Following the Cold: Geographic Differentiation between Interglacial Refugia and Speciation in Arcto-Alpine Species Complex Bombus monticola (Hymenoptera: Apidae)

Abstract

Cold-adapted species are expected to reach their largest distribution range during a part of the Ice Ages while the post-glacial warming leads to their range contracting toward high latitude and high altitude areas. This results in extant allopatric distribution of populations and possibly to trait differentiations (selected or not) or even speciation. Assessing an inter-refugium differentiation or speciation remains challenging for such organisms because of sampling difficulties (several allopatric population) and disagreement on species concept. Here, we assessed post-glacial inter-refugia differentiation and potential speciation among populations of one of the most common arcto-alpine bumblebee species in European mountains, Bombus monticola Smith, 1849. Based on mitochondrial (mt) DNA/nuclear (nu) DNA markers and eco-chemical traits, we performed integrative taxonomic analyses to evaluate alternative species delimitation hypotheses and to assess geographic differentiation between interglacial refugia and speciation in arcto-alpine species. Our results show that trait differentiations occurred between most of South European mountains (i.e. Alps, Balkan, Pyrenees and Apennines) and Arctic regions. We suggest that the monticola complex actually includes three species: B. konradini sp. nov. status distributed in Italy (Central Apennine mountains), B. monticola with five subspecies, including B. monticola mathildis ssp. nov. distributed in the North Apennine mountains and B. lapponicus. Our results support that post Ice-Age periods can lead to speciation in cold-adapted species through distribution range contraction. We underline the importance of an integrative taxonomy approach for rigorous species delimitation and for evolutionary study and conservation of taxonomically challenging taxa.

Full text

Systematic Entomology (2018), 43, 200– 217 DOI: 10.1111/syen.12268
Following the cold: geographical differentiation
between interglacial refugia and speciation in the
arcto-alpine species complex Bombus monticola
(Hymenoptera: Apidae)
BAPTISTE MARTINET1, THOMAS LECOCQ1,2,
NICOLAS BRASERO1, PAOLO BIELLA3,4, KLÁRA URBANOVÁ5,6,
IRENA VALTEROVÁ5, MAURIZIO CORNALBA7,JAN OVE
GJERSHAUG8, DENIS MICHEZ1andPIERRE RASMONT1
1Laboratory of Zoology, Research Institute of Biosciences, University of Mons, Mons, Belgium, 2Research Unit Animal and
Functionalities of Animal Products (URAFPA), University of Lorraine-INRA, Vandoeuvre-lès-Nancy, France, 3Faculty of Science,
Department of Zoology, University of South Bohemia, ˇ
Ceské Budˇ
ejovice, Czech Republic, 4Biology Centre of the Academy of
Sciences of the Czech Republic, v.v.i., Institute of Entomology, ˇ
Ceské Budˇ
ejovice, Czech Republic, 5Academy of Sciences of the
Czech Republic, Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic, 6Faculty of Tropical AgriSciences,
Department of Sustainable Technologies, Czech University of Life Sciences, Prague, Czech Republic, 7Department of Mathematics,
University of Pavia, Pavia, Italy and 8Norwegian Institute for Nature Research, Trondheim, Norway
Abstract. Cold-adapted species are expected to have reached their largest distribution
range during a part of the Ice Ages whereas postglacial warming has led to their range
contracting toward high-latitude and high-altitude areas. This has resulted in an extant
allopatric distribution of populations and possibly to trait differentiations (selected or
not) or even speciation. Assessing inter-refugium differentiation or speciation remains
challenging for such organisms because of sampling difculties (several allopatric
populations) and disagreements on species concept. In the present study, we assessed
postglacial inter-refugia differentiation and potential speciation among populations of
one of the most common arcto-alpine bumblebee species in European mountains,
Bombus monticola Smith, 1849. Based on mitochondrial DNA/nuclear DNA markers
and eco-chemical traits, we performed integrative taxonomic analysis to evaluate
alternative species delimitation hypotheses and to assess geographical differentiation
between interglacial refugia and speciation in arcto-alpine species. Our results show
that trait differentiations occurred between most Southern European mountains (i.e.
Alps, Balkan, Pyrenees, and Apennines) and Arctic regions. We suggest that the
monticola complex actually includes three species: B. konradini stat.n. status distributed
in Italy (Central Apennine mountains), B. monticola with ve subspecies, including
B. monticola mathildis ssp.n. distributed in the North Apennine mountains ; and
B. lapponicus. Our results support the hypothesis that post-Ice Age periods can lead to
speciation in cold-adapted species through distribution range contraction. We underline
the importance of an integrative taxonomic approach for rigorous species delimitation,
and for evolutionary study and conservation of taxonomically challenging taxa.
Correspondence: Baptiste Martinet, Laboratory of Zoology, Research Institute of Biosciences, University of Mons, Mons, Belgium.
E-mail: baptiste.martinet@umons.ac.be
200 © 2018 The Royal Entomological Society

Following the cold: speciation in B. monticola 201
Introduction
Past climatic oscillations have led to signicant changes in
distributions of species. However, species responses to cli-
mate change depend mainly on their eco-climatic requirements
and tolerances (Hewitt, 2004a,b; Thuiller, 2004; Stewart et al.,
2010). Pleistocene and Quaternary climatic cycles triggered
massive population movements resulting in periods of species
range reductions (i.e. during cold periods when populations are
restricted to refuge areas) for temperate species followed by
periods of species range expansions (i.e. during warmer peri-
ods when populations recolonize at least portions of their ini-
tial range) (Reinig, 1937; Stewart et al., 2010; Hewitt, 2004a).
These population dynamics have fostered intraspecic diver-
gence processes leading to differentiation and possibly specia-
tion (Avise, 2000; Hewitt, 2004a). Alternative demographic his-
tories and subsequent differentiation patterns can be expected
for cold-adapted species. Assessing accurately consequences of
past climate change on differentiation and speciation process is
a key element for better understanding and predicting the evo-
lution of future biodiversity and to propose evidence-based mit-
igation strategies (Rasmont et al., 2015).
Although population dynamics of temperate species fostered
by past climatic events and their consequences have been the
focus of abundant research (Zagwijn, 1992; Taberlet, 1998;
Hewitt, 1999; Stewart et al., 2010), cold-adapted species have
received comparatively little attention to date (Mardulyn et al.,
2009). Contrary to temperate taxa, cold-adapted species are
thought to have reached their largest distribution range during
the Ice Ages (Hewitt, 2011). The postglacial warming and sub-
sequent interglacial period is thought to have led to range con-
traction of such cold-adapted species toward the high-latitude
and-altitude areas (Barnes et al., 2007; Fedorov et al., 2008;
Hewitt, 2011). Such a population dynamic scenario can explain
current allopatric patterns of species distributed in the Arctic
and in southern mountains (i.e. arcto-alpine species) acting as
interglacial refugia. These taxa have their current distribution
in the relicts (refugia) of a widespread distribution fragmented
by postglacial warming (Reinig, 1937; Mardulyn et al., 2009;
Dellicour et al., 2014a,b). In Europe, due to interglacial periods,
arcto-alpine species exhibit a strong pattern of allopatry between
southern mountains (Pyrenees, Alps, Apennines, Balkans, and
Caucasus) and northern areas (arctic regions of North Scandi-
navia and Russia). Such allopatric patterns have fostered and still
foster gene ow disruptions, leading to divergence and possibly
speciation of cold-adapted species (Avise, 2000; Hewitt, 2004b).
However, assessing species delimitation remains challenging
because it requires the arbitrary selection of variable traits whose
accuracy continues to be debated (Mayr, 1942; De Queiroz,
2007; Lecocq et al., 2015a,d). Moreover, it is quite difcult
to comprehensively sample specimens for phylogeographical
or speciation studies across vast inhospitable areas such as
high-altitude mountains and Arctic areas (Hewitt, 2011). This
could lead to the underestimation of the variability within each
allopatric population and to misunderstanding of the allopatric
differentiation process.
The integrative taxonomy based on the unied species con-
cept (De Queiroz, 2007) aims to overcome limitations due
to unsettled adequacy of selected diagnostic traits and lim-
ited sampling. First, the approach considers multiple inde-
pendent lines of evidence to evaluate interpopulation differ-
entiation processes and taxonomic statuses (Schlick-Steiner
et al., 2010; Lecocq et al., 2015a,d). This reduces the likeli-
hood of false taxonomic conclusions driven by single trait.
Second, analysing multiple traits to investigate interpopulation
differentiation facilitates an increase in the amount of infor-
mation available despite a limited sample size (Lecocq et al.,
2011).
Among potential organisms of interest for studying climatic
oscillation consequences on cold-adapted species, bumblebees
(Hymenoptera, Apidae, Bombus) represent a relevant biological
system because some of them (i) live in the coldest areas inhab-
ited by insects and (ii) have undergone diversication processes
during the Pleistocene and Quaternary climatic cycles (Mich-
ener, 2007; Hines, 2008; Duennes et al., 2012; Martinet et al.,
2015a; Rasmont et al., 2015; Dellicour et al., 2016). Their inter-
specic and interpopulation differentiations have been studied
for a long time (e.g. Reinig, 1939). However, different diagnos-
tic traits (morphological traits, DNA sequences, eco-chemical
traits) have been used, resulting in conicting biological con-
clusions (e.g. Gjershaug et al., 2013; Williams et al., 2015).
Over the past few years, the efciency of available diagnos-
tic characters has been critically discussed and a merging of
these traits in an integrative taxonomic framework has been pro-
posed (e.g. Lecocq et al., 2015d). This provides the opportu-
nity to efciently delimit species for a common cold-adapted
bumblebee species with a strong pattern of allopatry. Moreover,
integrative taxonomy can help to dene the subspecies status
of allopatric populations (Lecocq et al., 2015a,b,d). In bum-
blebees, subspecies denition is traditionally based on colour
pattern variation, notwithstanding that this diagnostic character
requires an extensive overview of the interindividual variability
(Bertsch & Schweer, 2012a). However, colour pattern has been
shown to be unsuitable for taxonomic delimitation (Vogt, 1909;
Bertsch & Schweer, 2012a; Carolan et al., 2012; Williams et al.,
2015) as well as for intraspecic variation study (Lecocq et al.,
2015b,d).
Here, we investigated the potential inter-refugium differentia-
tion and speciation within one of the most common arcto-alpine
bumblebee species in European mountains (Rasmont et al.,
2015): Bombus (Pyrobombus)monticola Smith, 1849. We sam-
pled all of the allopatric regions where the species is known
(infraspecic taxa). We analysed interpopulation differentia-
tion through multiple diagnostic traits: (i) a mtDNA marker
(cytochrome oxidase I, COI), (ii) a nuDNA marker (phospho-
enolpyruvate carboxykinase, PEPCK), and (iii) eco-chemical
traits (cephalic labial gland secretions, CLGS). Based on
these traits, we developed an integrative taxonomic approach
sensu Lecocq et al. (2015a,d) to assess the taxonomic sta-
tus of major clades. In this approach, all taxonomic criteria
used must be signicantly differentiated to assign the species
status.
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

