGURE 7 Unweighted pair group method with arithmetic mean cluster based on a Pearson's r correlation distance matrix calculated from the
cephalic labial gland secretions matrix of six Bombus taxa: eversmanniellus (in purple), mollis (dark green), mucidus (light green), mocsaryi (blue),
aliceae (dark blue) and laesus (light blue). The values near the node are multiscale bootstrap resampling
Concern.” Nevertheless, reductions of the populations across
the range of the species have been observed (criterion A2c
in Nieto etal.,2014) and population reductions can be sus-
pected in a near future (Rasmont, Franzen, etal.,2015) with
a clear decline in habitat quality, calling the IUCN criterion
A3c. Altogether and awaiting for more studies on the conser-
vation of the species, we propose a “Near Threatened” cat-
egorisation of Bombus laesus (A2c+3c).
The highly polytypic nature of bumblebees makes their
taxonomy especially complex (Williams, 1998). Although
the increasing use of genetic markers (Ghisbain, Lozier,
et al., 2020; Williams et al., 2020), semio- chemical traits
(Martinet etal.,2018, 2019) sometimes combined with other
tools (e.g., geometric morphometrics on the wings, Gérard
etal., 2020) is significantly refining our global comprehen-
sion of this diverse group of bees, some taxa have remained
overlooked. Here, we clarified the taxonomic status of several
uncommon bumblebee taxa belonging to the former subgen-
era Eversmannibombus, Laesobombus and Mucidobombus,
now gathered in the monophyletic genus Thoracobombus
(Williams etal.,2008).
Taxonomic implication for the
Thoracobombus group
Firstly, our integrative taxonomic approach highlighted
the conspecific status of the two taxa eversmanniel-
lus and persicus, as part of the species Bombus persicus
Radoszkowski 1881 (refer decision framework Table 3).
The distribution of B. persicus is limited to the north by the
Caucasus but no geographical barrier seems to occur to the
east between the different populations from Turkey to Iran.
Secondly, our decision framework concluded that all
taxa within Mucidobombus are conspecific despite their
geographic isolation and marked differences in colour pat-
tern (Cantabrian Range, Pyrenees, Alps, Apennines, and
Balkans) (Table3). Although we have not been able to anal-
yse the CLGS of specimens from the Balkans and Cantabrian
mountain ranges, the genetic structure observed in our mark-
ers (Figure4) and the analysed CLGS strongly support the
conspecificity of all studied populations, as part of a single
species, Bombus mucidus Gerstaecker 1869. As for many
other bumblebee species, the current disjoint distribution
of B. mucidus can be explained by past climatic oscillations
of the Quaternary in Europe. During these most recent ice
ages, a plain of permafrost as well as tundra and cold steppe
extended between the mountains of southern Europe to
TABLE 3 Decision framework including former (according to Rasmont et al. 2008 and Williams et al. 2012b) and proposed taxonomic status
Former taxonomic status COI/bGMYC PEPCK CLGS
taxonomic status
Laesobombus mocsaryi aliceae
+(A)a / - +(A)a B. laesus aliceae
comb. nov
laesus (Turkey,
− (A) / − − (A) B. laesus
laesus (Iran) − (A) / − − (A) /
mocsaryi (Hungary) − (A) / − − (A)
mocsaryi (France) − (A) / − − (A) /
mocsaryi (Kyrgyzstan) − (A) / − − (A) /
mocsaryi (Mongolia) − (A) / − − (A) /
Mucidobombus mollis (Pyrenees) − (B) / − − (B) B. mucidus
mollis (Cantabrian) − (B) / − − (B) /
mucidus (Alps) − (B) / − − (B)
mucidus (Apennines) − (B) / − − (B)
pittioniellus (Balkans) − (B) / − − (B) /
Eversmannibombus eversmanniellus (Turkey) − (C) / − − (C) B. persicus
eversmanniellus (Iran) − (C) / − − (C) /
persicus (Iran) − (C) / − − (C) /
COI (cytochrome oxidase 1) and PEPCK (phosphoenolpyruvate carboxykinase) columns indicate if a taxon is strongly supported as monophyletic group (± means that
the taxon is/is not a monophyletic group). When the taxon is not a distinct monophyletic group, the latter group together with taxa included in the same monophyletic
group. CLGS (cephalic labial gland secretions) indicates if the taxon has/has not specific composition of cephalic labial gland secretions (±means that the taxon has/
has not a specific CLGS composition; ++ means that the specific composition involved main compounds).
aSpecimens from Morocco are well supported but do not constitute a reciprocal monophyletic group with the other specimens.
the Urals. These habitats have facilitated the movement of
cold- adapted species like B. mucidus as well as a constant
gene flow between populations (Hewitt, 1999; Steward
etal., 2003). Following the global climate warming of the
inter- glacial period, the lineage mucidus has been trapped in
the mountains of Southern Europe, a pattern also observed
in some plants, birds and mammals (Angus,1983; Steward
etal.,2003, 2010). Because ice age periods are longer than
inter- ice age periods (Hansen,2004), the genetic homogene-
ity found among all populations of B. mucidus could be ex-
plained by these long periods of continuous gene flow. This
pattern has been already observed in another mountain bum-
blebees (B. mendax) from an analysis combing (a) dating of
mountain orogeny, (b) modelling of regional climate change,
(c) modelling of evolution of climate preferences along spe-
cies' lineages, and (d) estimation of species’ dispersal/estab-
lishment potential (Williams etal.,2017).
