Abstract and Figures

Our grasp of biodiversity is fine-tuned through the process of revisionary taxonomy. If species do exist in nature and can be discovered with available techniques, then we expect these revisions to converge on broadly shared interpretations of species. But for the primarily arctic bumblebees of the subgenus Alpinobombus of the genus Bombus, revisions by some of the most experienced specialists are unusual for bumblebees in that they have all reached different conclusions on the number of species present. Recent revisions based on skeletal morphology have concluded that there are from four to six species, while variation in colour pattern of the hair raised questions as to whether at least seven species might be present. Even more species are supported if we accept the recent move away from viewing species as morphotypes to viewing them instead as evolutionarily independent lineages (EILs) using data from genes. EILs are recognised here in practice from the gene coalescents that provide direct evidence for their evolutionary independence. We show from fitting both general mixed Yule/coalescent (GMYC) models and Poisson-tree-process (PTP) models to data for the mitochondrial COI gene that there is support for nine species in the subgenus Alpinobombus. Examination of the more slowly evolving nuclear PEPCK gene shows further support for a previously unrecognised taxon as a new species in northwestern North America. The three pairs of the most morphologically similar sister species are separated allopatrically and prevented from interbreeding by oceans. We also find that most of the species show multiple shared colour patterns, giving the appearance of mimicry among parts of the different species. However, reconstructing ancestral colour-pattern states shows that speciation is likely to have cut across widespread ancestral polymorphisms, without or largely without convergence. In the particular case of Alpinobombus, morphological, colour-pattern, and genetic groups show little agreement, which may help to explain the lack of agreement among previous taxonomic revisions.
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RESEARCH ARTICLE
Genes Suggest Ancestral Colour
Polymorphisms Are Shared across
Morphologically Cryptic Species in Arctic
Bumblebees
Paul H. Williams
1
*, Alexandr M. Byvaltsev
2
, Björn Cederberg
3
, Mikhail V. Berezin
4
,
Frode Ødegaard
5
, Claus Rasmussen
6
, Leif L. Richardson
7
, Jiaxing Huang
8
, Cory
S. Sheffield
9
, Suzanne T. Williams
1
1 Department of Life Sciences, The Natural History Museum, London, United Kingdom, 2 Department of
General Biology and Ecology, Novosibirsk State University, Novosibirsk, Russia, 3 Swedish Species
Information Centre, Swedish University of Agricultural Sciences, Uppsala, Sweden, 4 Entomological
Department (Insectarium), The Moscow Zoo, Moscow, Russia, 5 Norwegian Institute for Nature Research,
Trondheim, Norway, 6 Department of Bioscience, Aarhus University, Aarhus, Denmark, 7 Gund Institute for
Ecological Economics, University of Vermont, Burlington, Vermont, United States of America, 8 Key
Laboratory for Insect-Pollinator Biology of the Ministry of Agriculture, Institute of Apicultural Research,
Chinese Academy of Agricultural Sciences, Beijing, China, 9 Royal Saskatchewan Museum, Regina,
Saskatchewan, Canada
* paw@nhm.ac.uk
Abstract
Our grasp of biodiversity is fine-tuned through the process of revisionary taxonomy. If spe-
cies do exist in nature and can be discovered with available techniques, then we expect
these revisions to converge on broadly shared interpretations of species. But for the primar-
ily arctic bumblebees of the subgenus Alpinobombus of the genus Bombus, revisions by
some of the most experienced specialists are unusual for bumblebees in that they have all
reached different conclusions on the number of species present. Recent revisions based on
skeletal morph ology have concluded that there are from four to six species, while variation
in colour pattern of the hair raised questions as to whether at least seven species might be
present. Even more species are supported if we accept the recent move away from viewing
species as morphotypes to viewing them instead as evolutionarily independent linea ges
(EILs) using data from genes. EILs are recognised here in practice from the gene coales-
cents that provide direct evidence for their evolutionary independence. We show from fitt ing
both general mixed Yule/coalescent (GMYC) models and Poisson-tree-process (PTP) mod-
els to data for the mitochondrial COI gene that there is support for nine specie s in the subge-
nus Alpinobombus. Examination of the more slowly evolving nuclear PEPCK gene shows
further suppor t for a previously unre cognised taxon as a new species in northwestern North
America. The three pairs of the most morphologically similar sister species are separated
allopatrically and prevented from interbreeding by oceans. We also find that most of the
species show multiple sha red colour patterns, giving the appearance of mimicry among
parts of the different species. However, reconstructing ancestral col our-pattern states
PLOS ONE | DOI:10.1371/journal.pone.0144544 December 10, 2015 1/26
OPEN ACCESS
Citation: Williams PH, Byvaltsev AM, Cederberg B,
Berezin MV, Ødegaard F, Rasmussen C, et al. (2015)
Genes Suggest Ancestral Colour Polymorphisms Are
Shared across Morphologically Cryptic Species in
Arctic Bumblebees. PLoS ONE 10(12): e0144544.
doi:10.1371/journal.pone.0144544
Editor: Zuogang Peng, SOUTHWEST UNIVERSITY,
CHINA
Received: June 18, 2015
Accepted: November 19, 2015
Published: December 10, 2015
Copyright: © 2015 Williams et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All sequences are
available from the online gene-sequence databases:
GenBank (ncbi.nlm.nih.gov/genbank/) and BOLD
(boldsystems.org). A detailed list of accession
numbers for the sequence numbers deposited in
these databases is given in the supporting
information (S2 File) in the manuscript.
Funding: The database of species distributions in
Russia was created with the financial support from a
Grant of the President of the Russian Federation for
young Russian scientists (grant MK-6176.2015.4) (to
AB). DNA barcodes for some of the material were
shows that speciation is likely to have cut across widespread ancestral polymorphisms,
without or largely without convergence. In the particular case of Alpinobombus, morphologi-
cal, colour-pattern, and genetic groups show little agreement, which may help to explain the
lack of agreement among previous taxonomic revisions.
Introduction
Recently authors have sough t to reduce subjectivity in the practice of species discovery or
delimitation [1] by comparing the results from applying multiple criteria in an explicitly inte-
grative taxonomy [2]. This is a search for congruent results from multiple criteria as cor robora-
tion, even though the different sets of characters employed (from different disciplines) may
have different evolutionary constraints requiring different evolutionary explanations [2].
Indeed, not all studies using different criteria can be expected to agree, because relationships
between criteria and speciation events differ [1, 3, 4]. It has even been acknowledged that those
criteria that relate most closely to speciation could form a minority pattern in some cases [2].
But for some arctic bumblebees, taxonomic revisions by experienced specialists have shown
unusually poor agreement on the number of species, we suggest in part because of poor agree-
ment among results from applying different criteria.
Bumblebees (genus Bombus Latreille) have been described as morphologically homoge-
neous compared with other bees [5]. It is therefore unsurprising that bumblebee species were
diagnosed initially in terms of the striking colour patterns of the hair on the dorsum of the
body [6]. But then, more than a century ago, it was realised that the groups supported by skele-
tal morphology are actually more clearly circumscribed, and that within these morphological
species, bumblebees are highly variable in colour patterns [710]. This is supported by recent
genetic studies [1114], reaffirming the long-held view that bumblebee colour patterns do not
necessarily diagnose species [15]. Detailed studies of bumblebee variation have led to the reali-
sation that species can be cryptic in both morphology and colo ur pattern [16, 17], now con-
firmed by gen etic studies [18, 19].