202 B. Martinet et al.
Material & methods
Model species
Bombus (Pyrobombus)monticola Smith, 1849 is an
arcto-alpine species widespread in the alpine and sub-alpine
stages of the highest mountain ranges of Europe with isolated
populations in Northern Europe and Mediterranean moun-
tains (Cantabrian Mountains, Pyrenees, Alps, Apennines and
Balkans, but not Caucasus) (Svensson, 1979; Kuhlmann et al.,
2014; Rasmont et al., 2015) (Fig. 1). Bombus monticola was
conrmed as an unique taxonomic unit by chemical (cephalic
labial gland secretion and enzymology) and genetic analy-
sis (Svensson, 1979; Gjershaug et al., 2013) in comparison
with its most similar taxon B. lapponicus (Fabricius, 1793).
The analysis of Hines (2008) suggested that B. monticola
diverged from its sister species B. lapponicus about 3 Ma. The
species displays geographically differentiated colour patterns
(Reinig, 1965) that have been used to dene ve pheno-
typically diagnosable allopatric subspecies (Table 1; Fig. 2,
Rasmont et al., 2015): (i) B. monticola scandinavicus Friese,
1912 (Fennoscandia), (ii) B. monticola monticola Smith, 1849
(British Islands), (iii) B. monticola alpestris (=hypsophilus,
Tkalcu, 1992) Vogt, 1909 (Alps, the Balkans and the Olympus
Mount), (iv) B. monticola rondoui Vogt, 1909 (Cantabrian
Mountains and Pyrenees) and (v) B. monticola konradini
Reinig, 1965 (Apennine Mountains) (Figs 1, 2). We dene
‘monticola complex’ as B. monticola ssp. +B. lapponicus
and only ‘monticola’ gathering exclusively all subspecies of
B. monticola.
Sampling
We sampled 70 specimens including all B. monticola taxa
(Appendix S1) from the entire known distribution area:
B. monticola scandinavicus (n=11) from North Scandi-
navia, B. monticola monticola (n=10) from the British Isles,
B. monticola rondoui (n=9) from the Pyrenees, B. monticola
alpestris from the Alps (n=9), Balkans (n=3) and Mount
Olympus (n=1), and B. monticola konradini (sensu Reinig,
1965) from the Central Apennines (Sibilini Mountains) (n=5)
and from the North Apennines (n=2). The North Apennines
population, whose geographical distribution includes the high-
est peaks in the Apuan Alps, is separated by wide gaps not
only from the Central Apennines populations, but also from
alpine alpestris (almost 230 km). We used the phylogenetically
closely related species B. (Pyrobombus) lapponicus (n=10)
for comparison (see Cameron et al., 2007) and B. bimaculatus
(Cresson, 1863) (n=10) to root trees in our genetic analyses.
All specimens were killed by freezing at −20∘C. We considered
all taxa without aprioritaxonomic status and referred to them
as scandinavicus,monticola,rondoui,alpestris,konradini,
lapponicus,andbimaculatus (Table 2). We further split kon-
radini into konradini-N to indicate the Northern Apennines
population and konradini-C to indicate the Central Apennines
population.
Fig. 1. Distribution map (Gall projection) of Bombus monticola (Ras-
mont & Iserbyt, 2014) and its traditional subspecies in Europe according
to (Rasmont, 1983). (A) Bombus monticola scandinavicus queen, red
area on the map; (B) B. monticola monticola queen, dark green area;
(C) B. monticola rondoui queen, purple area; (D) B. monticola alpestris
queen, blue area; (E) B. monticola mathildis ssp.n. Holotype male, pink
area; (F) B. konradini stat.n. Lectotype queen, yellow area. White dots
indicate the occurrence of the taxon in the region. [Colour gure can be
viewed at wileyonlinelibrary.com].
Genetic differentiation analyses
In order to investigate the potential genetic differentiation
between B. monticola taxa, we sequenced two genes that are
commonly used in bee phylogenetic and phylogeographic stud-
ies (e.g. Pedersen, 2002; Cameron et al., 2007; Williams et al.,
2012; Dellicour et al., 2015): the mitochondrial gene COI and
the nuclear gene PEPCK. We performed DNA extraction pro-
tocol, PCR (COI primers Apl2013/Aph2931, Pedersen, 2002;
PEPCK primers FHv4/RHv4, Cameron et al., 2007), sequenc-
ing procedures and DNA sequence alignment using the method
described in Lecocq et al. (2013a,b). We uploaded the result-
ing COI (938 bp) and PEPCK (925 bp) sequences in GenBank
(accession numbers Appendix S1).
We investigated the potential genetic differentiation within
B. monticola through haplotype network analyses and phyloge-
netic inference. We carried out the analyses for each gene indi-
vidually. We used the median-joining method to produce haplo-
type networks with N 4.6.1.0 (www.uxus-engineering
.com). We weighted transversions twice as high as transitions to
reconstruct the network (Lecocq et al., 2015a,b).
In phylogenetic analyses, we analysed each gene with
maximum parsimony (MP), maximum-likelihood (ML), and
Bayesian (MB) methods. We carried out MP analyses (heuristic
method) using S 3.2 (Galtier et al., 1996) with 1000 000
replicas. Only high-quality trees and the majority rule 50%
consensus tree were conserved. For ML and MB, each gene
was partitioned as follows: (i) the nuclear gene (PEPCK)into
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