Finally, our integrative taxonomic decision framework
supports the conspecificity of all lineages within the group
Laesobombus (Table3). This result confirms the hypothesis
proposed by Williams (1998, 2009) who did not find discrete
morphological differences between laesus and mocsaryi, ex-
cept an obvious difference in colour pattern. The genetic di-
vergences based on COI and PEPCK sequences did not reflect
the current separation between the taxa laesus and mocsaryi
based on coat colour pattern (Figures 4 and 5). Although
no genetic structuring was found in COI analyses (MB and
bGMYC), the analyses of the PEPCK fragment highlighted a
well- supported clade including all mocsaryi, therefore mak-
ing the laesus group paraphyletic (Figures4 and 5).
The eco- chemical traits bring new information about
Moroccan populations originally described by Cockerell
(1931) (Figure7). While non- significant at the specific level,
the CLGS of the Moroccan population differed from other
populations encountered in Turkey and Hungary in light
weight compounds (e.g., octadec- 9- enyl acetate and octadec-
9- enoic acid; Appendix S2). These compounds are known to
have a long- distant attractive effect (Ayasse etal.,2001). This
differentiation could, therefore, lead to the establishment of
a reproductive isolation barrier as previously already sug-
gested in other subgenera including Megabombus, Psithyrus,
Pyrobombus and Thoracobombus (reviewed in Valterová
et al., 2019). Unfortunately, we could not analyse the CLGS
composition of the French population exhibiting unique hap-
lotypes in both COI and PEPCK markers (Figures4 and 5).
Since allopatric taxa of this species are found between south-
ern France and Hungary, further analyses are needed to as-
sess the hypothesis whether the French population is closer
or not to that of Morocco.
Even if unique haplotypes stand out from our analyses (lae-
sus from Iran; mocsaryi from France, Kyrgyzstan, Mongolia
and aliceae from Morocco), they are not strongly supported as
reciprocal monophyletic groups in our phylogenies (Figures4
and 5). All taxa within the Laesobombus group can, therefore,
be considered as infraspecific to B. laesus Gerstaecker1869.
Based on its unique haplotype, a subspecies status is proposed
for the population of Morocco. The name aliceae was origi-
nally used by Cockerell (1931) to describe what he called a “va-
riety” of the taxon mocsaryi as Bombus mocsaryi var. aliceae
Cockerell 1931, based on a single worker collected in 1930
in Asni, Morocco. Given that the oldest available name for
the species is Bombus laesus, the Moroccan subspecies must
rather be regarded as Bombus laesus aliceae Cockerell1931.
As previously mentioned, some populations could not be
analysed in the light of their attractive chemical secretions.
We can, however, safely propose species delineation hypoth-
eses according to our decision framework on the basis of the
unambiguous genetic data. Overall, we can conclude that the
CLGS data does not conflict with the genetic data for the
slightly reduced set of samples for which it was collected. A
broader sampling of CLGS would be still useful to further
investigate any eco- chemical differentiation within these un-
common species at the population level, notably for B. persi-
cus persicus and B. mucidus pittioniellus.
Implications for conservation
All species studied here are known to be restricted to open-
field environments (either steppes, sub- alpine or alpine
meadows) (Rasmont, Franzen, etal.,2015). It appears clear
that the current fragmentation of their populations is the
result of multiple threats including climate change, intensi-
fication and extension of agricultural practices to the detri-
ment of open- field environments (Iserbyt & Rasmont,2012;
Manino etal.,2007). Among the studied species, only B.
mucidus has been assessed to the International Union for
Conservation of Nature Red List as “Near Threatened”
(Rasmont etal.,2015). Based on the taxonomic knowledge
at that time, Bombus laesus and B. mocsaryi had been as-
sessed as two different species by Nieto etal.(2014), with
B. laesus assessed as “Near Threatened” and B. mocsaryi
as “Endangered”. Both subspecies mocsaryi and laesus had
also been assessed as separate species by Rasmont, Franzen,
etal.(2015), and the current status of B. laesus as a species
including all taxa laesus, mocsaryi and aliceae implies the
need for a re- evaluation of the species’ conservation status.
Based on our results, we propose such a re- evaluation of
the conservation status of B. laesus as “Near Threatened”
according to the same methodology of Nieto etal.(2014).
The effective conservation of bumblebees ultimately re-
lies on both the precise knowledge of their distribution and an
unambiguous identification (Ghisbain etal.,2020). Although
bumblebees constitute one of the most studied groups of bees
worldwide, much work remains to clarify the taxonomy of
the genus. New species are continuously described all over
the world (Martinet et al., 2019; Williams et al., 2020),
highly polymorphic lineages thought to be conspecific can
hide multiple species (Ghisbain, Lozier, etal.,2020; Martinet
etal.,2018; Williams etal.,2019) and in contrast taxa thought
to be distinct can result in being conspecific (Williams
etal.,2020; present study). Much work is still required to dis-
entangle the phylogenetic relationships and taxonomic status
of widespread, polymorphic bumblebee taxa to ensure their
correct identification and eventually implement geographi-
cally and taxonomically adapted conservation strategies.
The authors are particularly grateful to Christophe Praz
(University of Neuchâtel) for all his advice and comments.