Particularly intriguing is the observation that bumblebee colour patterns are often impres-
sively similar among species [8, 2026]. In many cases, a single species may show different col-
our patterns in different geographical regions, in each of which they may resemble closely
other species as members of regional colour-pattern groups [24, 27]. Several possible explana-
tions have been proposed (reviewed in [26, 28]), including Müllerian mimicry [22, 29]. Recent
advances in understanding evolutionary relationships have begun to clarify the chronology of
the evolution of some of these resemblances [30], an important pre-requisite to discerning
causes [31]. In cases of regional colour-pattern resemblance, the similar species are often only
distantly related, so that the similarity has been interpreted as the result of evolutionary conver-
gence [26]. We explore another possible explanation for the evolution of colour-pattern resem-
blance, as an alternative to convergence: that in some situations a polymorphism within an
ancestral species may have been inherited in parallel by several descendent species.
The bumblebees of the subgenus Alpinobombus Skorikov are a small group of closely related
species [32]. Previous taxonomic revisions (including global revisions and broader regional
faunal revisions with global referencing) of Alpinobombus by some of the most experienced
bumblebee specialists have relied on both morphology (especially the more variable characters
of the male penis valve, volsella and gonostylus, and of the female oculo-malar area and hind
tibia) and on the colour patterns of the hair and have all differed in their conclusions on the
number of species present (Table 1). Several recent faunal lists have accepted as valid just the
Cryptic Arctic Diversity
PLOS ONE | DOI:10.1371/journal.pone.0144544 December 10, 2015 2/26
funded by the Saskatchewan Ministry of Agriculture
and the Canada-Saskatchewan Growing Forward 2
bi-lateral agreement (grant 20130112) (to CS). Work
in Greenland was supported by the Carlsberg
Foundation (grants 2009-01-0534 and 2011-01-0463)
(to CR). DNA barcodes for some of the Norwegian
material were funded by the Research Council of
Norway (grant 266134/F50) and the Norwegian
Biodiversity Information Centre (grant 70184209).
The Norwegian part of the project was funded by the
Taxonomy Initiative of the Norwegian Biodiversity
Information Centre (grant 70184228) (to FO). DNA
sequencing for some of the material was funded by
the Agricultural Science and Technology Innovation
Program, China (grant CAAS-ASTIP-2015-IAR) (to
JH). Part of the database of species distributions in
North America was created with financial support
from grants from the National Science Foundation to
John Ascher, Douglas Yanega, and Jerome Rozen
(grants NSF-DBI 0956388 and NSF-DBI 0956340) (to
LR). The funding agencies had no role in the study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. The other
authors received no specific funding for this work.
Competing Interests: The authors have the
following interests: work in Greenland was supported
by the Carlsberg Foundation (grants 2009-01-0534
and 2011-01-0463). There are no patents, products in
development or marketed products to declare. This
does not alter the authors' adherence to all the PLOS
ONE policies on sharing data and materials, as
detailed online in the guide for authors.
five more clearly morphologically-diagnosable species (interpreted in a broad sense, from the
shape of the female head, male genitalia, etc.): B. alpinus, B. polaris, B. balteatus, B. neoboreus,
and B. hyperboreus [10, 33 35]. But variation in colour pattern prompted some authors to
question whether at least two more species might be added to this list: B. pyrrhopygus (but
using the junior synonyms B. diabolicus Friese and B. alpiniformis Richards for bees with the
Scandinavian colour pattern of this species) and B. kirbiellus, even though both lacked clearly
discrete diagnostic morphological characters [3638]. The colour patterns of Alpinobombus
species have been described as being both variable within species while showing close similari-
ties among species [36, 37, 3943]. These species are unusual even among bumblebees [10, 44]
for occupying extreme arctic or alpine habitats [40, 43, 45]. Therefore , at least in the far north,
they co-occur with few or no other bumblebee species [36, 37, 43, 46], which simplifies the sys-
tem for analysis if mimicry were involved.
We use sequences for parts of the COI and PEPCK genes from freshly collected specimens
and from museum specimens to recognise the evolutionarily independent lineages of the sub-
genus Alpinobombus as species and to estimate the phylogenetic relationships among these
species. We then test whether the colour-pattern polymorphisms are more likely either to be
ancestral, or to have arisen more recently and convergently within the daughter species. From
our analyses we conclude: (1) that coalescents for the COI gene support nearly twice as many
species (nine cf. five) as have been recognised from skeletal morphology and that three of these
species, including one taxon not recognised previously, are also supported by PEPCK polymor-
phisms; and (2) that similarity in colour pattern among parts of these species is likely to have
arisen from broadly conserved ancestral polymorphisms, not from convergence. The work
reported here builds on a survey of the morphology and colour patterns of the North American
Alpinobombus [42] and is part of a revisionary study of Alpinobombus bumblebees world-wide,
which will treat type specimens and nomenclatural issues separately.
Table 1. Lists and numbers of Species of the Subgenus Alpinobombus from previous taxonomic revisions.
a
Richards 1931 [39] Skorikov 1937 [43] Milliron 1973 [36]Løken 1973 [37] This study
alpinus alpinus alpinus alpinus alpinus
pyrrhopygus
arcticus arcticus polaris arcticus polaris
kincaidii kincaidi
balteatus balteatus balteatus balteatus balteatus
tristis
kirbyellus kirbiellus
neoboreus neoboreus ? neoboreus
strenuus strenuus strenuus ?
unnamed
natvigi
hyperboreus hyperboreus hyperboreus hyperboreus hyperboreus
Total 7 8 6 4 9
Split
b
2300-
Lumped
b
3323-
a
Species names and spellings are those used in the original publications. The revision by Løken did not cover the New World fauna and so did not treat B.
neoboreus / B. strenuus.
b
Counts of split and lumped species compared to this study exclude the unnamed taxon that we treat (conservatively) as unsampled previously.
Authors of the taxon names: alpinus (Linnaeus), arcticus Kirby, balteatus Dahlbom, hyperboreus Schönherr, kincaidii Cockerell, kirbiellus Dahlbom, natvigi
Richards, neoboreus Sladen, polaris Curtis, pyrrhopygus Friese, strenuus Cresson, tristis Friese.
doi:10.1371/journal.pone.0144544.t001
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Materials and Methods
Ethics statement
No humans or vertebrate animals were used in this research. Field permits: (Russia) Bumblebees
of the subgenus Alpinobombus are not included in any Red Lists and are not listed under the law
On environmental protection in Russia. None of the areas where field work was carried out are
private. Only Meduza Bay in West Taimyr is a part of the Great Arctic Reserve, where the
research was conducted with the permission of the Reserve management. Specimens from the
research collection of Novosibirsk passed the State veterinary control before export on loan.
(Norway) No permits were needed for collecting of the Norwegian data.
(Sweden) None of the bees were collected within National Parks or Nature Reserves. They
are not subject to any collection restrictions and therefore permits are not required. At present
there is no legislation or restrictions that regulates the export of genetic samples from Sweden.
There is no other restriction on the transfer of scientific material of this sort bet ween EU mem-
ber states.
(Denmark / Greenland) The Greenland Ecosystem Monitoring Coordination Group at the
National Environmental Research Institute, Aarhus University, approved our research propos-
als for access and research activities in both 2010 and 2011.
(Canada) No permits were required for collection sites visited.
(USA) Collections from the Rocky Mountain Biological Laboratory in Colorado were made
according to an agreemen t between the RMBL and the United States Forest Service.
We bring together samples of taxa of the subgenus Alpinobombus from throughout their
global distributions. Taxonomic and geographical breadth is essential in a taxonomic revision
for achieving a representative sample in order to assess the full range of patterns of variation
and relationship [47, 48]. This is especially important for reducing the bias that might other-
wise result if sampling were restricted to just one or two isolated regions or were to exclude
major lineages. To cover all variation, we made a preliminary survey of specimens from
museum collections (Fig 1; see the supporting information).