Following the cold: speciation in B. monticola 203
Table 1. Range, conservation status, and main morphological and colour pattern differences (male and female) between Bombus konradini stat.n.,monticola subspecies including mathildis ssp.n.
and the similar species B. lapponicus according to Gjershaug et al. (2013), Løken (1973), Pittioni (1939) and original observations.
scandinavicus monticola rondoui alpestris mathildis ssp.n. konradini stat.n. lapponicus
Range Fennoscandia British Isles Pyrenees Alps, Balkans, Mt.
Olympus
North Apennines Central Apennines Fennoscandia
Conservation status No regression was
mentioned
In decline Evans &
Potts (2013) and
Fitzpatrick et al.
(2006)
In decline Iserbyt &
Rasmont (2012)
Few data show a
decline in Italy
Manino et al.
(2007)
No regression was
mentioned
Rare and localized
Ricciardelli & Piatti
(2003)
Stable Nieto et al. (2014)
Female
Morphology
Furrow of gena The surface between the punctures on vertex is shiny, and there is a slight depression with some punctures near
the compound eye
Similar to monticola The surface between the
punctures on vertex is
rugose and dull and the
furrow is distinct,
nearly reaching the
compound eye
Hind meta-basitarsus Slight pubescence and the maximal width of the basitarsus is high (sensu Gjershaug et al., 2013). The length of
the metabasitarsus of these taxa is large (Appendix S3)
Strong pubescence and
the maximal width of
the basitarsus is low
(sensu Gjershaug et al.,
2013) as in lapponicus.
The ratio maximum
length/maximum width
of the metabasitarsus of
this taxon is
intermediate
(Appendix S3).
Strong pubescence and
the maximal width of
the basitarsus are low
(sensu Gjershaug et al.,
2013). The length of
the metabasitarsus of
this taxon is short
(Appendix S3).
Coat colour variation Dark Dark Light Relatively dark Light and colorful Large and light Reinig
(1965)
Varies from very light and
colorful in Northern
Fennoscandia, to rather
dark in Southern
Fennoscandia
(Southern Norway)
Colour pattern
Face Black Black Yellow Black Yellow or sometimes
black (Figure S1)
Yellow (Figure S1) Black
Collare and scutellare Small dark yellow Small dark yellow
and black
Light yellow/yellow Small dark
yellow/dark yellow
Wide light yellow with a
black line near the
tegulae/yellow
Wide yellow band to the
tegulae/yellow
Yellow
Tergite 1 Black/Red Black Yellow Yellow/black Yellow/black (center of
tergite)
Yellow/red/black Yellow/red/black
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

204 B. Martinet et al.
Table 1. Continued
scandinavicus monticola rondoui alpestris mathildis ssp.n. konradini stat.n. lapponicus
Tergite 4 Dark red Dark red Red Light red Dark red with sometimes
yellow (few)
Yellow Yellow
Tergite 5 Dark red Light red Light red Light red Dark red with sometimes
yellow (few)
Yellow Yellow
Male
Colour pattern
Face Dark yellow Yellow Yellow Yellow Yellow Yellow Yellow
Collare/scutellare Dark yellow/NO Yellow/dark yellow Yellow/large yellow Yellow/large yellow Yellow/large yellow Yellow/large yellow Yellow/large yellow
Tergite 1 Black and red Black and red Yellow and black Yellow and black Yellow and black Yellow Yellow
Tergite 4 Dark red Dark red Dark red Dark red Light red Red/yellow Yellow/red
Tergite 5 Dark red Red Red Red Light red Red/yellow Yellow/red
two exons and two introns and (ii) each nuclear exon and (iii)
the mitochondrial gene (COI) by base positions (rst, second
and third nucleotide) to dene the best substitution model with
JMT S 2.0 (Posada, 2008) using the corrected
Akaike information criterion. Best-tting substitution models
(i) for COI:GTR+I (rst position), TIM2 +I (second posi-
tion), TrN +G (third position); (ii) for PEPCK intron 1: TPM1
uf +I; (iii) for PEPCK exon 1: HKY +I (rst position), JC
(second position), TrN +I (third position); (iv) for PEPCK
intron 2: TrN +I; (v) for PEPCK exon 2: JC (rst position),
JC (second position), JC (third position). For ML analyses,
we performed ten independent runs in GARLI 2.0 for both
genes (Zwickl, 2006); the topology and −ln L was the same
among replicates. Only the run with the highest likelihood
was saved. We assessed statistical signicance of nodes with
10 000 nonparametric bootstrap replicates. We considered a
topology to be well supported (high condence) whenever the
bootstrap value (branch supports) was greater than 85% (Hillis
& Bull, 1993). We carried out Bayesian inference analyses
(MB) with  3.1.2 (Ronquist & Huelsenbeck, 2003). We
achieved ten independent analyses for each gene (100 million
generations, four chains with mixed models, default priors,
saving trees every 100generations). Then we removed the
rst 10 000 000 generations as burn-in procedure. Then a
majority-rule 50% consensus tree was constructed. Only branch
supports (topologies) with high posterior probabilities (≥0.95)
were considered to be statistically signicant (Wilcox et al.,
2002). We (re) rooted all trees with the taxon B. bimaculatus.
In order to recognize species threshold, we used a Bayesian
implementation of the general mixed Yule-coalescent model
(bGMYC) for species delimitation based on the COI tree (Reid
& Carstens, 2012; see an example of the use of the approach
in Lecocq et al., 2015d). These analyses were performed with
‘bGMYC’ R packages (Reid & Carstens, 2012). The stationar-
ity and the modal coalescent/Yule ratio have been assessed to
continue the analysis. A range of probabilities >0.95 was con-
sidered as strong evidence that taxa were conspecic, whereas a
range of probabilities <0.05 suggested that taxa were heterospe-
cic (Reid & Carstens, 2012). Because bGMYC required ultra-
metric trees, we performed a phylogenetic analysis with 
1.7.2 (Drummond & Rambaut, 2007) using a phylogenetic clock
model to generate a posterior distribution of trees [length of
the Markov chain Monte Carlo (MCMC): 1 billion generations],
with the rst million sampled trees as burn-in, using the maxi-
mum clade credibility method and setting the posterior probabil-
ity limit to 0. We based the bGMYC analysis on 1000 trees sam-
pled every 10000 generations. For each of these 1000 trees, the
MCMC was made of 100 000 generations, discarding the rst
90 000 as burn-in and sampling every 100 generations. Posterior
probability distribution was applied against the rst sample tree
to provide a ‘heat’ map’.
Molecular clock – estimating divergence time
Following the approach of Duennes et al. (2012) and Lecocq
et al. (2013a), we analysed the COI dataset in  v1.7.2
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