The authors are particularly grateful to Thomas Wood for cor-
recting the English. The authors thank the Parco Nazionale
dei Monti Sibillini for granting permission to collect in their
respective territories to the author PB. Special thanks go to
M. Rami (University of Mons), A. Cetkovic and A. Popovic
(University of Belgrade), V. Cyriaque (University of Mons) and
R. DeJonghe for their help in the sampling. Computational re-
sources have been provided by the Consortium des Equipements
de Calcul Intensif (CECI), funded by the FRS- FNRS (Fonds de
la Recherche Scientifique, Brussels, Belgium). GG contributes
as a PhD student granted by the FRS- FNRS (grant “Aspirant”)
and BM as a postdoctoral researcher for the FRS- FNRS (grant
“Chargé de Recherches”). Part of this work (Eco- chemical
trait differentiation) was supported by the Institute of Organic
Chemistry and Biochemistry of the Academy of Sciences of the
Czech Republic (#61388963). The research has received funding
from the European Community's Seventh Framework Program,
STEP Project (Status and Trends of European Pollinators, www.
step-, grant agreement no 244090, FP7/2007- 2013).
NB, GG, TL, IV, PR and BM: conceived and designed the
experiments. NB, AM, PR, PB and BM: performed sampling.
NB, GG and BM: analysed the data. NB, GG and BM: wrote
the paper. All authors discussed the results, edited and ap-
proved the content of the manuscript.
Nicolas Brasero
Guillaume Ghisbain https://orcid.
Thomas Lecocq
Denis Michez
Irena Valterová
Paolo Biella
Alireza Monfared
Paul Hugh Williams
Pierre Rasmont
Baptiste Martinet
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Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Brasero N, Ghisbain G,
Lecocq T, et al. Resolving the species status of
overlooked West- Palaearctic bumblebees. Zool Scr.
2021;00:1– 17.
... Martinet et al. 2018;Potapov et al. 2019;Williams et al. 2019), we expect a clear genetic structure between the Nearctic and Palearctic populations of B. jonellus. Because polytypic bumblebee taxa can show signs of population structuring in the COI barcode fragment Brasero et al. 2021; but see Lecocq et al. 2015), some degree of genetic differentiation in the phenotypically divergent subspecies B. jonellus hebridensis is expected (Potapov et al. 2018). Finally, the isolated Pyrenean population of B. jonellus is also expected to appear as a distinct genetic cluster, as observed in other bumblebee whose distributions encompass isolated high-altitude habitats in the southern part of their range (Martinet et al. 2018;Brasero et al. 2021). ...
... Because polytypic bumblebee taxa can show signs of population structuring in the COI barcode fragment Brasero et al. 2021; but see Lecocq et al. 2015), some degree of genetic differentiation in the phenotypically divergent subspecies B. jonellus hebridensis is expected (Potapov et al. 2018). Finally, the isolated Pyrenean population of B. jonellus is also expected to appear as a distinct genetic cluster, as observed in other bumblebee whose distributions encompass isolated high-altitude habitats in the southern part of their range (Martinet et al. 2018;Brasero et al. 2021). ...
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While the Arctic and boreal bumblebee fauna is increasingly studied worldwide, information is missing about the genetic connections between circum-boreal populations of some widespread species, especially those living in remote regions like North-East Siberia and Alaska. Here, we study one of the most common boreal bumblebee species, Bombus (Pyrobombus) jonellus (Kirby, 1802), as a model to investigate current circum-boreal genetic connections and relations with relict populations in its post-glacial refugia in Southern Europe. Our haplotype network analysis based on a fragment of the cytochrome oxidase 1 gene reveals two main conspecific lineages, one in Europe including the Southern relictual populations from the Pyrenees and the second comprising Eastern Palearctic and Nearctic populations. However, West-Siberian populations of Bombus jonellus share haplotypes with the two distinct lineages. These results could indicate a postglacial, multidirectional, and circum-boreal recolonisation both in Europe and East-Palearctic from refugia in Siberia, in addition to other recolonisation ways from Southern European refugia and Beringia. These findings highlight that a priori distant and isolated conspecific populations of B. jonellus could presently remain connected or have only presented a recent break in gene flow.
... wing shape and size, colour pattern) or ecological traits (e.g. unique trophic association with a particular resource, divergence in semio-chemical signals to attract a conspecific mate) (Hawlitschek et al., 2012;Lecocq et al., 2015a;Martinet et al., 2019;Brasero et al., 2021;Lhomme et al., 2021). Our choice to formally recognise subspecies has the combined advantage of drawing attention to distinctive populations that vary in potentially adaptive traits and to prevent an artificial taxonomic inflation at the specific level (Isaac et al., 2004). ...
... The level of differentiation of these subspecies can vary widely, ranging from (i) no genetic or semio-chemical significant differentiation (e.g. based on allopatry and colour pattern, Lecocq et al. 2015); (ii) a low genetic differentiation accompanied with a dialectic semio-chemical signal (e.g. Brasero et al., 2020Brasero et al., , 2021; (iii) a significant genetic differentiation with a dialectic semio-chemical signal (Martinet et al., 2018, but see Williams et al., 2019. The present taxon reinigiellus falls in the first case where the taxon is isolated, presents tenuous but extant morphological and colour differences (Rasmont, 1983;Castro, 1988) and a distinct wing phenotype and a unique wing size/ITD ratio but shows no molecular differentiation based on our two other traits (COI and CLGS). ...