To recognize species of the subgenus Alpinobombus, we accept the broadly held concept of
species as evolutionarily independent lineages (EILs) [1]. To recognize these species in practice,
we use statistical tests designed to identify gene coalescents, which provide direct evidence for
EILs [49]. Coalescence methods are suggested to be especially useful compared to less direct
indicators (including criteria related to mate recognition and reproductive isolation) because
they can detect lineage separation even in its early stages [1, 3]. We use one of the fastest evolv-
ing, most highly variable mitochondrial genes, coding for COI (cytochrome oxidase subunit I).
This is particularly appropriate for detecting species because of its rapid coalescence within lin-
eages [3, 50]. From among recently collected specimens showing the most obvious morpholog-
ical and colour variation, we sequenced the barcode region of the COI gene (Fig 1).
Most of the extraction, amplification, and sequencing work was done at the Biodiversity
Institute of Ontario, Guelph, using standard protocols [51] and primers [52]. A minority of
specimens were processed at the Institute of Apicultural Research, Beijing. Because the geo-
graphic range occupied by Alpinobombus is large and many areas are difficult to access, mito-
chondrial DNA with many copies per cell has the advantage of being more easily extracted, so
that even pinned museum specimens can be sampled. Although 70% of Alpinobombus speci-
mens surveyed are known to be less than a century old, only 8.6% are less than 10 years old. A
problem with older museum specimens is that the sequences obtained are often not of full
length [53]. Sequence alignments were made using the ClustalW function in MEGA (version
6.06, accessed 2014: megasoftware.net [54]) and amino acid translations were checked with
EMBOSS Transeq (accessed 2014: ebi.ac.uk/Tools/st/emboss_transeq/).
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PLOS ONE | DOI:10.1371/journal.pone.0144544 December 10, 2015 4/26
To check for possible mismatches between trees for different genes, and hence for possible
mismatches between gene trees and species trees [49, 55], we also sequenced the nuclear gene
PEPCK (phosphoenolpyruvate carboxykinase; accession numbers for sequences listed in
Table A in S2 File, obtained using primers from [32]). This gene has been popular for studies
of bumblebees [11, 30, 32].
To recognise species, single-threshold general mixed Yule/coalescent (GMYC) models seek
a transition expected in the tree-branching rates between interspecific branches and intraspe-
cific branches [5661]. GMYC analysis requires an ultrametric estimate of the phylogenetic
tree that includes only unique haplotypes [59, 62] (T. Barraclough, pers. comm.) in order to
avoid spurious inflation of estimates of terminal branching rates. After sorting the sequences
by decreasing length, the longest examples of each unique haplotype are recognised using Col-
lapse (version 1.2, accessed 2011: softpedia.com/get/Science-CAD/Collapse.shtml), which
ignores sites with missing data. As outgroups for rooting the tree with this fast gene, we include
species from closely related bumblebee subgenera [32]: B.(Cullumanobombus) rufocinctus
Cresson, B.(Pyrobombus) vagans Smith, B.(Bombus) ignitus Smith, B.(Bombus) terrestris
(Linnaeus), and B.(Bombus) cryptarum (Fabricius). The best nucleotide-substitution model
for COI according to the Bayesian information criterion (BIC) obtained from MEGA is the
general time-reversible model with a gamma-frequency distribution of changes among sites
(GTR+Γ). BEAST (version 1.8.0, accessed 2013: beast.bio.ed.ac.uk) was used for Bayesian anal-
ysis of multiple model trees [63]. The clock model was set to the uncorrelated lognormal
(relaxed clock), the tree-speciation prior was set to a constant-size coalescent process (consis-
tent with the null hypothesis that there is a single species in the data), and the chain length for
the Markov-chain Monte Carlo (MCMC) algorithm was set to two billion generations, with
sampling of the trees every 200,000 generations. The sample of resulting trees from the MCMC
Fig 1. Global Sampling of the Subgenus Alpinobombus. Grey spots show sample sites for the 4345
specimens examined (Alpinobombus species are unknown from the tropics or from the southern
hemisphere; collections listed in Table A in S1 File); black spots show the sites for the 173 samples with COI
barcodes; and question marks show the sites for doubtful records (specimens supposedly from sites that
combine low latitude with low elevation). Polar projection, North Pole (starred) at the centre of the map.
Terrestrial national boundaries and the Arctic Circle are shown as narrow grey lines. Image created in ArcGIS
using World_Shaded_Relief basemap which is Copyright 2014 ESRI.
doi:10.1371/journal.pone.0144544.g001
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PLOS ONE | DOI:10.1371/journal.pone.0144544 December 10, 2015 5/26
algorithm was examined using Tracer (version 1.6.0, accessed 2013 [63]), which showed that
stationarity had been achieved within the first 1% of the total MCMC generations. The large
number of MCMC generations was needed with our data to increase effective sample sizes. A
maximum clade-credibility tree was obtained from the post burn-in tree sample after rejecting
the first 1% of sampled trees using TreeAnnotator (version 1.8.0, as for BEAST). After removal
of the outgroups, the GMYC model was applied to the tree using the SPLITS library (version
1.011, accessed 2011: r-forge.r-project.org/projects/splits/) running on the R platform (ver-
sion 2.12.1, accessed 2011: www.r-project.org). The oldest available name [64] is applied to
each coalescent group as a prospective species. Multiple runs of all analyses were made and the
stability of the results checked.
To check the robustness of the GMYC results by taking into account the uncertainty in esti-
mates of phylogeny, we applied a Bayesian GMYC analysis [59] using the bGMYC library (ver-
sion 1.0.2, accessed 2015: sites.google.com/site/noahmreid/home/software) on the R platform.
bGMYC was applied to the last 100 trees from the sample of 10,000 trees from the BEAST run
of two billion MCMC generations (a sample representing the last 20 million trees) that had
been checked previously with Tracer for stationarity. bGMYC was run with the default settings
(MCMC 50,000 generations, burn-in 40,000, thinning 100).
As another check on the number of species recognised, we apply another related approach
for discovering species designed for use with single-locus data, looking for changes in the num-
bers of nucleotide changes along branches from between- to within-species relationships,
based on Poisson-tree processes (PTP: [65]). PTP analysis is less demanding of tree informa-
tion than the GMYC approach and there is some evidence that it can perform better [65]. We
apply the Bayesian implementation on the bPTP server (accessed 2015: species.h-its.org)toa
metric maximum-clade-credibility tree from COI barcodes obtained with MrBayes (version
3.1.2, accessed 2011: [66]), using th e same data, outgroups, and nucleotide-substitution model,
four chains (temperature 0.2), and 100 million generations of the MCMC algorithm with a 1%
burn-in selected using Tracer. We used the default bPTP options after removing outgroups
from the rooted tree.