Following the cold: speciation in B. monticola 205
Fig. 2. Photos of the studied bumblebee taxa. (A) Bombus monticola scandinavicus queen; (B) Bombus monticola monticola queen; (C) Bombus
monticola rondoui queen; (D) Bombus monticola alpestris queen; (E) Bombus monticola mathildis ssp.n. Holotype male; (F) Bombus konradini stat.n.
Lectotype queen. All photographs are by P. Rasmont. [Colour gure can be viewed at wileyonlinelibrary.com].
(Drummond & Rambaut, 2007) to estimate the divergence time
among different clades. Using the GTR +I model selected by
jModeltest, we ran MCMC simulations with the coalescent
constant population size tree model and the relaxed clock
model. Considering that no fossils of Pyrobombus species are
available, the phylogeny is calibrated with a range date from a
molecular study. We specied a range of possible substitution
rates which includes the extreme rate for insect mitochondrial
genes recorded in the literature (e.g. Duennes et al., 2012) using
a at prior ranging from 1 ×10–9 to 1 ×10–7 substitutions site-1
and year-1. Simulations were run for 300 million generations,
sampling every 1000 generations. Four independent runs were
assessed in T v1.4.1 (Rambaut & Drummond, 2013)
to conrm convergence, determine burn-in and examine the
effective sample size of all posterior parameters. Log les from
each run were combined in LC v1.6.1 (Rambaut &
Drummond, 2013) for nal parameter estimates.
Eco-chemical trait differentiation
We focused on CLGS, the most studied eco-chemical trait
involved in bumblebee pre-mating recognition (Baer, 2003;
Ayasse & Jarau, 2014). These secretions are complex mixtures
of mainly aliphatic compounds synthesized de novo by male
cephalic labial glands (Coppée et al., 2008; Lecocq et al., 2011;
Žacek et al., 2013). We identied the main component as the
Table 2. Summary of sampling table with genetic and eco-chemical
criteria for Bombus species and subspecies used in this study.
Taxa Sampling site PEPCK COI CLGS
B. lapponicus
(Fabricius 1793)
North Sweden 5M 5M 10M
B. bimaculatus
(Cresson 1863)
East Canada 3M 5M 10M
B. monticola
scandinavicus
Friese 1911
North Sweden 5M 5M 11M
B. konradini stat.n.
Reinig, 1965
Italy (Central
Apennines)
3M, 2F 2M, 2F 2M
B. monticola
mathildis ssp.n.
Martinet, Cornalba
& Rasmont 2016
Italy (North
Apennines)
2M 2M 2M
B. monticola alpestris
Vogt, 1909
Alps, Balkans, Mt.
Olympus
6M 6M 13M
B. monticola
monticola Smith
1849
Scotland 5M 4M 10M
B. monticola rondoui
Vogt, 1909
France (Pyrenees) 2F 4M 7M
PEPCK, Phosphoenolpyruvate carboxykinase gene; COI: Cytochrome
oxidase 1 gene; CLGS, cephalic labial gland secretions; M, male; F,
female.
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

206 B. Martinet et al.
compound that had the highest relative area (RA) among all
compounds of CLGSs at least in one specimen of the taxon. The
CLGS are species-specic blends with some interpopulation
variations and are, subsequently, commonly used for species
discrimination and assessment of intraspecic variability in
bumblebees (review in Lecocq et al., 2015a,d). We extracted the
CLGS with 400 𝜇L of n-hexane, according to De Meulemeester
et al. (2011) and Brasero et al. (2015). Samples were stored at
−40∘C prior to the analyses.
We qualied the CLGS composition of each sample by gas
chromatography–mass spectrometry (GC/MS) using a Focus
GC (Thermo Scientic) with a nonpolar DB-5 ms capillary
column [5% phenyl (methyl) polysiloxane stationary phase;
column length 30 m; inner diameter 0.25 mm; lm thickness
0.25 μm] coupled to a DSQ II quadrupol mass analyser (Thermo
Scientic, Waltham, MA, U.S.A.) with 70eV electron impact
ionization. We identied each compound using the retention
times and mass spectra of each peak, in comparison to those
from the National Institute of Standards and Technology library
(NIST, U.S.A.) database. We determined double bound positions
(C=C) by dimethyl disulde (DMDS) derivatization (Vincenti
et al., 1987).
We quantied the CLGS compounds with a gas chromato-
graph Shimadzu GC-2010 system (GC-FID) equipped with
a nonpolar SLB-5 ms capillary column [5% phenyl (methyl)
polysiloxane stationary phase; column length 30 m; inner diam-
eter 0.25 mm; lm thickness 0.25 𝜇m] and a ame ionization
detector. We quantied the peak areas of compounds in GC solu-
tion postrun (Shimadzu Corporation) with automatic peak detec-
tion and noise measurement. The relative areas (RAs, expressed
in %) of compounds in each sample were calculated by dividing
the peak areas of compounds by the total area of all compounds.
We excluded compounds for which RA were less than 0.1% for
all specimens (De Meulemeester et al., 2011). The data matrix
for each taxon was based (Appendix S2) on the alignment of
each relative proportion of compound between all samples per-
formed with GCA 1.0 (Dellicour & Lecocq, 2013a,b).
For GC/MS and GC-FID analyses, we injected 1 𝜇L, using
a splitless injection mode (injector temperature of 220∘C) and
helium as carrier gas (1 mL/min, constant velocity of 50 cm/s).
The oven temperature (of the column) was programmed isother-
mally, starting at 70∘C for 2min and then rising from 70 to
320∘C at a rate of 10∘C/min. The temperature was then held at
320∘C for 5 min.
In order to facilitate the alignment of compounds and their
identication, before each sample injection, a standard (Kovats)
was injected containing a mix of hydrocarbons (alkanes) from
C10 (decane) to C40 (tetracontane). Kovats indices were calcu-
lated with GCK 1.0 according to the method described by
Dellicour & Lecocq (2013a,b).
We performed statistical comparative analyses of the CLGSs
using R environment (R Development Core Team, 2013) to
detect CLGS differentiations between B. monticola taxa. We
used a clustering method, computed with the unweighted
pair-group method with average linkage (UPGMA) based on
Canberra distance matrices (RA of each compound) (R package
ape; Legendre & Legendre, 2004; Paradis et al., 2004), to detect
the divergence between taxa in the CLGS composition. We
assessed the uncertainty in hierarchical cluster analysis using
P-values calculated by multiscale bootstrap resampling with
100 000 bootstrap replications (signicant branch supports
>0.85) (R package pvclust; Suzuki & Shimodaira, 2011). We
assessed CLGS differentiations between taxa by performing
a permutation multivariate analysis of variance using distance
matrix (er) (R package vegan; Oksanen et al., 2011).
When a signicant difference was detected, we performed a
pairwise multiple comparison with an adjustment of P-values
(Bonferroni correction) to avoid type I errors. We determined
specic compounds of each taxon (indicator compounds) with
the indicator-value (IndVal) method (Dufrene & Legendre,
1997; Claudet et al., 2006). This value is the product of relative
concentration and relative occurrence frequency of a compound
within a group. The statistical signicance of an indicator com-
pound (threshold of 0.01) was evaluated with a randomization
procedure.
Morphological analyses
In order to investigate diagnostic morphological characters for
species identication and new taxa description (not for species
delimitation), a total of 60 worker bees were analysed morpho-
logically to discriminate between B. lapponicus,B. konradini,
and B. monticola. We included only workers to have a suf-
cient sampling (minimum 15 specimens) and because the differ-
ences in metabasitarsus measurements were more pronounced
in females than males. We selected the maximum length and
width of metabasitarsus following the work of Gjershaug et al.
(2013) and we calculated the ratio (max length:max width) of
these two measures to reduce the effect of body size on this
morphological analysis. One picture was taken for each mea-
surement and specimen using a binocular coupled with a digital
camera (Nikon D70). The specimen was positioned in such a
way as to maximize focus on the metabasitarsus. The maximum
metabasitarsus distance was measured on the picture with the
software I 1.5 (Abràmoff et al., 2004) (Table 1, Appendix
S3). Kruskal–Wallis analyses (Kruskal-Wallis test and multi-
ple comparison test after Kruskal-Wallis; ‘pgirmess’ R-package,
Siegel & Castellan, 1988) were performed using R (R Develop-
ment Core Team, 2013) to compare the different studied taxa.
Data integration and decision framework
Assuming that species diagnosis and interpopulation dif-
ferentiation are more efcient in a multiple evidence-based
approach (De Queiroz, 2007; Schlick-Steiner et al., 2010), we
proposed a species delimitation hypothesis according to our
genetic and CLGS criteria based on the method performed by
Lecocq et al. (2015a,d) derived from the approach established
by Schlick-Steiner et al. (2010). In this method, all criteria used
in the integrative approach must be convergent to assign spe-
cic status. This strict approach can lead to underestimation of
the species differentiation but reduces the taxonomic ination
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