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• Against the context of global wildlife declines, targeted mitigation strategies have become critical to preserve what remains of biodiversity. However, the effective development of conservation tools in order to counteract these changes relies on unambiguous taxonomic determination and delineation. • In this study, we focus on an endemic bumblebee species recorded only from the highest altitudes of the Sierra Nevada (Spain), Bombus reinigiellus (Rasmont, 1983). The species has the smallest range of any European bumblebee, along with a restricted diet and an inability to disperse because of its isolated montane distribution, making it an appropriate conservation target. However, through an integrative taxonomic approach including genetics, morphometrics and semio-chemistry, we demonstrate the conspecificity of this taxon with one of the most common and widespread bumblebee species of Europe, Bombus hortorum (L. 1761). We assign a subspecies status to this endemic taxon (Bombus hortorum reinigiellus comb. nov.) shown to be different in colour and morphology but also in wing shape and relative wing size compared to the other conspecific subspecies. • Following our taxonomic revision, we reassessed the IUCN conservation status of Bombus hortorum both at the continental and Spanish scale. We then propose how historic climatic oscillations of the last Ice age could explain such a phenotypic divergence in a post-glacial refugium and highlight the critical role of establishing unambiguous taxonomic revision prior to any conservation assessment.
... The taxon mocsaryi Kriechbaumer, 1877 (Figs 5A, B) was re-assessed as a subspecies of Bombus laesus Morawitz (1875) (Figs 5C, D) by Brasero et al. (2021) based on genetic and semio-chemical analyses. ...
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At a time when nature conservation has become essential to ensure the long-term sustainability of our environment, it is widely acknowledged that conservation actions must be implemented within a solid taxonomic framework. In preparation for the upcoming update of the IUCN Red List, we here update the European checklist of the wild bees (sensu the IUCN geographical framework). The original checklist, published in 2014, was revised for the first time in 2017. In the present revision, we add one genus, four subgenera and 67 species recently described, 40 species newly recorded since the latest revision (including two species that are not native to Europe), 26 species overlooked in the previous European checklists and 63 published synonymies. We provide original records for eight species previously unknown to the continent and, as original taxonomic acts, we provide three new synonyms, we consider two names as nomina nuda, ten names as nomina dubia, three as species inquirenda, synonymize three species and exclude 40 species from the previous checklist. Around a hundred other taxonomic changes and clarifications are also included and discussed. The present work revises the total number of genera for IUCN Europe to 77 and the total number of species to 2,138. In addition to specifying the taxonomic changes necessary to update the forthcoming Red List of European bees, we discuss the sampling and taxonomic biases that characterise research on the European bee fauna and highlight the growing importance of range expansions and species invasions.
... This study seeks to clarify genetic patterns within the flavescens-complex, to establish the species status of geographically separated lineages and to infer biogeographic scenarios for bumblebees in Southeast Asia more generally. Species-level entities in bumblebees have been delimited by morphological, molecular (e.g., Williams et al. (2019Williams et al. ( , 2020), and chemical approaches, the latter using Cephalic Labial Gland Secretions or CLGS (e.g., Brasero et al. (2021), Ghisbain et al. (2021)). Given the challenges of morphology for taxonomic assessment of the flavescens-complex, molecular data are required to resolve the species status of sub-lineages and their relationships. ...
Full-text available
Bombus flavescens Smith is one of the most widespread bumblebee species in the Oriental region. Due to colour polymorphisms, this species or species-complex has been a challenge for taxonomy. This study aims to assess the taxonomic status of the flavescens-complex using evidence from COI barcodes and morphology. We then reconstruct its biogeographic history from a phylogenetic analysis of populations across the current range, combining COI with 16S and nuclear PEPCK data. Despite a large range of polymor-phisms across its distribution, the results show that B. flavescens is a single species based on algorithmic species delimitation methods, and it is clearly separated from its sister species, B. rotundiceps Friese. We suggest that B. flavescens diverged from its sister lineage in the Himalaya and dispersed into Southeast Asia in the Pleistocene. Conservation of the widespread B. flavescens will need to consider its several unique island populations.
... In the last two decades, investigation into cryptic species' diversity has increased rapidly, revealing that the DNA barcoding (Hebert et al., 2003) and species molecular delimitation methods provide irreplaceable complements to the morphological taxonomy (DeSalle & Goldstein, 2019;Kress et al., 2015;Lukhtanov, 2019). Among Hymenoptera, an integration of molecular methods allowed the differentiation of cryptic species with different habitat preferences, ecological niche, and behaviour divergence in bees (Mayr et al., 2021), including bumblebees (Brasero et al., 2021;Ghisbain et al., 2020;Waters et al., 2011), ants (Cordonnier et al., 2019;Schlick-Steiner et al., 2006) and wasps (Darwell & Cook, 2017;Kurushima et al., 2016;Orlovskytė et al., 2016;Somavilla et al., 2021;Wilson et al., 2012;Zhang et al., 2022). ...