The variation in colour patterns for the subgenus Alpinobombus includes many subtle dif-
ferences [42], which are similar in the two sexe s. In all cases colour patterns refer to the colour
of the hair on various body regions, not to the colour of the body surface, which is brown or
nearly black throughout. Generally, we observe that the presence of non-black hair on the dif-
ferent body segments is not independent, but appears to be conditionally dependent among
segments [8] (e.g. there are no Alpinobombus bees with the hair on metasomal tergum 2 black
and on tergum 3 yellow, or on tergum 4 orange and on tergum 5 black, although in both cases
the reverse conditions are common). Therefore it should be reasonable to begin by coding
bumblebee colour patterns for the presence or absence of areas of pale hair and then later add-
ing characters as modifiers for the extent of the pale hair [26, 67]. As a first approximation, we
assume that Alpinobombus colour patterns can be reduced to a representation as two principal
characters, each with two states. Character (1): whether there are transve rse yellow bands in
the hair on the body, usually anteriorly and often posteriorly on the thoracic dorsum and/or on
metasomal terga 12 (state B, banded: minimally with an obvious yellow band on metasomal
tergum 2, varying from pale straw yellow to darker orange brown), or whether the hair in these
areas is black or nearly black (state U, unbanded). Character (2): whether there is obvious pale
hair (most often red or orange, but sometimes yellow or white) on the tail, consisting of meta-
somal terga 46 (state P, pale: minimally covering tergum 5 or 6), or whether the hair of the tail
is black or nearly black (state D, dark). The most widespread colour pattern both among all
bumblebees [26] and among the sister-group to the subgenus Alpinobombus, the subgenus
Bombus sensu stricto [19], has both the yellow bands and a pale tail. Replacement of the pale
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bands and the pale tail with black can also be polymorphic within some Bombus s. str. species,
although this is relatively rare (occurring in 3/17 and 1/17 species for the two characters respec-
tively). Colour characters were scored for a sample of 1646 Alpinobombus specimens of both
sexes from across the distribution range. To reconstruct the history of colour-pattern charac-
ters, we estimate the species tree using BEAST linked trees from COI and PEPCK exon and
intron sequences. The best (unlinked) nucleotide-substitution models according to the MEGA
BIC are HKY for both the PEPCK exon and introns, the tree-speciation prior was set to a
birth-death process appropriate for a multi-species tree, and from examination of the results
with Tracer, the MCMC algorithm was set to run for 1.5 billion generations. Outgroups and
other settings were as for the first analysis. No fossils of Alpinobombus species are known for
dating parts of the tree, so the phylogeny was calibrated with a date from a molecular study:
Hines [68] estimated the age of divergence between the subgenus Alpinobombus and the subge-
nus Bombus s. str. to be 13 Ma. Mesquite (version 3.02, accessed 2015: mesquiteproject.org
[69]) was used to reconstruct ancestral characters states on this tree. We used parsimony analy-
sis because we wanted to retain information on alternative explanations rather than just the
most likely. We coded polymorphic populations as a third intermediate ordered state, allowing
state changes in either direction ([70]; W. Maddison, pers. comm.).
Results
We found 46 unique haplotypes among 173 COI-barcode samples of the subgenus Alpinobom-
bus (accession numbers listed in Table A in S2 File). These COI data are information rich, with
92/658 nucleotide sites phylogenetically informative. The GMYC models show a significant
change in branching rate through time in the COI barcode tree (likelihood ratio 14.03 between
the GMYC multiple-species model and the null model that there is a single species in the
group, p = 0.0028). This threshold (at -0.0041 substitutions per nucleotide) leads us to recog-
nise nine candidates for prospective species (with a 95% confidence interval of 811 species)
within Alpinobombus as in Fig 2, which also shows that each of these species is strongly sup-
ported as a monophyletic group.
The bGMYC analysis showed stationarity and a high modal coalescent/Yule ratio consistent
with success. The posterior probability distribution is shown against the first sample tree in Fig 3
to provide a heat map of the probabilities that the haplotypes are conspecific. Adopting a thresh-
old probability of 0.5 from the posterior mean from the analysis (corresponding to a moderate
position, midway between a spli tter and a lumpe r) recognises nine species within the subgenus
Alpinobombus on the diagonal (unsurprisingly, the species with more haplotypes have more struc-
ture within them). These are the same nine species as are identified in the GMYC analysis in Fig 2.
The Bayesian PTP solution with the most support (Fig 4) recognises the same nine prospec-
tive species (with a range of 730 species), although the individual Bayesian support values for
the species are not high (0.480.79).
As expected, we found much less variation in the PEPCK gene among species of the subge-
nus Alpinobombus. Only 4/903 nucleotide sites in our sequences are uniquely diagnostic for
species, with unique PEPCK nucleotide changes supporting the species B. alpinus (base posi-
tion 351), B. neoboreus (bp 755), and two for the unnamed species (bp 360, 533) (Table 2).
Additional PEPCK nucleotide differences give further support for the distinction between the
species pair B. neoboreus / unnamed (bp 769). The estimate of phylogeny for Alpinobombus
species from the combined gene data is shown in Fig 5. This shows strong support for all
groups except for the position of B. alpinus, which remains uncertain.
The principal colour patterns recorded for the subgenus Alpinobombus show three of the
four possible combinations of the two states for the two characters we survey (Table 3, Fig 6):
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PLOS ONE | DOI:10.1371/journal.pone.0144544 December 10, 2015 7/26
Fig 2. Recognising Species of the Subgenus Alpinobombus with GMYC. The 173 sequences reduced to 46 unique COI-barcode haplotypes on a
BEAST ultrametric gene tree (outgroups not shown). The single threshold (T = -0.0041) from the GMYC model is shown by the vertical grey bar and the
intersecting lineages are interpreted as subtending nine prospective species (black spots show the coalescent node for each species). The tree is the
Bayesian ultrametric maximum-clade-credibility tree from a sample of trees after 1% burn-in from 2 billion generations of the MCMC algorithm in BEAST.
Values next to the nodes are Bayesian posterior probabilities showing branch support. Each haplotype is represented by one of the longest sample
sequences, labelled with a species name, a code that consists of an identifier from the project database and (after the hyphen) from the BOLD database,
followed by its geographic origin.
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UP, unbanded pale tails; BP, banded pale tails, and BD, banded dark tails. The fourth state,
entirely black (UD), is not recorded here, although one specimen of B. balteatus from Kara-
ginskiy Island (Kamchatka, Russia) comes close, with only a narrow and inc omplete yellow
band on metasomal tergum 2. Fig 7 shows the distributions of the three principal colour pat-
terns. The UP colour pattern is the most restricted geographically and is concentrated primar-
ily in Europe; UP also occurs in isolated pockets in Russia and in North America but is
unrecorded from Greenland. In contrast, the BP and BD patterns co-occur broadly, both taxo-
nomically (Table 3 ) and geographically (Fig 7). The frequency of the BD colour pattern relative
to BP colour pattern may increase towards the north (Fig 7). Nonetheless, the three principal
colour-pattern groups are each shared by three of the nine prospective species (Table 3; B.
Fig 3. Recognising Species of the Subgenus Alpinobombus with Bayesian GMYC. The 173 sequences reduced to 46 unique COI-barcode haplotypes
on one BEAST ultrametric gene tree (outgroups not shown). The posterior probability distribution (right, colour scale far right) is plotted against a sample tree
from BEAST (left) to provide a heat map of the probabilities that haplotypes are conspecific by bGMYC. Black spots show the coalescent node for each
species from Fig 2.
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pyrrhopygus, B. polaris, and B . balteatus), as well as being recorded from both the Old World
and New World regions, as widespread polymorphisms. While there are often small differences
between the most frequent patterns for each species, nonetheless in all cases it is possible to
find specimens that resem ble other species so closely that diagnosis by colour pattern is not
always possible.
Fig 4. Recognising Species of the Subgenus Alpinobombus with Bayesian PTP. The 46 unique COI-barcode haplotypes as in Fig 2 on a MrBayes
metric gene tree with the Bayesian PTP solution with highest support, showing nine prospective species (black spots show the coalescent node for each
species; outgroups not shown). The tree is the maximum-clade-credibility tree after 1% burn-in from 100 million generations of the MCMC algorithm in
MrBayes. Values above the nodes are PTP Bayesian support values that all daughter haplotypes belong to a single species population; values below the
nodes are Bayesian posterior probabilities showing branch support. Haplotype selection and labels as in Fig 2. The scale bar for branch lengths shows 0.02
substitutions per base position.