Following the cold: speciation in B. monticola 207
(Lecocq et al., 2015d) We assigned the species status to a taxon
(with a high degree of certainty) if this taxon: (i) was geneti-
cally differentiated in all genetic markers (unique haplotype);
(ii) constituted a monophyletic group with high branch sup-
port; and (iii) was signicantly differentiated in CLGS com-
position (by indices including IndVal indicator compounds,
PERMANOVA, high bootstrap values >0.85) (Lecocq et al.,
2015a). We assigned the subspecies taxonomic status to pheno-
typically distinct allopatric populations with differentiation in
some but not all traits, in order to highlight those populations
displaying such differentiation (originality) and to reduce the
‘underestimate’s risk’ of our strict approach to assigning species
status by naming them as a subspecies (Zink, 2004; Hawlitschek
et al., 2012; Ennen et al., 2014; Lecocq et al., 2016).
Identication and type revision
The type series of Bombus lapponicus konradini Reinig, 1965
are presently at the Zoologische Staatssammlung München and
have been revisited for this study. The identication of other
studied taxa was checked with traditional identication keys
such as Løken (1973) and Gjershaug et al. (2013).
Results
Intertaxa differentiation
Haplotype network analysis revealed six unique haplotypes
for COI and two for PEPCK (Fig. 3) within the B. monticola
taxa complex. Konradini-C was the only taxon displaying
unique COI (6.8% sequence difference from monticola alpestris
and 5.3% from lapponicus)andPEPCK (0.7% sequence differ-
ence from monticola and 0.97% from lapponicus) haplotypes
in the ingroup. All phylogenetic analyses (MP, ML and MB)
of each single gene showed a similar topology with clades cor-
responding to haplotype groups found in the networks. Anal-
yses showed strong support for all groups, but the position
of konradini-C was variable in the clade in our phylogenetic
analyses, and hence remains uncertain (Fig. 3). Phylogenetic
analyses on PEPCK showed two main lineages within ‘monti-
cola’ (Fig. 3): the central Apennines lineage (konradini-C, here-
after referred to simply as konradini) and the main lineage (all
other taxa). COI-based trees resolved konradini as the sister
group to the outgroup B. lapponicus rather than to other lin-
eages of B. monticola. Among these last ones, COI phylogenetic
trees underlined some geographical subgroups within ‘monti-
cola’ (Fig. 3): (i) the northern Apennine lineage of ‘monticola’
(described hereafter as mathildis ssp.n.), (ii) a western group
including taxa from Pyrenees (rondoui) and Scotland (monti-
cola); and (iii) an eastern-northern group including specimens
from Sweden (scandinavicus) and Alps +Balkans +Mt. Olym-
pus (alpestris).
In comparison to the ML, MP, and MB analyses for COI
data, the tree generated for bGMYC analysis displayed differ-
ence (not biologically signicant) mainly in the branching of
mathildis ssp.n. As discussed in the literature, these differences
were probably due to the different parameters used in the 
1.7.4 software to calculate the bGMYC model and because this
pairwise matrix (heat map) was plotted against a sample tree
(Barraclough et al., 2003; Lecocq et al., 2015d). The bGMYC
analysis (Fig. 4) highlighted several entities with low probabili-
ties (<0.05) to be conspecic with the other ones. These results
match with the same taxa recognized in the COI tree (MP, ML,
MB analyses; Fig. 3). Overall, the bGMYC suggested the delim-
itation of four prospective species (P<0.05) within the monti-
cola complex (and the comparison group) as in Fig. 3: (i) one
group including all lapponicus (bGMYC conspecicity proba-
bilities between individuals included in the group, P>0.98– 1),
(ii) a group with all konradini from the Central Apennines
(P>0.99–1), (iii) one group with all bimaculatus (P>0.98 – 1),
(iv) all monticola subspecies (P>0.13– 0.95) including ron-
doui (p>0.95–1), alpestris (P>0.95 – 1), scandinavicus
(P>0.98–1), monticola (P>0.99 – 1) and mathildis ssp.n.
(P>0.99–1) which are signicantly conspecic. The pairwise
matrix (Fig. 4) shows more structure within B. monticola ssp.
where the group displays different haplotypes. These intermedi-
ate values of bGMYC (Fig. 4) between the different monticola
lineages (genetic differentiation below the species differentia-
tion threshold) are useful to discuss of subspecies concept.
In chemical analyses, 103 compounds were detected; 82 in the
CLGSs of B. monticola taxa (Appendix S2) except for konradini
for which we detected only 50 compounds. The differentiation
of CLGS composition between B. monticola taxa and outgroup
species (B. lapponicus and B. bimaculatus) was conspicuous
(IndVal; PERMANOVA F=115.63 and F=122.52, P<0.05;
Fig. 3). Except konradini, all other B. monticola taxa shared
the same compounds with similar relative concentration (RA)
(PERMANOVA F=6.00 –13.20, P>0.05) (Appendix S2). Dif-
ferences between konradini and other B. monticola taxa were
particularly marked in the rst half of the spectrum repre-
senting the most volatile molecules. The relative abundance
of several compounds was different compared with the rel-
ative abundance in other taxa of B. monticola. The IndVal
method highlighted several unique and diagnostic compounds
of konradini (Table 3; i.e. ethyl tetradecenoate, ethyl tetrade-
canoate, hexadec-7-en-1-ol ethyl octadecadienoate, dotriacon-
tane, ethyl octadec-9-enoate). In particular, konradini was char-
acterized by ethyl octadec-9-enoate with a relative abundance
of 8.28% although it had very low relative abundance in other
subspecies (median 0.57%). The discrimination between kon-
radini and other B. monticola taxa was supported by maximal
bootstrap support values (100%) (Fig. 3). This differentiation
was conrmed by statistical analysis (PERMANOVA F=29.36
P<0.05, between konradini and other B. monticola taxa).
Taxonomic status
Species status was conrmed for the comparison group
B. bimaculatus and B. lapponicus. According to the mtDNA
and nuDNA divergence along with the CLGS composition
differentiation (including main compounds) (Table 4), species
status was assigned to konradini (detailed information is given
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

208 B. Martinet et al.
Fig. 3. Genetic and chemical analyses within the monticola complex. (A) Majority rule (50%) consensus tree based on maximum-likelihood (MB)
analyses of COI. Values above tree branches are parsimony bootstrap values/ML bootstrap values/Bayesian posterior probabilities. Only ML and
parsimony bootstrap values >70% and posterior probabilities >0.95 are shown. (B) Majority rule (50%) consensus tree based on ML analyses of
PEPCK (MB). Values above tree branches are parsimony bootstrap values/maximum likelihood bootstrap values/Bayesian posterior probabilities. Only
ML and parsimony bootstrap values >70% and posterior probabilities >0.95 are shown. (C) (1) Dendrogram of cephalic labial gland secretion (CLGS)
differentiation within monticola complex and Bombus bimaculatus. This cluster was obtained by hierarchical clustering using an unweighted pair-group
method with arithmetic mean (UPGMA) based on a Canberra matrix calculated from the CLGS matrix of B. bimaculatus (red), B. lapponicus (dark blue),
B. konradini stat.n. (green), B. m. rondoui (pink), B. m. scandinavicus (yellow), B.m. monticola (light blue), B.m. alpestris (black), B. m. mathildis
ssp.n. (orange). The values near nodes represent multiscale bootstrap resampling values (only values >80 of main groups are shown except nodes
between B. monticola subspecies). (2) Principal component analysis (PCA) of CLGS differentiation within monticola complex and B. bimaculatus:
B. bimaculatus (red circles), B. lapponicus (dark blue circles), B. konradini stat.n. (green circles), B. m. rondoui (pink circles), B. m. scandinavicus
(yellow circles), B.m. monticola (light blue circles), B.m. alpestris (black circles), B. m. mathildis ssp.n. (orange circles). PC1, PC2 and PC3 are the
rst, the second and the third axes. [Colour gure can be viewed at wileyonlinelibrary.com].
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