The aim of the study was to clarify the phylogenetic relationships among Northern European Ancistrocerus and comparison of the applicability of evolutionarily neutral and non-neutral markers for reconstruction of phylogeny. We used a 19,400 bp long dataset that included parts of mitochondrial DNA, nuclear rDNA operon, and 10 nuclear protein-coding genes. Application of molecular barcoding and species delimitation algorithms unveiled a presence of cryptic species, A. balticus sp. n., in the trap-nesting wasp communities of the centre of Europe. We assessed the morphological, biological, and ecological differences of it from the sibling A. trifasciatus and updated the regional identification key. Phylogeny reconstruction using the neutral and the presumably non-neutral markers resulted in different tree topologies. Evolutionary congruence of the rDNA operon with the other markers was relatively low. Evolutionary rate of the mitochondrial genes was 7–8 times as high as that of the exons of the nuclear genes, therefore, the mitochondrial markers overshadowed the nuclear ones in the phylogeny reconstructions. We assumed that at the speciation level, we might consider two different patterns of phylogeny: one based on evolutionary time and neutral changes, and the other based on adaptive evolutionary pathways under directional selection pressures. We assessed the effect of directional selection on the nuclear protein-coding genes, applying the Spearman's rank correlation between pairwise phylogenetic distances among species, estimated using exons, and these distances, estimated using introns. One of these markers demonstrated a lack of positive correlation, implying a variable directional selection pressure on the coded protein. The publication has been registered on ZooBank:
... Due to the taxonomic works that have occurred in the last few years on European bumblebees, the extinction risk of several species will need to be reassessed. Examples include B. laesus (shown to be conspecific with B. mocsaryi from DNA evidence in Brasero et al. 2021) and the Spanish taxon reinigiellus (shown to be conspecific with B. hortorum in Ghisbain et al. 2021) among others. ...
Technical Report
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The BBSG exists to foster the conservation of bumblebees and their habitats around the world especially through the IUCN Red-Listing process. In this ninth report of the BBSG’s activities, 2022 has been another unusual year as the pandemic has continued to interfere with field work for some people. But despite the difficulties, there has been progress towards our goal of evaluating the extinction risk of all species of bumblebees worldwide using the IUCN Red List Criteria.
... mocsaryi Kriechbaumer, 1877, which vary in hair colour and are present in sympatry across much of Eastern Europe. Though varying in their hair colour, these taxa were recently found to belong to the same broad taxon (Brasero et al. 2021). ...
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Iran is a huge but understudied Middle Eastern country with a rich but chronically understudied bee fauna, including for the highly-speciose bee genus Andrena. Examination of unidentified museum material combined with recent field collections and a critical review of the literature has revealed a total of 197 species of Andrena in the Iranian fauna, of which 65 are newly reported for the country, with an additional 16 species new for science. Andrena (Aciandrena) deminuta Wood sp. nov., Andrena (Euandrena) boustaniae Wood sp. nov., Andrena (Euandrena) oblata sp. nov., Andrena (Euandrena) sani sp. nov., Andrena (Micrandrena) elam Wood sp. nov., Andrena (Micrandrena) subviridula Wood sp. nov., Andrena (Notandrena) idigna Wood sp. nov., Andrena (Planiandrena) flagrans Wood sp. nov., Andrena (Planiandrena) sella Wood sp. nov., Andrena (Ulandrena) bulbosa Wood sp. nov., Andrena (incertae sedis) hosseiniiae Wood & Monfared sp. nov., and Andrena (incertae sedis) rostamiae sp. nov. are described from Iran, Andrena (Micrandrena) extenuata sp. nov. is described from Iran and Syria, Andrena (Micrandrena) tabula Wood sp. nov. and Andrena (Micrandrena) obsidiana Wood sp. nov. are described from Iran and Turkey, and Andrena (Planiandrena) huma sp. nov. is described from Iran, Syria, and the Golan Heights. Eight taxa are synonymised (valid name first): Andrena (Melandrena) assimilis Radoszkowski, 1876 = Andrena (Melandrena) gallica Schmiedeknecht, 1883 syn. nov.; Andrena (Notandrena) emesiana Pérez, 1911 stat. resurr. = Andrena (Notandrena) recurvirostra Warncke, 1975 syn. nov.; Andrena (Plastandrena) eversmanni Radoszkowski, 1867 = Andrena (Plastandrena) peshinica Nurse, 1904 syn. nov.; Andrena (incertae sedis) hieroglyphica Morawitz, 1876 = Andrena (Carandrena) cara Nurse, 1904 syn. nov. and Andrena (Carandrena) halictoides Nurse, 1904 syn. nov.; Andrena (Melandrena) induta Morawitz, 1894 = Andrena (Melandrena) patella Nurse, 1903 syn. nov.; Andrena (incertae sedis) minor Warncke, 1975 stat. nov. = Andrena (Carandrena) splendula Osytshnjuk, 1984 syn. nov.; Andrena (Notandrena) zostera Warncke, 1975 = Andrena (Carandrena) subsmaragdina Osytshnjuk, 1984 syn. nov. Overall, these results considerably improve our understanding of the Iranian Andrena fauna, and suggest that overall bee diversity in this country is substantially more than 1000 species.
... Several recent revisions of particular subgenera or of faunas worldwide have sought clarifications on species' status, enabled specimen identification from both morphology and barcodes, as well as providing published barcodes (Brasero et al., 2021;Williams, 2021Williams, , 2022Williams et al., 2012Williams et al., , 2014Williams et al., , 2016Williams et al., , 2019Williams et al., , 2022. Bombus rubriventris Lepeletier is considered probably extinct (Williams, 2015). ...