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Table 2. PEPCK polymorphisms.
a
Species 88 96 114 187 223 309 310 347 351 360 467 504 533 546 548 621 626 732 741 755 762 763 764 769 794 801 803 814 842 884 902
alpinus (2) T A T G G T C G GCCGG C G A T G/A G C AAGT GTTAAGG
pyrrhopygus (3) . . . . . . . . A ..A.....G...........G/T.
polaris (2) . . . G/A . . . . A . C/T G/A . . . . . . . . . . . . . . . . . . .
balteatus (3) . . T/G . G/A T/A . . A . . . . C/A . . T/G . . . . . . . . T/G T/A . . . .
kirbiellus (3) . . . . . . . G/A A .....G/A............A/G...
neoboreus (2) . . . . . . . . A ..........A ....A......
unnamed (2) . . . . . . C/G . AA..T . G/A A/G . . G/A . A/G A/G G/C A G/A . . A/G A/G . G/C
natvigi (2) T/C A/T . . . . . . A .C/T............A .......
hyperboreus (2) . . . . . . . . A .T............A .......
a
Numbers in the top row refer to nucleotide positions within a condensed alignment of the sequences with minimal gaps (903 base pairs), letters are FASTA codes for nucleotides but with
additional polymorphisms shown explicitly. Dots indicate a nucleotide matching the rst (or for position 732, the second) sequence. Species-diagnostic sites (positions 351 360 533 755 769)
are shown in bold, and other polymorphic sites are shown without bold. Numbers next to species names are the numbers of sequences examined.
doi:10.1371/journal.pone.0144544.t002
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Reconstructions of the most parsim onious ancestral states for the two colour-pattern char-
acters scored for the subgenus Alpinobombus are shown in Fig 8. For the banding pattern, Fig 8
supports either a root polymorphism with two subsequent reversals to a monomorphic banded
pattern (to kirbiellus as well as to the ancestor of the group neoboreus-hyperboreus), or alterna-
tively two forward changes to a polymorphic banded/unbanded pattern (to balteatus as well as
to the ancestor of the group alpinus-polaris). For tail colour, Fig 8 supports a root ancestor with
Fig 5. Estimate of phylogeny for species of the subgenus Alpinobombus. Estimated using a linked-tree BEAST analysis of COI-barcode and PEPCK
exon and intron sequences for each species. Values below the nodes are Bayesian posterior probabilities showing branch support. Numbers above the
nodes show the estimated dates of divergence events in Ma (millions of years before the present) calibrated with a molecular estimate for the date of
divergence between the subgenus Alpinobombus and the subgenus Bombus s. str. from Hines (2008) and grey bars show the 95% confidence limits on the
estimated dates of divergence.
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a polymorphism for tail colour. There is a reversal to a monomorphic pale tail for alpinus and
a second forward change to a monomorphic black tail in the ancestor of the group natvigi-
hyperboreus. Therefore we infer that both colour-pattern characters are likely to have had poly-
morphisms within the common ancestors of multiple species, which have then persisted in sev-
eral of the descendent species as trans-species polymorphisms.
Discussion
Bumblebees have long been used in discussions of the nature of species [8, 21, 23]. Many of the
different ideas of what constitutes species [71] have been applied to bumblebees, including the
biological species concept [72, 73] and specific-mate recognition systems [67, 74]. Among
these ideas, pattern-based and process-based views have sometimes appeared to be irreconcil-
able [10, 75]. But more recently a consensus has emerged in favour of thinking of species gener-
ally as EILs, which provides a framework that is sufficiently broad to accommodate the many
different criteria used to recognise species in practice, including both pattern-based and pro-
cess-based approaches [1].
The ability of the concept of species as EILs to accommodate multiple criteria has led to the
idea of integrative taxonomy [2]. The integrative approach is based on the premise that the
best way to discover species is to undertake multiple studies of the same set of individuals using
different character sets and criteria, to compare the results, and then the best answer is found
where different studies agree, i.e. through corroboration. Arguments that integratio n provides
a total evidence approach to bring out a shared underlying signal are less well founded,
because the different character sets may have phylogenetic histories that are not shared [4].
Table 3. Principal Colour Patterns for each Species of the Subgenus Alpinobombus.
a
alpinus pyrrhopygus polaris balteatus kirbiellus neoboreus unnamed natvigi hyperboreus
BD BD BD BD BD BD BD BD
BP* BP BP BP BP BP BP
UP UP UP UP
a
Species are those recognised in Figs 24. Colour-pattern codes: UP, unbanded pale tail; BP, yellow-banded pale tail; BD, yellow-banded dark (black)
tail. No unbanded dark tails have been recorded.
*For B. alpinus, males from the Alps often have a yellow-banded pattern although this is rare among females.
Pale tails are most often orange-red, but additional yellow- or white-tailed colour patterns of B. balteatus and B. kirbiellus are included.
doi:10.1371/journal.pone.0144544.t003
Fig 6. Colour Patterns of the Subgenus Alpinobombus. Simplified diagrams [26] representing the principal variation in colour patterns of the hair on the
female dorsum coded as two two-state characters: UP, unbanded pale tail; BP, banded pale tail; BD, banded dark tail; with hair colours: yellow, yellow or
yellow-brown hair; orange, orange-red or white hair; dark grey, black hair. Only female patterns are shown because females make up the majority of samples
for social bumblebees, although male patterns are similar. Photos show: (left) B. alpinus and (right) B. hyperboreus. Photos by (left) A. Staverløkk and (right)
G. Holmström.
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The integrative approach has been applied previously for recognising bumblebee species [73, 76,
77]. However, our analysis of the subgenus Alpinobombus shows unusually weak agreement for
bumblebees among groups from morphological, colour pattern, and genetic data. Morphology
has been used in earlier studies to recognise five groups. Genetic data are used here to recognise
nine groups, which although they are nested within the morphological groups, are mostly incon-
gruent with them (Fig 9). Colour patterns can be used to recognise three principal groups,
although most of the morphological or genetic groups overlap with more than one colour-pattern
group (Fig 9). Within the framework of integrative taxonomy, this poor agreement among the
groups supported by all three different character sets (the best that is achieved is nested sets: Fig
9) would require invoking special evolutionary explanations for each [2]. In this situation, the
explanations might involve time lags between the evolution of some colour, genetic, and morpho-
logical characters [1] and this needs further investigation. This lack of congruency among groups
based on different character sets may help to explain why previous taxonomic revisions have
failed to agree on the number and identity of Alpinobombus species (Table 1).
Fig 7. Distribution of Colour Patterns of the Subgenus Alpinobombus. Pie diagrams (UP/BP/BD) show
relative frequencies of colour patterns (black, UP; white, BP; grey, BD) from a sample of 1646 colour-coded
specimens with coordinates by regions (clockwise from the top): Greenland (n = 27), Canada and Alaska
(n = 257), southern Rockies (n = 25), Russia excluding Murmansk Province (Kola Peninsula) (n = 654), Altai
and Sayan (n = 6), Scandinavia and Murmansk (n = 643), and Alps (n = 34). Spot diagrams show the
occurrence of each colour pattern: grey circles show all sites represented in the sample, while black spots
show those sites in which each colour pattern is recorded: unbanded pale tail (UP n = 329); yellow-banded
pale tail (BP n = 1029); yellow-banded dark tail (BD n = 288). Map projection and other symbols as for Fig 1.
Image created in ArcGIS using World_Shaded_Relief basemap which is Copyright 2014 Esri.
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Can we find other character sets that might support groups showing better agreement?
Another approach for bumblebees considers characterising chemical extracts of male labial
gland secretions. These extracts include sex-attractant or arrestant pheromones [7780], which
are expected to be important in specific-mate recognition systems [74]. These extracts can
Fig 8. Evolution of Colour Patterns of the Subgenus Alpinobombus. Reconstruction of ancestral states by parsimony in Mesquite for two colour-pattern
characters each with two states (Fig 6) among species based on the estimate of phylogeny in Fig 5, several of which are polymorphic and show both states
for both characters. Above: banding colour pattern, with yellow spots showing yellow-banded (B) populations, black spots showing unbanded (U)
populations, mixed yellow/black spots showing polymorphic populations, mixed yellow/grey spots showing uncertain polymorphic/monomorphic populations
(*for B. alpinus, males from the Alps often have a yellow-banded pattern although this is rare among females). Below: tail colour pattern, with orange spots
showing populations with pale (P: orange or white) hair on the tail, black spots showing populations with dark (D: black) hair on the tail, mixed orange/black
spots showing polymorphic populations.