Following the cold: speciation in B. monticola 209
Fig. 4. Species recognition pairwise matrix. Species recognition pairwise matrix based on ultrametric tree of cytochrome oxidase 1 (COI) sequences
with bGMYC pairwise probability of conspecicity plotted on a sample tree from . The coloured matrix corresponds to the pairwise probabilities
of conspecicity returned by the Bayesian implementation of the general mixed Yule-coalescent model (bGMYC) method (colour scale on the right of
the gure). Black spots show the coalescent node for each species. [Colour gure can be viewed at wileyonlinelibrary.com].
Table 3. List of indicator compounds (IndVal method, compounds >70%a) and main compoundsbidentied for Bombus konradini stat.n. within
cephalic labial gland secretions.
alpestris
(n=13)
monticola
(n=10)
rondoui
(n=7)
scandinavicus
(n=11)
mathildis
ssp. nov
(n=2)
konradini
nov status
(n=2)
lapponicus
(n=10)
bimaculatus
(n=10)
Compounds MW M M M M M M M M
Citronellola156 - - - - 0.08 0.17 - -
Ethyl tetradecenoatea,c 254 - - - - - 0.13 - -
Ethyl tetradecanoatea256 - - - - - 0.18 - -
Hexadec-7-en-1-ola240 0.11 0.08 - - 0.25 1.84 - -
Ethyl hexadec-9-enoatea282 - - 1.00 - - 1.41 0.03 -
Hexadec-9-enyl acetateb282 52.34 55.15 57.05 53.96 35.27 51.53 0.08 32.95
Geranyl citronellolc292 - - - - - - 71.32 -
Ethyl octadecadienoatea,b308 - - - - - 0.37 - -
Ethyl octadec-9-enoatea310 0.68 0.35 0.57 0.46 1.73 8.28 - -
Geranyl geranyl acetated332 - - - - - - - 31.61
Dotriacontanea451 - - - - - 0.09 - -
Hexadecyl hexadecanoatea,c 480 0.04 0.07 0.09 0.22 0.25 0.94 0.19 0.09
cMain compound identied for B. lapponicus.
dMain compound identied for B. bimaculatus.
The full matrix is presented in Appendix S2.
MW, molecular weight; n, number of specimens; M, median of compound relative concentration (%); -, absent compounds.
in Supporting information, Appendix S3). Bombus konradini
was originally described by Reinig (1965) as a subspecies
typical of the northern and central Apennines, ranging from
the provinces of Genova and Parma to L’Aquila. All other
taxa were included in B. monticola but their colour pattern
(Table 1) and/or differentiation in CLGS composition (minor
quantitative differences) and/or in COI marker implied their
assignation to the subspecies status. It is important to note
the distinction of the North Apennines (province of Genoa
and Parma to the provinces of Bologna and Lucca) monticola
population (B. monticola mathildis ssp.n.) from the Central
Apennines taxon (B. konradini stat.n.) and the population from
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

210 B. Martinet et al.
the Alps (B. monticola alpestris). Indeed, considering the slight
differentiation in COI (0.53% of divergence from alpestris)and
the strong divergence in coat colour from alpestris (Table 1,
Fig. 3), the North Apennines population should have a new
subspecies status: B. monticola mathildis (detailed information
is given in Supporting information, Appendix S3).
Divergence times among clades
Based on the COI data, the divergence between B. konradini
(Central Apennines) and B. lapponicus was estimated with
a median of 0.79 Ma (min 0.25 – max 1.9 Ma) at the end
of the Günz-Mindel interglacial period. The divergence
time between the outgroup bimaculatus and the clade
‘monticola–lapponicus – konradini’ was estimated with a
median of 2.40 Ma (min 1.14 – max 3.88 Ma). In contrast, the
divergence time between lapponicus-konradini and the clade
‘monticola’ was estimated with a median of 2.30 Ma (min
1.23 – max 4.11 Ma. These last two divergence times corre-
spond approximately to the onset of glaciation events and the
formation of the Bering Strait. The other monticola subspecies
have diverged recently with an estimated time of 40 000– 18 000
(min 7500 – max 548 000) yr ago.
Morphological analysis
Measurements of the ratio between the maximum length
and width of the metabasitarsus show signicant differences
(Kruskal–Wallis multiple comparison 𝜒2=32.757; all P-values
<0.05) between lapponicus and monticola alpestris,monti-
cola mathildis ssp.n. The ratio is also signicantly different
between konradini and monticola ssp. but not between mon-
ticola alpestris and monticola mathildis (Figure S2, Appendix
S3). However, between konradini and lapponicus, although our
results present a clear trend which highlights a larger ratio for
konradini, there is no signicant differentiation. According to
these results, konradini appears as intermediate between mon-
ticola s.s. (large metabasitarsus ratio) and lapponicus (small
metabasitarsus ratio). Diagnostic morphological characters are
summarized in Table 1.
Impact of new taxa in zoological nomenclature
Bombus konradini stat.n. (more information in
Appendix S3).
Original taxonomic combination: Bombus lapponicus konra-
dini Reinig, 1965: 105.
Locus typicus: Monti Sibillini, Central Apennine Mountains
(Italy).
Syntypes: 13 queens, 93 workers, 28 males.
Lectotype (present designation): 1 queen, labelled: 1) ‘Italia,
Monti Sibillini, Nh. M. Vettore, Baumgrenze, 15–1600m,
14.6.61, Reinig’.; 2) (on red paper) ‘LECTOTYPE’; 3)
‘det. P. Rasmont 2015 Bombus (Pyrobombus) monticola
konradini Reinig’ (Fig. 1).
Paralectotype: 2 queens, 41 workers, 16 males have been
located, designated and labelled as paralectotypes (Table 2).
In this series, only 1 queen (lectotype) and 21 workers from
Marche, Umbria, Lazio and Abruzzo have been identied as
Bombus konradini. The remaining paralectotypes (2 queens,
20 workers, 16 males) from Liguria, Emilia-Romagna and
Toscana have been assumed as Bombus monticola mathildis.
Bombus monticola mathildis Martinet, Cornalba & Rasmont
ssp.n. (more information in Appendix S3).
Locus typicus: North Apennines, Emilia Romagna, Reggio
Emilia, Villa Minozzo, Mt Cusna (Italy).
Holotype (present designation): 1 male, labelled: 1) ‘Italy,
Emilia Romagna, Reggio Emilia, Villa Minozzo, Mt
Cusna, 44.283492∘N 10.401028∘E, 2057m, 05.VIII.2015,
S/Scabiosa sp, Rec. M. Cornalba, BMAR0431’; 2) (on
red paper) ‘Holotype’; 3) ‘det. B. Martinet 2016 Bombus
(Pyrobombus) monticola mathildis Martinet, Cornalba &
Rasmont’ (Fig. 1).
Paratype: Two males have been located, designated
and labelled as paralectotypes: ‘Italy, Emilia Romagna,
Reggio Emilia, Villa Minozzo, Mt Cusna, 44.283492∘N
10.401028∘E, 2057m, 05.VIII.2015, S/Scabiosa sp, Rec.
M. Cornalba, BMAR0432’ and ‘Italy, Emilia Romagna,
Reggio Emilia, Villa Minozzo, Mt Cusna, 44.282288∘N
10.401603∘E, 2055m, 12.VIII.2015, S/Carduus carlini-
folius, Rec. M. Cornalba, BMAR0433’.
Discussion
Interpopulation differentiation of B. monticola
The concordance between genetic differentiation, geographic
distribution, and CLGS divergence of populations suggests
a strong intraspecic structure between the subspecies of
monticola (Fig. 3). The western subspecies (B. monticola
rondoui from Pyrenees and B. monticola monticola from
British Islands), the North Apennines population (B. monticola
mathildis ssp. nov.) and the eastern-northern subspecies
(B. monticola scandinavicus from Sweden and B. monticola
alpestris from the Alps, Balkans and Mt. Olympus) con-
stitute, with the COI marker, ve differentiated groups in
three main lineages (Fig. 3) which diverged recently (about
40 000 – 18 000 year ago based on molecular clock estimates)
during the Pleistocene/Quaternary. This could explain the
weak divergence of the PEPCK marker between monticola
subspecies (recent divergence) because nuclear genes have
a lower mutation rate than mitochondrial genes (Lunt et al.,
1996; Trunz et al., 2016). Such a time of divergence matches
with the start of the last postglacial warming. Thus, it appears
that the geographical pattern is most likely a consequence of
allopatric differentiation and genetic drift triggered by a range
fragmentation subsequent to the last postglacial warming. We
speculate that, at the beginning of the current interglacial period,
taxa found refuge in southern mountainous areas (the Alps and
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