A problem for understanding bumblebee biogeography is that if bumblebees dispersed from Asia through North America to South America, if they are poor at long-distance dispersal with establishment over sea, and if the land bridge between North and South America was not established until c. 3Ma BP, then there is an apparent conflict with the divergence among currently endemic South American lineages having been dated as early as 15–17 Ma. Using the first complete phylogenetic trees for all known and accepted extant species of the groups involved, we show how this conflict could be resolved. We suggest that characterizing bumblebees as being associated generally with temperate flower-rich meadows conflates divergent habitat specializations between two early lineages, associated with northern lowland grasslands and with southern montane grasslands respectively, which may have driven divergences in behaviour and in biogeographic processes. First, for most of the lowland grassland group of bumblebees, estimated dates of divergence are consistent with dispersal to South America via the land-bridge corridor that opened at c. 3Ma, followed by extant endemic lineages diverging in situ within South America. In contrast, for the second group that occupies montane grassland habitats (and for a few montane lineages of the ‘lowland’ group), we suggest that dispersal to South America at c. 3Ma could be consistent with older divergence for currently endemic species if: (1) many of the extant South American lineages had already diverged outside the region before 3Ma in neighbouring Mesoamerica; and (2) they had been constrained within the high mountains there, dispersing southwards into South America only once the isthmus corridor had become established; and (3) some of those ancestral montane lineages had become extirpated from Mesoamerica during subsequent warm climatic fluctuations. This interpretation re-emphasizes that biogeographic studies need to consider habitat-specific dispersal models that change through time.
... In a study based on genetic and semio-chemical traits, Brasero et al. (2021) conducted an integrative taxonomic analysis to evaluate species-delimitation hypotheses within a monophyletic group of bumblebees including the formerly recognised subgenera Eversmannibombus, Laesobombus and Mucidobombus, which are now included in the subgenus Thoracobombus. ...
Technical Report
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Annual report on activities of the regional groups of the IUCN Bumblebee Sub Group of the Wild Bee Specialist Group for 2021
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Habitat degradation and climate change are globally acting as pivotal drivers of wildlife collapse, with mounting evidence that this erosion of biodiversity will accelerate in the following decades. Here, we quantify the past, present and future ecological suitability of Europe for bumblebees, a threatened group of pollinators ranked among the highest contributors to crop production value in the northern hemisphere. We demonstrate coherent declines of bumblebee populations since 1900 over most of Europe and identify future large-scale range contractions and species extirpations under all future climate and land use change scenarios. Around 38–76% of studied European bumblebee species currently classified as ‘Least Concern’ are projected to undergo losses of at least 30% of ecologically suitable territory by 2061–2080 compared to 2000–2014. All scenarios highlight that parts of Scandinavia will become potential refugia for European bumblebees; it is however uncertain whether these areas will remain clear of additional anthropogenic stressors not accounted for in present models. Our results underline the critical role of global change mitigation policies as effective levers to protect bumblebees from manmade transformation of the biosphere.
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Species are often presumed to be apparent in nature, but in practice they may be difficult to recognise, especially when viewed across continents rather than within a single site. Coalescent-based Poisson-tree-process (PTP) models applied to fast-evolving genes promise one quantitative criterion for recognising species, complete with the estimates of uncertainty that are required of a scientific method. Such methods face challenges especially in discerning between widespread polytypic species and complexes of closely related, restricted-range species. In particular, ‘over-sampling’ of many closely related individuals within one species could risk causing groups of less closely-related individuals within other species appearing relatively more distinct and consequently could risk them being interpreted falsely as separate species. Some of the most persistent taxonomic problems among bumblebees (genus Bombus Latreille, 1802) are within the subgenus Melanobombus von Dalla Torre, 1880. For a global revision of Melanobombus species, we use COI barcodes and seek to reduce the risk from localised over-sampling by filtering the data to include only unique haplotypes. Unique haplotypes give more conservative results than unfiltered data, but still increase the number of species in comparison with recent morphological treatments. After integrative assessment of COI coalescents in comparison with morphological groups, the number of accepted species shows a non-linear increase with sample size that plateaus to an increase of 47% (to 25 species) compared with a previous estimate (of 17) based on morphology alone. For the most widespread and variable species-complexes, our revised species improve the match to the patterns expected of species, both for genetic divergence-with-distance and for sympatry, leading to three main inferences. (1) The particularly widespread polytypic Bombus sichelii Radoszkowski, 1859, is a single species. (2) We detect two candidates for species within previous broad concepts of each of the former B. lapidarius (Linnaeus, 1758), B. miniatus Bingham, 1897, and B. rufofasciatus Smith, 1852. Within B. lapidarius s. lat. we find insufficient evidence to corroborate the candidate species, with no coalescent or morphological support for a recent claim for a separate species, B. bisiculus Lecocq, Biella, Martinet & Rasmont, 2019 described from southern Italy, but rather we find a weak and uncorroborated coalescent for a different and much broader group of samples from across south-eastern Europe but excluding Turkey. Within the former broad concepts of B. miniatus s. lat. and B. rufofasciatus s. lat. the coalescent evidence is stronger and subtle evidence from morphology corroborates recognising B. miniatus s. str. and B. eurythorax Wang, 1892 stat. rev. as separate species as well as B. rufofasciatus s. str. and B. prshewalskyi Morawitz, 1880 stat. rev. as separate species. (3) Our coalescent and morphological results ‘split’ more clearly what has long been interpreted as a single polytypic B. keriensis Morawitz, 1887, s. lat., by supporting novel concepts of the restricted range species: B. alagesianus Reinig, 1930 stat. rev., B. incertoides Vogt, 1911 stat. rev., B. keriensis s. str., B. qilianensis sp. nov., B. separandus Vogt, 1909 stat. rev., and B. tibeticus sp. nov. A lectotype is designated for the name B. keriensis and a neotype is designated for the name B. alagesianus. We estimate the phylogeny of Melanobombus species by including three slower-evolving genes to provide more evidence for deeper relationships, to estimate the time calibration of this phylogeny, and to estimate ancestral distributions, all within a Bayesian framework. We provide the first keys for identifying all of the species of Melanobombus.