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work well as indicators of species [7982]. However, there is also evidence that within some
bumblebee species there can be both divergence in these male secretions across different parts
of a single species distribution, as well as divergence in the female preferences among these
secretions [83]. In addition, and unfortunately for the case of Alpinobombus, when closely
related bumblebee species have geographical distributions that do not overlap, at least some-
times the mate-recognition pheromones do not differ between these species [73]. We would
anticipate that pheromones could be undifferentiated between the three recent east-west hemi-
sphere pairs of sister species recognised in Figs 24, because these species are already prevented
from interbreeding and given evolutionary independence through lack of gene flow, but caused
in this case by ocean barriers (see below). Consequently there would be no selection for further
reinforcement of barriers to interbreeding by causing divergence in sex pheromones.
Interbreeding and hybridisation are often seen as criteria central to species concepts [71
73]. Interbreeding is unlikely between the populations that we recognise here as sister species
from their gene coalescents (e.g. between B. polaris and B. pyrrhopygus, between B. balteatus
and B. kirbiellus, and between B. hyperboreus and B. natvigi) because interbreeding would have
disrupted these coalescents. In each case, the distributions of these sister species are now sepa-
rated so that they are prevented from dispersing and interbreeding by oceans, by 82 km at the
Bering Strait and by >1000 km at the Greenland Sea. It is possible that bumblebees might be
capable of dispersing across 82 km of sea, but it is unlikely that enough of them would arrive in
a suitable physiological condition either to establish colonies or to mate successfully. But more
persuasively, we see no evidence of dispersal, interbreeding, or introgression in this group from
conflicts between the genetic and geographical data. In these cases, allopatric speciation is likely
to have followed the repeated loss of the Bering land connection (which took place over as much
as the last 57Ma[84, 85]) according to dates estimated from our phylogenetic tree (Fig 5).
Another response to conflicting evidence has been to seek to accommodate the conflict
within a hierarchy, by interpreting the broader groups as species and the narrower groups as
subspecies [77, 86] (i.e. by choosing to reject the narrower groups as species). However, not
only were gene-coalescent-based methods like GMYC specifically designed to discover species
[49, 57, 60], not subspecies, but the justification for recognising subspecies is now far from uni-
versally accepted. Subspecies were introduced into bumblebee taxonomy origin ally as a typo-
logical concept, long before the biological species concept, and as a way of giving names to
groups of individuals with differing colour patterns within a species [10]. Unfortunately, in
practice the concept of subspecies has been applied with even less consistent meaning than the
concept of species, to the point where subspecies have been considered to have hindered prog-
ress in taxonomy, evolutionary studies, and conservation [8789]. For example, a recent review
of the well-known bumblebee B. terrestris defines several subspecies from across Europe that
include both discrete colour-pattern groups on islands, as well as dividing segments from more
continuous colour-pattern clines across the continent [90]. It is unavoidable that dividing con-
tinuous clines is essentially arbit rary. This distinction between islands and the continent in col-
our-pattern variation is paralleled in evidence from normally highly variable DNA
microsatellites, which also support many island groups as distinct, but which show no
Fig 9. Disagreement among Groups of the Subgenus Alpinobombus Based on Three Character Sets.
Above, set diagrams for the different but nested groups within Alpinobombus based on exoskeletal
morphology (in grey) and based on genes (in black). Below, set diagrams for the different and mostly
overlapping groups based on the colour patterns of the hair of the dorsum (in grey) and based on genes (in
black). Areas of intersection of sets occupied by bumblebee samples are shown in grey. Abbreviations for
colour patterns: BP, yellow-banded pale-tailed; UP, unbanded pale-tailed; and BD, yellow-banded dark-
tailed.
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significant differentiation within the continental population [91]. We wish to avoid sometimes
weakly-differentiated colour-pattern groups being over-interpreted in terms of (e.g.) ecology
when this has not been justified by a thorough geographical analysis. At the same time, COI-
barcode variation shows a single coalescent for the entire species [19] (see below regarding
Corsica). To help avoid ambiguous differences in the meaning of subspecies in different cases,
we agree that subspecies and trinomials should be replaced, either by direct informal descrip-
tions of the particular colour patterns on which th ey are usually actually based and which they
are used to indicate [67], or preferably by direct descriptions of the underlying genetic patterns
from phylogeographic studies [30, 78].
Alternatively, when called upon to make taxonomic decisions in the face of conflicting evi-
dence, we are prompted to consider which criterion within our framework is most appropriate
and reliable for recognising species. If we accept the recent move from viewing species as mor-
photypes towards viewing them as EILs, then gene coalescents, which are very direct evidence
of evolutionary independence, may be considered a special case as evidence for recognising
species [1, 3, 49]. We still need to consider carefully whether sufficient conditions are likely to
have been met to expect that fixed coalescents will have formed in each of these cases, for
example whether populations are too large, or whether there is residual gene flow. We also
accept that making the choice to view species differently (i.e. not as morphotypes) might alter
the shape of taxonomy to some degree [2]. It has been argued that coalescence methods could
reduce investigator-driven biases in species delimitation [49] and this should increase the pre-
cision in the groups recognised as species.
Both the GMYC and PTP methods have potential pitfalls and have to be applied with care
[3, 5961, 65]. For example, both are likely to split any samples isolated on trees by long termi-
nal branches. These branc hes can become exaggerated as artefacts arising from a variety of
causes, including sequencing errors [65], short sequences [59, 92], as well as unrepresentative
sampling. For example, when samples from only Corsica and adjacent Europe were analysed
with GMYC models, B. xanthopus Kriechbaumer was interpreted as an endemic Corsican
bumblebee species separate from the mainland B. terrestris [86]. But when samples from
throughout the known global distribution of B. terrestris were analysed [19], including samples
from Madeira, the Canary Islands, North Africa, Europe, Russia, Iran, Central Asia, and from
as far east as the indigenous eastern limit of the species in Mongolia, then the Corsican xantho-
pus samples were found to be part of the species B. terrestris, closely related to particular sub-
groups within that species (consistent with the results of an earlier analysis [91]). Sampling can
never be complete, but best practice in morphological taxonomy [48] accepts that it is essen-
tial to sample from across the breadth of global ranges of all included taxa when revising any
taxonomic group. The same principle holds when sampling genes, as we have attempted to do
here (Fig 1). GMYC may also not work well when there has been extremely rapid speciation,
either very recently or if populations were separated over a very short period long ago followe d
by a long slow divergence [59]. There is no reason to believe that these conditions apply in this
case ( Fig 5).
Some of our prospective species are currently known from few haplotypes, even though
they represent larger samples. For the Old World B. hyperboreus, we have only two unique hap-
lotypes shown in our trees (Figs 2 and 4), although these come from a sample of 13 Old World
sequences. Six morphologically similar individuals from Greenland share the haplotype of B.
natvigi from Canada, the oldest available name [39] for the New World sister species to the
Old World species B. hyperboreus. In contrast, more material is needed to clarify the status of
the unnamed taxon, represented here by sequences from five individuals collected from high
elevations in southern Alaska (Alaska range) and the Yukon (Saint Elias Mountain s),
sequences which differ from one another at two nucleotide positions. These sequences are
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unlikely to be paralogous nuclear pseudogenes of mtDNA, or numts [93], because they have
no indels, no stop codons, and have a higher variability with a strong bias towards high A/T
frequency at codon position 3 [94]. Heteroplasmy has been found in bees [95], but we exam-
ined the trace files for these sequences and found no evidence of double or irregular peaks.