Following the cold: speciation in B. monticola 211
Table 4. Taxonomic decision table with all criteria used for Bombus species delimitation.
Former taxonomic status
Morphology
(Gjershaug
et al., 2013)
Private haplotypes
(COI/PEPCK)CLGS
COI gene/
bGMYC
PEPCK
gene
Proposed taxonomic
status
B. monticola scandinavicus −+/−−(B) −LS (B)/−− B. monticola scandinavicus
B. monticola monticola −+/−−(B) −LS (A)/−− B. monticola monticola
B. monticola rondoui −+/−−(A) −LS (A)/−− B. monticola rondoui
B. monticola alpestris −+/−−(C) −LS (B)/−− B. monticola alpestris
B. monticola konradini (North Apennines) −+/−−(C) −LS (C)/−− B. monticola mathildis ssp. nov.
B. monticola konradini (Central Apennines) ++/+++/++B. konradini stat.n.
B. bimaculatus ++/+++/++B. bimaculatus
B. lapponicus ++/+++/++B. lapponicus
CLGS, cephalic labial gland secretions; COI, cytochrome oxidase 1; PEPCK, phosphoenolpyruvate carboxykinase. Morphology indicates if a taxon has
a diagnostic morphological character (+/−means that morphology is/is not diagnostic). Private haplotypes indicate if a taxon has a specic haplotype
(+/−means that the taxon has/has not only private haplotype (s). When the taxon shares haplotype with other ones, the letters group together taxa that
share haplotypes). CLGS indicates if the taxon has/has not diagnostic composition of CLGSs with different main compounds (+/−means that the taxon
has/has not a specic CLGS composition. When the taxon shares CLGS composition with other ones, the letters group together taxa that share similar
CLGS. COI and PEPCK columns indicate if a taxon forms a strongly supported monophyletic group (+/−means that the taxon is/is not a monophyletic
group) with maximum parsimony, maximum-likelihood and Bayesian methods. When the taxon is not a distinct monophyletic group, the letters group
together taxa included in the same monophyletic group). LS, low supported differentiation.
southern peninsulas of Europe) and Northern Europe by con-
traction of their distribution areas or range shifting (Hewitt,
1999; Hewitt & Ibrahim, 2001; Petit et al., 2003; Stewart
et al., 2010), in a similar way to the boreo-montane leaf beetle
(Chrysomelidae), Gonioctena pallida (Mardulyn et al., 2009).
The resulting allopatry has fostered mtDNA differentiation
along with minor differentiation of chemical reproductive traits
similar to what has already been shown for insular populations
of bumblebees (Lecocq et al., 2013b, Lecocq et al., 2015a).
Despite their relative geographical isolation, all other
B. monticola allopatric taxa previously recognized by Reinig
(1965) are considered as conspecic based on our diagnos-
tic criteria with a low geographical genetic and phenotypic
differentiation (decision framework Table 4; Fig. 3). Overall
they shared the same CLGS composition (except for some
low relative concentration differences) and are characterized
by only slight genetic differentiation. These low differen-
tiations, particularly in CGLS composition, can simply be
explained by the short time of divergence due to geograph-
ical isolation and intraspecic variability (Lecocq et al.,
2011, 2016). Within B. monticola alpestris, the three sampled
populations (Alps, Balkans and Mt. Olympus) are clearly
consubspecic.
Mountaintop speciation: Bombus konradini stat.n.
Contrary to the situation within B. monticola,B. konradini
stat.n. displays greater genetic and chemical trait differentia-
tion (Fig. 3). Allopatry has most likely shaped the reproductive
trait (CLGS) differentiation as observed in other species
(Lecocq et al., 2013a,b; Lecocq et al., 2015c). The strong
genetic differentiation of B. konradini could be explained by
an earlier divergence from the common ancestor with other
B. monticola lineages, most likely temporally close to the
B. monticola –B. lapponicus complex divergence. Indeed,
based on genetic differences in the 16S gene, Hines (2008)
suggested that B. lapponicus and B. monticola diverged from
each other about 3 Ma. In temperate species, the post-Ice Age
recolonization of territories by relict populations (from refu-
gia), could have led to a new shufing of the genetic pool by
re-contacting these populations without speciation (Coyne &
Orr, 2004; Hewitt, 2004a,b). The modication of geographical
range could trigger genetic and CLGS differentiation. Indeed,
it has been shown that reproductive traits including CLGS
can differentiate from both sides of the physical barriers that
may exist between refuge areas (Lecocq et al., 2013a). The
case of the new species status of B. konradini lends strength to
the hypothesis that for cold-adapted taxa, climatic oscillations
(i.e. interglacial periods) have led to species differentiation
in mountain refuges after geographical separation. Further
phylogeographical and phylogenetic studies, based on larger
sampling (including additional closely related species) and
other genetic markers, are needed to accurately assess these
hypotheses.
Our integrative taxonomic decision framework supported
and conrmed the species status of B. monticola compared
with its morphologically closely related species (B. lapponicus)
(Cameron et al., 2007; Løken, 1973; Svensson, 1979; Gjershaug
et al., 2013). Our results also supported the species status of
konradini which is endemic at high altitudes (>1800 m a.s.l.) of
the Central Apennines (Manino et al., 2007) (Fig. 3, Table 4).
Concerning eco-chemical traits (CLGS), konradini differed
from the other B. monticola taxa by lightweight compounds
(volatile molecules) which could have a long-distance attractive
role (Ayasse et al., 2001). Therefore, the differentiation of these
compounds may be a signicant pre-mating reproductive bar-
rier or may simply reect divergence times and drift. Besides,
according to the results for the COI marker, konradini could be
more closely related to B. lapponicus (Fig. 3) than B. monticola
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