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Aim: Among the numerous anthropogenic pressures threatening biodiversity, habitat destruction and climate change are pointed to as dominant. In response, a number of mitigation strategies are elaborated to save endangered living organisms. However, the taxonomic level and geographical extent at which conservation strategies should be designed and implemented remain generally unclear. Here, we aim to assess and discuss the importance to apply conservation strategies at an appropriate taxonomic scale. For this purpose, we focus our analyses on bumblebees (genus Bombus), a group of critically important and endangered pollinators. Location: West-Palaearctic. Methods: We use a species distribution modelling approach to investigate and compare climatic and habitat-related variables associated with the distribution of West- Palaearctic bumblebees. Our analyses are based on a data set gathering more than 125,000 unique observation points for 68 species. Results: We highlight species-specific associations with climatic and land cover variables, depicting the strong relevance of taxon-specific mitigation strategies for the conservation of those key pollinators. We also identify that the occurrence probability of localized and widespread species is mostly predicted by specific land cover characteristics and climatic conditions, respectively. Finally, we report the general absence of phylogenetic signal associated with the relative importance of each environmental variable in species distribution models, underlining the difficulty to predict species-specific environmental requirements based on evolutionary relationships. Main conclusions: In the light of these results, we conclude that climate change and landscape destruction are not expected to drive the fate of all bumblebee species in a same direction, even for phylogenetically close lineages. We argue in favour of geographically and taxonomically adapted conservation strategies and discuss the limitations of untargeted action plans for species with different climatic/habitat requirements.
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Since the beginning of taxonomy, species have been described based on morphology, but the advent of using semio-chemicals and genetics has led to the discovery of cryptic species (i.e. morphologically similar species). When a new cryptic species is described, earlier type specimens have to be re-evaluated, although this process can be challenging as only non-destructive methods ought to be used in order to preserve the integrity of the type specimens. Methods should allow comparison with recently collected specimens clustered based on chemical, ethological and/or genetic traits with old specimens (i.e. type specimens) where only morphological traits are available. Here we develop a method based on geometric morphometric analyses of wing shape for a taxonomically challenging group of bumblebees, the subgenus Alpinobombus. We consider nine monophyletic taxa (including several cryptic species) to (i) assess the accuracy of this method to discriminate the taxa based on their wing shape and then (ii) to attribute type specimens using a Leave-One-Out cross-validation procedure. We show that for these bees wing shape is taxon-specific, except for two sister taxa for which the species status is still debated. Moreover, for most of the taxa, type specimens were correctly attributed with high posterior probabilities of attribution, except for a few type specimens corresponding to the same two sister taxa where taxa delimitation based on wing shape was previously the subject of discussion. Our study highlights the potential of geometric morphometric analyses to help in the re-attribution of type specimens when the existence of cryptic species is revealed.
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Phenotypic polymorphism can constitute an inherent challenge for species delimitation. This issue is exemplified in bumble bees ( Bombus ), where species can exhibit high colour variation across their range, but otherwise exhibit little morphological variation to distinguish them from close relatives. We examine the species status of one of the most abundant North American bumble bees, Bombus bifarius Cresson, which historically comprised two major taxa, bifarius s.s. and nearcticus . These lineages are recognized primarily by red and black variation in their mid‐abdominal coloration; however, a continuum from black ( nearcticus ) to red ( bifarius s.s.) variation has led to their historic synonymization. Integrating mitochondrial and nuclear data and whole‐genome sequencing, we reveal a high level of both mitochondrial and nuclear divergence delimiting two morphologically cryptic species – the red bifarius s.s. and the colour‐variable (black to red) nearcticus . Population genomic analysis supports an absence of recent genomic admixture and a strong population structure between the two clades, even in sympatry. Species distribution models predict partially differentiated niches between the genetically inferred clades with annual precipitation being a leading differentiating variable. The bifarius s.s. lineage also occupies significantly higher elevations, with regions of sympatry being among the highest elevations in nearcticus . Our data also support a subspecies‐level divergence between the broadly distributed nearcticus and the island population vancouverensis . In this paper, we formally recognize the two species, Bombus bifarius Cresson and Bombus vancouverensis Cresson, the latter including the subspecies B. vancouverensis vancouverensis comb.n. and B. vancouverensis nearcticus comb.n ., with vancouverensis the name bearer due to year priority.
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Recent improvements in taxonomy considers multiple operational criteria. The integrative taxonomy provides a methodological framework merging these multisource approaches. Bumblebees are considered as a uniform group where their taxonomy remains one of the most difficult. Here, we investigate the taxonomic statuses inside a monophyletic group including six taxa (B. inexspectatus, B. mlokosievitzii, B. ruderarius, B. sylvarum, B. velox and B. veteranus) in the most diverse subgenus of bumblebees: Thoracobombus. We used an integrative approach based on mitochondrial and nuclear genetic makers and eco-chemical traits commonly used in bumblebee taxonomy. For all species, our study shows a clear differentiation in DNA and the eco-chemical traits. However, we conserve the subspecies status of B. ruderarius simulatilis and B. sylvarum daghestanicus from the east of Turkey and Iran according to their degree of genetic and eco-chemical differentiation.