Another possible explanation for distinguishing unnamed might be incomplete lineage sort-
ing [ 3], resulting in what might appear in our trees as paraphyly between B. neoboreus and the
taxon unnamed. The counter argument is that the six B. neoboreus sequences (three of them
sequenced twice, spanning a range from very pale to very dark specimens, with three unique
haplotypes) differ from the five unnamed sequences at 19 nucleotide positions (two of them
causing amino acid changes at translation). This is sufficient for the GMYC analysis to recog-
nise them as separate species. Independent corroborative support for the unnamed taxon as a
separate species is shown by unique diagnostic base changes in the PEPCK gene (Table 2: 454
specimens have been examined for the two taxa combined, although most are too old for us to
sequence). The biotic history of the Arctic and of the Beringian refuge may be complex [84,
96]: it is possible that B. neoboreus and the unnamed species could represent relict populations
left from successive waves of range expansion and contraction following the cycles of climate
change associated with the Pleistocene glaciations. For comparison, high elevations of the
Yukons Kluane mountains are unusual for having outlying disjunct and genetically divergent
populations of an otherwise arctic moth, Gynaephora groenlandica (Wocke) (Erebidae), and of
the endemic arctic plants, Oxytropis arctica R. Br. (Fabaceae) and Puccinellia vahliana (Liebm.)
Scrib. and Merr. (Poaceae), which have been interpreted as isolated relict populations [97].
Our results stand out compared with other revisionary studies of entire monophyletic bum-
blebee subgenera using genes [19, 76] in finding evidence that supports nearly twice as many
species as had been recognised in morphology-based revisions. Most of the taxa of the subge-
nus Alpinobombus have been known for more than a century and only the one unnamed spe-
cies may not have been examined for previous revisions (Table 1). The other species
recognised here for the first time from gene coalescents differ from the species splits in previous
revisions (Table 1 ), which were based on differences in colour pattern. A large increase in spe-
cies number is all the more surprising because the Arctic is expected to have relatively low bio-
diversity [98, 99]. Nonetheless, both the GMYC and PTP results (Figs 24) agree in separating
Old World from New World sister populations for B. polaris
/ B. pyrrhopygu s, B. balteatus / B.
kirbiellus, and B. hyperboreus / B. natvigi. Discontinuity of populations as geographical disjunc-
tion is also a feature of morphologically cryptic species in other genetic studies of insects [100].
These patterns are at odds with ideas in some earlier revisions of the subgenus Alpinobombus,
which had concluded that both sister species in some of these species pairs might occur either
within the Old World (both of B . polaris and B. pyrrhopygus)[37] or within the New World
(both of B. balteatus and B. kirbiellus)[36]. But, for B. kirbiellus, even the rare individuals with
black hair on the side of the thorax (resembling many Old World B. balteatus) from as far
north in the New World as Ellesmere Island do belong to B. kirbiellus according to our genetic
results. Similarly, we infer that both banded and unbanded individuals belong to the Eurasian
B. pyrrhopygus. The same nine species were also recognised by the refined single-linkage
(RESL) clustering procedure (single-linkage clustering followed by Markov clustering) that
generates BOLDs(boldsystems.org) Barcode Index Number (BIN) system from COI barcodes
[101]. The RESL procedure is related to the empirical barcoding gap idea. Consequently,
while it is computationally fast, it lacks the theoretical justification of the coalescence-based
approach, which is likely to cause results to diverge in some cases.
Unexpected and morphologically cryptic diversity has also been detected from genetic evi-
dence in other groups of insects that had previously been studied intensively and had been con-
sidered taxonomically mature [100, 102]. Among the species recognised here but not in the
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most recent revisions, B. kirbiellus has been regarded as a separate species by just one reviser
[36], although not with quite the same concept (the population from northern Canada was
excluded). In contrast, B. pyrrhopygus, B. natvigi, and the unnamed species have no closely cor-
responding concepts of species in earlier revisions (Table 1). Intensity of sampling may be a
factor in the discovery of the unnamed species, but for the others, the geographical breadth of
this study is likely to be a key factor (along with the use of genetic data) allowing critical assess-
ment of the variation.
Our results for the subgenus Alpinobombus are the first we know of for bumblebees to dem-
onstrate support for close resemblance among parts of different species populations arising
from shared ancestral polymorphisms (Fig 8). Many other cases of resemblance among more
distantly related bumblebee species have been ascribed to evolutionary convergence [25, 26,
30]. Whereas spatial segregation (by latitude, longitude, or elevation) is characteristic of many
of these convergent bumblebee-colour-pattern groups elsewhere in the world [26], we find
broad spatial overlap between the individuals of Alpinobombus with the banded pale-tailed
(BP) and the banded dark-tailed (BD) patterns. There may be some variation in the relative fre-
quency of the two patterns with latitude (Fig 7). The unbanded pale-tailed (UP) pattern is
more highly concentrated (lo ngitudinally) in Europe, although it is also present in a few loca-
tions in both Asia and North America. The majority of species show more than one of these
principal colour patterns, and one third of the species show all three (Table 3). This analysis
uses our combined COI and PEPCK tree as an estimate of the species tree, which matches a
tree for five of the species from five genes (includin g PEPCK but not COI) [32]. Therefore we
feel it is reasonable at present to treat the combined COI and PEPCK tree as an estimate of the
species tree. In any case, no estimate of phylogeny from more genes could alter the inference
that ancestral polymorphisms are most likely to explain the pattern of extant polymorphisms
within Alpinobombus, as long as a similar set of species is supported.
The principal weakness of our analysis of colour-pattern characters is that we do not yet
know how colour pattern s are inherited for bumblebees [103]. A diversity of possibilities for
genes and molecular-development mechanisms is known to exist even among closely related
species of flies within the genus Drosophila and among some mimetic butterflies, although
within the butterf ly genus Heliconius there is also evidence of a broadly conserved genetic basis
for mimicry [104 ]. The simplest explanation for the pattern of states for the two characters of
the subgenus Alpinobombus in Fig 8 is that in both cases divergence of the principal colour-pat-
tern states preceded the divergence of many of the species. However, even though this result is
more consistent with ancestral polymorphisms for both colour-pattern characters and this
result is necessary in order to support the idea, it is not sufficient to prove the case. Ultimately
proof will require identific ation of the genes governing these colour-pattern elements and the
demonstration of homology of these genes among the species.
Why would an ancestra l polymorphism persist, without one colour pattern becoming fixed
in all populations by drift or selection? Local samples of the subgenus Alpinobombus we have
examined do appear to show that local populations of a single species are often polymorphic
(e.g. within B. polaris and Russian B. pyrrhopygus). Are these different colour patterns selec-
tively neutral, or could there be heterogeneity in selection that actually favours the polymor-
phism, perhaps depending on temporal or spatial variation in thermal constraints or in the
abundance of predators or mimics in different microhabitats? Curiously, entirely black indi-
viduals of
Alpinobombus are not present in our sample. Within Scandinavia, B. balteatus shows
a tendency towards an increased frequency of darker colour patterns in the more southern
mountains ([37]: her figure 69), which Løken associated with areas that have high humidity
near the ground. Pekkarinen [41] recorded almost completely black Scandinavian males of B.
balteatus and described melanism in this species as typical high-altitude or high-latitude
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melanism. Contrary to this assertion, the highest frequency of bumblebee species with many
entirely black individuals world-wide occurs in the tropical lowlands ([26]; see also [105]). Our
observations here show that for Alpinobombus, individuals from the highest latitudes (northern
Greenland and Novaya Zemlya) are characterised by extensive yellow bands, although often
with black tails, and there does appear to be some increase in the frequency of black-tailed indi-
viduals towards the north (Fig 7). The thermal properties of bumblebee colour patterns need
further study [26].