212 B. Martinet et al.
taxa, as suggested in the original description of Reinig
(1965). However, the phylogenetic position of konradini is not
completely resolved because of the different tree topologies
between COI and PEPCK results.
The species status of B. konradini suggests that interglacial
periods can lead to species differentiation in mountain refugia in
cold-adapted taxa. Unlike the populations of the Alps, Pyrenees
and Balkans, where the interconnection and thus the possibility
of exchanges and conspecicity are likely, the population of the
central Apennines is much more isolated from other mountain
chains with a possible endemic speciation (Martín-Bravo et al.,
2010). Several studies have shown the presence of endemic
taxa in the Central Apennines (e.g. in amphibians, Canestrelli
et al., 2008; Canestrelli et al., 2012 and Mattoccia et al., 2011;
in reptiles, Joger et al., 2007; in turtles, Fritz et al., 2005; in
plants, Conti et al., 2005, Fuente et al., 2011 and Frattaroli
et al., 2013; and in bumblebees, Lecocq et al., 2013a). For
example, Lecocq et al. (2013a) provided evidence that the
population of B. lapidarius (a Palaearctic polytypic species)
from the Southern Italian refugia has experienced genetic and
CLGS differentiation during Quaternary glaciations leading
to an incipient speciation process. Populations inhabiting the
Mediterranean mountains (e.g. the Apennines, one of the few
mountain ranges in Europe arranged on a north–south axis) are
characterized by a high genetic diversity (hotspot) with endemic
taxa (Ruiz-Labourdette et al., 2012).
The sympatry of two different species, dened by divergent
taxonomic traits, reinforces the ‘species’ status because indi-
viduals co-inhabit the same area without hybridization. Our
results suggest that B. monticola (s.s.) is absent in the Cen-
tral Apennines unless this could be due to a sampling bias.
Such an absence could have resulted in a lack of sympa-
try between B. monticola and B. konradini. Several hypothe-
ses could explain the potential absence of B. monticola (s.s.)
in the Central Apennines: (i) for eco-climatic constraints, his-
torical or competition reasons, this taxon has never inhabited
this region or has disappeared; and (ii) despite the signi-
cant observed differences (genetic, morphological and chem-
ical traits), a limited hybridization between monticola and
konradini still could be possible. Following this second hypoth-
esis, along the contact zone between monticola and konradini,
the subspecies mathildis could represent an intermediate pop-
ulation resulting from some introgressions of the population
living in the Alps (alpestris). Our COI results suggest that
the subspecies mathildis (low branch support) is closer to
B. konradini than all other subspecies of B. monticola (s.s.)
(Fig. 3). However, phenotypic and chemical trait results do not
support this hypothesis (Figs 3C, Figure S2). Although distinct,
B. konradini could be the ‘replacement species’ to B. monticola
(s.s.) with similar eco-climatic constraints and lling the eco-
logical niche in Apennines or a relict population of a near
relative of B. lapponicus in Italy considering our COI results.
Additional ethological experiments (hybridization tests) and
further genetic analyses (e.g. Microsatellite, SNPs, RAD-seq)
are necessary to test these hypotheses of intermediate pop-
ulations or replacement species in the context of taxonomic
implications.
Conservation remarks on the B. monticola complex and the
practice of integrative taxonomy
Considering all taxonomic criteria in our integrative approach
(Fig. 3, Table 4), we propose conservation of the subspecies sta-
tus for ve monticola taxa (Hawlitschek et al., 2012; Lecocq
et al., 2015a,d, 2016): B. monticola rondoui from the Pyrenees,
B. monticola monticola from the British Isles, B. monticola
scandinavicus from Fennoscandia, B. monticola alpestris from
the Alps, Balkans and Mt. Olympus, and B. monticola mathildis
ssp.n. from the North Apennines (formerly included by Reinig
within konradini). Although the usefulness of subspecies sta-
tus in bumblebees has been criticized and debated (Ebach
& Williams, 2009) during recent decades (Williams, 1991;
Bertsch & Schweer, 2012a), we propose that these allopatric
subspecies (partially isolated lineages) represent an important
component and a useful pragmatic taxonomical unit for evolu-
tionary biology and biological conservation of the evolutionary
legacy of B. monticola (i.e. Waples, 1995; Patten & Unitt, 2002;
Phillimore & Owens, 2006; Rasmont et al., 2008; Patten, 2009;
Crowhurst et al., 2011; Braby et al., 2012; Sackett et al., 2014).
These differentiations could be local adaptations to particular
environments (Avise, 2000; Frankham et al., 2010; Braby et al.,
2012; Lecocq et al., 2013a). Therefore, subspecies classication
seems suitable to reect the intraspecic differentiation within
B. monticola taxa.
The monticola complex is a stunning example of the dif-
culty, in taxonomy, of dening the species or subspecies sta-
tus of a population. Here the integrative taxonomy, consider-
ing several criteria independently, provide strong pieces of evi-
dence to take decision concerning species status of taxa. We
assigned subspecies taxonomic status to phenotypically distinct
allopatric groups of populations with differentiation in some but
not all criteria used in the integrative decision framework (i.e.
conict in selected criteria) (Hawlitschek et al., 2012; Ennen
et al., 2014; Lecocq et al., 2015a,d; Lecocq et al., 2016). Tax-
onomical conclusions based only on the differentiation of one
mitochondrial marker (e.g. COI barcoding) can lead to weak
taxonomic hypotheses (Andriollo et al., 2015; Mutanen et al.,
2016; Trunz et al., 2016) as mitochondrial differentiation may
result from sex-specic characteristics, as lower dispersion for
females (Kraus et al., 2009; Lepais et al., 2010), or mtDNA
introgression or incomplete lineage sorting (Bensasson et al.,
2001; Lecocq et al., 2015a). Taxonomic diagnosis based on mul-
tiple evidence (integrative taxonomy) is the best approach to
avoid overestimation of species diversity which would lead to
taxonomic ination. Subspecies can be considered as a simple
allopatric differentiation (Mayr, 1942; Patten, 2010). This pro-
cedure allows the assignment of taxonomic status to any doubt-
ful bumblebee taxa and marks these taxa for further taxonomic
studies (Lecocq et al., 2015a). Moreover, despite the argument
advanced by Williams et al. (2015), there is no case in bum-
blebees where the CLGS (mate recognition system) was not
differentiated between two different species, even when closely
related bumblebee species have geographical distributions that
do not overlap [e.g. B. terrestris (L.) and B. ignitus Smith,
© 2018 The Royal Entomological Society, Systematic Entomology,43, 200– 217

Following the cold: speciation in B. monticola 213
De Meulemeester et al., 2011, or B. patagiatus and B. magnus,
Bertsch & Schweer (2012b).
Conservation implication of the new taxonomic status
of B. konradini stat.n.
The new taxonomical status has implication for the red
list assessments of the European bumblebees studied herein,
according to the IUCN criteria (Nieto et al., 2014). Although
Rasmont et al. (2015) assess all taxa lumped into B. monticola,
the new taxonomic status of B. konradini implies an evaluation
of its conservation status independently from other B. monticola
taxa.Bombus konradini was described as a rare, geographi-
cally very restricted taxon endemic to the central Apennines of
Marche, Umbria, Lazio, Abruzzo and mostly occurring exclu-
sively at elevations over 1800 m a.s.l. (Reinig, 1965; Riccia-
rdelli & Piatti, 2003; Manino et al., 2007, Rasmont et al., 2015).
The apparent scarcity of B. konradini could lead to signi-
cant genetic drifts (Ricciardelli & Piatti, 2003; Frankham et al.,
2010) that might signicantly increase the species extinction
risk (Rasmont et al., 2015). Indeed, according to Frankham et al.
(2010), small and isolated populations of a taxon are inher-
ently more vulnerable to local extinction due to environmental
and demographic stochasticity. It is therefore important to con-
sider this new taxonomic status in our models and in our future
back-up plans (mitigation measures).
Supporting Information
Additional Supporting Information may be found in the online
version of this article under the DOI reference:
10.1111/syen.12268
Appendix S1. Table of sampling. Sample code refers to the
sample labels used in different analyses. COI and PEPCK
are the GenBank accession numbers for each sample (when
consubspecic samples display the same gene sequence,
only one of them has been submitted to Genbank;).
Appendix S2. Data matrix of cephalic labial gland secretions
(CLGS) (relative concentration of each compound), list of
the identied compounds and IndVal analysis with specic
compounds in the monticola complex. Unknown x’s indicate
undetermined compounds.
Appendix S3. Description of the new subspecies Bombus
monticola mathildis ssp.n. and Bombus konradini stat.n.,
designation of the holotype and lectotype and morphological
differentiation.
Figure S1. Morphology and coloration variation of the face
of Bombus konradini stat.n. (Lectotype female, A) and
Bombus monticola alpestris (female, B). Photographs are by
P. Rasmont.
Figure S2. Comparison of the ratio maximum
length/maximum width metabasitarsus between workers
of B. lapponicus,B. konradini,B. monticola alpestris and
B. monticola mathildis. With n =number of used speci-
mens; * =signicant differences (Kruskal-Wallis multiple
comparison, p-value <0.05).
Acknowledgements
The authors thank the Abisko and Tarfala scientic sta-
tions (Sweden) for their welcome and their help in material
collection. We acknowledge all people that helped us in our
journey to the Abisko and Tarfala stations: P. Lakare and
G. N. Rosqvist (University of Stockholm), M. Augner and
L. Wanhatalo (Abisko Station), H. Savela (Oulu University,
INTERACT project administration), and J. Strand and T.
Wikstrom (Lansstyrelsen i Norrbottens lan Naturvardsen-
heten, Lulea). The authors also thank the Parco Nazionale
dei Monti Sibillini and the Parco Nazionale dell’Appennino
Tosco-Emiliano for granting permission to collect in their
respective territories. Special thanks go to P. Salvi (Sibillini),
W. Reggioni (Appennino Tosco-Emiliano), J. Devalez, and A.
Cetkovic (University of Belgrade) for their help in the sampling.
Computational resources have been provided by the Consor-
tium des Équipements de Calcul Intensif (CÉCI), funded by
the Belgian FRS (Fonds de la Recherche Scientique)-FNRS.
We thank also the two anonymous reviewers for their help in
improving this manuscript. BM contributes as a PhD student
granted by the Research Council of University of Mons and
by the FRS-FNRS. PB contributes as a PhD student funded
by the Czech Science Foundation (GA ˇ
CR GP14-10035P) and
by the University of South Bohemia (GA JU 152/2016/P).
Part of this work (Eco-chemical trait differentiation) was sup-
ported by the Institute of Organic Chemistry and Biochemistry
of the Academy of Sciences of the Czech Republic (project
No. 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) and
the research leading to these results has received funding from
the European Union’s Horizon 2020 project INTERACT, under
grant agreement No 730938.
BM, TL, NB, CU, IV and PR conceived and designed the
experiments; BM, NB, PB, MC and PR carried out the sampling;
BM analyzed the data; and BM, TL, NB, PB, MC, CU, IV, JOG,
DM and PR wrote the paper.
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