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Müllerian mimicry theory states that frequency-dependent selection should favour geographical convergence of harmful species onto a shared colour pattern. As such, mimetic patterns are commonly circumscribed into discrete mimicry complexes, each containing a predominant phenotype. Outside a few examples in butterflies, the location of transition zones between mimicry complexes and the factors driving mimicry zones has rarely been examined. To infer the patterns and processes of Müllerian mimicry, we integrate large-scale data on the geographical distribution of colour patterns of social bumblebees across the contiguous United States and use these to quantify colour pattern mimicry using an innovative, unsupervised machine-learning approach based on computer vision. Our data suggest that bumblebees exhibit geographically clustered, but sometimes imperfect colour patterns, and that mimicry patterns gradually transition spatially rather than exhibit discrete boundaries. Additionally, examination of colour pattern transition zones of three comimicking, polymorphic species, where active selection is driving phenotype frequencies, revealed that their transition zones differ in location within a broad region of poor mimicry. Potential factors influencing mimicry transition zone dynamics are discussed.
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The bumblebees of the subgenus Alpinobombus of the genus Bombus are unusual among bees for specialising in many of the most northerly vegetated arctic habitats on Earth. Most named taxa in this group (37 available names from a total of 67 names) were described originally from differences in the colour patterns of the hair. Previous revisions have shown unusually little agreement, recognising a range of 6‒9 species, in part because of pronounced intraspecific variation in both skeletal morphology and in the colour patterns of the hair. Here we examine variation among 4622 specimens from throughout the group’s global range. Bayesian inference of the gene tree for the fast evolving mitochondrial COI gene combined with Poisson-tree-process analysis of this tree shows support for 10 gene lineages as candidates for being putative species lineages. Integrative assessment shows that the interpretation of these results is not straightforward. Evidence from the fast evolving mitochondrial 16S ribosomal RNA gene supports two of the COI gene alleles (from the samples B. kluanensis s. str. and ‘unnamed2’) as being associated with just one 16S allele. Double COI bands on the PCR gels for these individuals and double peaks on sequence traces (in one case with both COI alleles sequenced from one individual) identifies this as a likely case of COI paralogy that has resulted in mitochondrial heteroplasmy. Evidence from morphology also supports only the remaining nine lineages as separate. Evidence from extracts of cephalic labial gland secretions (CLGS, with components believed to function as sex pheromones) reported by others shows small diagnostic differences between all of the candidate species examined (although B. kluanensis s. l. was not examined) and shows larger differences between all of the species pairs that we find are likely to have co-occurred at least in the past, revealing a likely limitation to the CLGS approach in cases of recent and continuously allopatric species. Consequently we infer nine species in the subgenus Alpinobombus (so that B. kluanensis s. str. and ‘unnamed2’ are interpreted as conspecific, as B. kluanensis s. l.). We provide distribution maps and identification keys for the nine species. The morphology of the male of B. kluanensis is described for the first time, including a unique, unusually dense pad of short hair on the mandible that may have a function involving CLGS in mate-searching behaviour. In seeking to identify the valid names for these species, seven new lectotypes are designated and support is provided for synonymizing 10 names as proposed in a recent summary table of names. The prevailing usage of Bombus balteatus Dahlbom is maintained as valid by proposing Bombus nivalis Dahlbom and Bombus tricolor Dahlbom as nomina oblita and by proposing Bombus balteatus Dahlbom as a nomen protectum. The prevailing usage of Bombus hyperboreus Schönherr is maintained as valid by supporting Apis arctica Quensel as a nomen oblitum and by supporting Bombus hyperboreus Schönherr as a nomen protectum. We then use sequence data from COI and 16S together with nuclear PEPCK and opsin genes to estimate dated phylogenetic relationships among the nine species, allowing for incongruent gene trees with *BEAST. If crown-group divergence within the subgenus Alpinobombus coincided with the global climate cooling and with the growth of the northern ice sheets at the end of the Miocene at ca 7.2 Ma, then divergences between each of the three pairs of sister species are likely to have coincided with fluctuations in vegetated land connections across the Bering Strait after ca 2.5 Ma.
Males of many bumblebee species exhibit a conspicuous premating behaviour with two distinct behavioural components: scent marking and patrol flying. The marking pheromone is produced by the cephalic part of the labial gland (CLG). As far as known, the CLG secretion is species-specific and it usually consists of two types of compounds: (i) straight chain aliphatic alcohols, aldehydes, or esters, and (ii) acyclic mono-, sesqui- and diterpenes (alcohols or acetates). Here we summarize data from the literature reporting chemical composition of the CLG secretions of more than 80 bumblebee species. Similarities and differences within and between subgenera are discussed in the context of biosynthetic pathways and evolution.
While bumblebees have been the focus of much research, the taxonomy of many species groups is still unclear, especially within circum-polar species. Delimiting species based on multisource datasets provides a solution to overcome current systematic issues of closely related populations. Here, we use an integrative taxonomic approach based on novel genetic and eco-chemical datasets to resolve the taxonomic status of Bombus lapponicus and B. sylvicola. Our results support the conspecific status of B. lapponicus and B. sylvicola and that the low gradual divergence around the Arctic Circle between Fennoscandia and Alaska does not imply speciation in this species complex. Therefore, based on our molecular and morphological analyses, we propose to assign the sub-specific status: B. lapponicus lapponicus comb. nov. from Fennoscandia and W-Siberia and B. lapponicus sylvicola comb. nov. from Alaska and Yukon. Moreover, our analyses reveal a new cryptic arctic species in the B. lapponicus complex: B. (Pyrobombus) interacti sp. nov. Martinet, Brasero & Rasmont from Alaska.