Resolving the species status of overlooked West-Palaearctic bumblebees

Abstract

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.

Full text

Zoologica Scripta. 2021;00:1–17. wileyonlinelibrary.com/journal/zsc
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1
© 2021 Royal Swedish Academy of Sciences
Received: 18 November 2020
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Revised: 6 February 2021
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Accepted: 12 March 2021
DOI: 10.1111/zsc.12486
ORIGINAL ARTICLE
Resolving the species status of overlooked West- Palaearctic
bumblebees
NicolasBrasero1
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GuillaumeGhisbain1
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ThomasLecocq1,2
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DenisMichez1
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IrenaValterová3,4
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PaoloBiella5
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AlirezaMonfared6
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Paul HughWilliams7
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PierreRasmont1
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BaptisteMartinet1,8
1Laboratory of Zoology, Research institute
for Biosciences, University of Mons, Mons,
Belgium
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
Republic
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,
Belgium
Correspondence
Baptiste Martinet, Evolutionary Biology &
Ecology, Université Libre de Bruxelles, C.P.
160/12, Avenue F.D. Roosevelt, 50, B- 1050
Brussels, Belgium.
Email: Baptiste.Martinet@umons.ac.be
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
ct.net, 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).
Abstract
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.
KEYWORDS
Bombus, cryptic species, DNA sequences, integrative taxonomy, male marking secretion

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BRASERO Et Al.
1
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INTRODUCTION
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

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BRASERO Et Al.
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.
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MATERIAL AND METHODS
2.1
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Sampling
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.
2.2
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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)

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BRASERO Et Al.
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

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BRASERO Et Al.
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-
its.org/) 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).
2.3
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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

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BRASERO Et Al.
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

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BRASERO Et Al.
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.
2.4
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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
Taxa
Sampling country
nuDNA marker
(PEPCK)
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
Spain
2M, 4F 3M, 4F 4M
mucidus Austria, France, Italy and
Switzerland
17M, 7F 14M, 4F 15M
pittioniellus Albania, Macedonia and
Montenegro
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
BOLD
1F 1F -

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BRASERO Et Al.
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).
2.5
|
Conservation
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

|
9
BRASERO Et Al.
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
etal.(2014).
3
|
RESULTS
3.1
|
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

10
|
BRASERO Et Al.
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).
3.2
|
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).
3.3
|
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:
3.9±0.2mm.
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

|
11
BRASERO Et Al.
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.
3.4
|
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

12
|
BRASERO Et Al.
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).
4
|
DISCUSSION
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).
4.1
|
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
Proposed
taxonomic status
Laesobombus mocsaryi aliceae
(Morocco)
+(A)a / - +(A)a – B. laesus aliceae
comb. nov
laesus (Turkey,
Kyrgyzstan)
− (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.

|
13
BRASERO Et Al.
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.
4.2
|
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

14
|
BRASERO Et Al.
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.
ACKNOWLEDGEMENTS
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- project.net, grant agreement no 244090, FP7/2007- 2013).
AUTHOR CONTRIBUTIONS
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.
ORCID
Nicolas Brasero https://orcid.org/0000-0001-9665-302X
Guillaume Ghisbain https://orcid.
org/0000-0003-2032-8081
Thomas Lecocq https://orcid.org/0000-0002-4947-0332
Denis Michez https://orcid.org/0000-0001-8880-1838
Irena Valterová https://orcid.org/0000-0001-5723-6143
Paolo Biella https://orcid.org/0000-0003-2297-006X
Alireza Monfared https://orcid.org/0000-0002-4465-3228
Paul Hugh Williams https://orcid.org/0000-0002-6996-5682
Pierre Rasmont https://orcid.org/0000-0003-0891-2189
Baptiste Martinet https://orcid.org/0000-0003-4369-8552
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SUPPORTING INFORMATION
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. https://doi.org/10.1111/zsc.12486