Conclusions
To examine gene coalescents as at least part of the evidence for discovering bumblebee species,
studies should focus on obtaining: (a) homologous gene sequences; (b) comparable-length
sequences; (c) sequences from multiple independent genes (which need to be rapidly evolving);
and should (d) apply coalescence-based methods such as GMYC analysis. These aims supple-
ment the more general sampling aims long recognised by morphological taxonomists [48]of
the essential need to: (e) study a taxonomic group throughout its entire global range so as to
include all constituent lineages or taxa; and (f) study many samples from throughout each con-
stituent taxons range, both of which are just as important in genetic studies. Much more work
is needed to elucidate the genetic control of bumblebee colour patterns and to assess the rela-
tive roles of ancestral polymorphisms and convergence in governing polymorphisms within
species.
Supporting Information
S1 File. Table A. Depositories. Collections from which pinned material of the subgenus Alpi-
nobombus has been examined.
(DOC)
S2 File. Table A. Sequences. Sequenced samples of bumblebees (genus Bombus) of the subge-
nus Alpinobombus and outgroups with their accession numbers for GenBank (G) and the pub-
lic project folder BBAL of the BOLD online database (B).
(DOC)
Acknowledgments
Thanks to all who generously donated, loaned, or photographed specimens, especially to J.
Ascher, Y. Astafurova, C. Buddle, S. Cannings, S. Cardinal, S. Colla, D. Currie, S. Droege, L.
Gall, A. Guidotti, B. Harris, K. Martins, L. Packer, M. Proshchalykin, S. Schmidt, V. Scott, D.
Sikes, J. Strange, J. Thomas, J. Thomson, J. Weintraub, T. Wheeler, D. Yanega; to the Biodiver-
sity Institute of Ontario for barcoding of North American material; to the Norwegian Barcode
of Life Network for barcoding of Norwegian material; to L. Bailey, T. Barraclough, S. Cameron,
S. Cannings, M. Kuhlmann, W. Maddison, and A. Vogler for discussion; and to P. Foster for
help in setting up the bGMYC software.
Author Contributions
Conceived and designed the experiments: PHW. Performed the experiments: PHW STW. Ana-
lyzed the data: PHW. Contributed reagents/materials/analysis tools: PHW AMB BC MVB
CR LLR JH CSS. Wrote the paper: PHW.
Cryptic Arctic Diversity
PLOS ONE | DOI:10.1371/journal.pone.0144544 December 10, 2015 21 / 26
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... Recently, the classification of Alpinobombus has been studied in detail by different authors (e.g. Williams et al. 2015Williams et al. , 2019Potapov et al. 2019). Based on morphological and molecular characterizations, the presence of at least eight species in this subgenus, namely Bombus alpinus (Linnaeus 1758), B. pyrrhopygus (Friese 1902), B. polaris (Curtis 1835), B. balteatus (Dahlbom 1832), B. kirbiellus (Curtis 1835), B. neoboreus (Sladen 1919), B. kluanensis (Williams and Cannings 2016) and B. hyperboreus (Schönherr 1809), has been suggested. ...
... Based on morphological and molecular characterizations, the presence of at least eight species in this subgenus, namely Bombus alpinus (Linnaeus 1758), B. pyrrhopygus (Friese 1902), B. polaris (Curtis 1835), B. balteatus (Dahlbom 1832), B. kirbiellus (Curtis 1835), B. neoboreus (Sladen 1919), B. kluanensis (Williams and Cannings 2016) and B. hyperboreus (Schönherr 1809), has been suggested. However, there is some disagreement regarding the taxonomic position of B. hyperboreus natvigi having been raised to species level by Williams et al. (2015Williams et al. ( , 2019) but seen as a geographic race within the B. hyperboreus complex by Potapov (2019). Of all the species of the subgenus Alpinobombus, only two, B. polaris and B. hyperboreus natvigi, are currently known to occur in Greenland (Potapov et al. 2019;Williams et al. 2019). ...
... Other species, even today, are distributed over vast areas and, in the case of B. polaris, which is known from Alaska, the Canadian Arctic and Greenland, can be considered at least semi-circumboreal (Richards 1973;Vilhelmsen 2015). Until the review of the subgenus Alpinobombus by Williams et al. (2015), in which B. polaris was divided into two taxa, namely B. polaris sensu stricto in North America and Greenland and B. pyrrhopygus in coastal northern Eurasia, the latter was treated as a member of B. polaris Iserbyt 2010-2014;Williams et al. 2015). ...
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The bumble bee Bombus polaris (Curtis 1835) is known from the northernmost region of Greenland. But how it can survive there, where in terms of geographic origin it came from, and which species in addition to B. pyrrhopygus (Friese 1902) genetically it is most closely related to are insufficiently answered questions that have motivated us to carry out this study. On the basis of a molecular analysis of the cytochrome oxidase I gene of a B. (Alpinobombus) polaris from North Greenland (82° 48′ N; 42° 14′ W), we conclude that the female specimen we analysed was most closely related to the Canadian populations of B. polaris . Geographic proximity, occurrence of B. polaris on Ellesmere Island and wind direction are likely factors that have aided B. polaris to establish itself in northern and eastern Greenland. The presence of five haplotypes in the studied sequences from Greenland indicates a moderately high level of genetic diversity of B. polaris in Greenland, reflecting the successful adaptation of B. polaris populations. In the broader context of entomological life in the high Arctic, our results on B. polaris allow us to conclude that the survival of pollinating species in the high Arctic under the changing climate scenario depends not only on the weather but also on an individual’s opportunity to continue to locate suitable food sources, i.e. pollen and nectar in the case of B. polaris . This aspect, briefly touched upon in this study, is of relevance not just to B. polaris , but the Arctic entomofauna generally.
... This can have substantial consequences, especially for conservation, because IUCN focuses Red List assessments of threat status, at least initially, primarily on species (IUCN, 2001). The splitting of species into multiple separate species in the sense used here is an issue separate from the subdivision of species into multiple constituent subspecies, which is not supported here because of inconsistencies in concepts and operational practice regarding subspecies (Wilson & Brown, 1953;Barrowclough, 1982;Zink, 2004;Williams et al., 2015). ...
... Accepting polytypic species was part of a move toward thinking of species as populations or metapopulations and, combined with 'the modern synthesis' of evolution with Mendelian genetics (Dobzhansky, 1937;Huxley, 1942), led to the rise in popularity of the interbreeding or 'biological' concept of species (Poulton, 1904;Mayr, 1963). In applying an interbreeding concept (evident in the writings of, for example : Vogt, 1909;Reinig, 1935;Krüger, 1951), increasing emphasis was given to coincident discontinuities in variation Source Smith, 1854von Dalla Torre, 1896Skorikov, 1923Skorikov, 1937Milliron, 1973Williams, 1998 The species listed as 'unnamed' by Williams et al. (2015) was subsequently described under the name Bombus kluanensis by Williams et al. (2016a). CLGS: male cephalic labial gland secretions, compared using data from Martinet et al. (2018). ...
Article
Splitting or lumping of species is a concern because of its potential confounding effect on comparisons of biodiversity and on conservation assessments. By comparing global lists of species reported by previous authors to lists of the presently recognized species that were known to those authors, a simple ratio can be used to describe their relative splitting or lumping of species. One group of ‘model’ organisms claimed for the study of what species are and how to recognize them is bumblebees. A comparison of four bumblebee subgenera shows: (1) an early phase (up to and including 1931) showing splitting, in which taxonomy was dominated by a typological concept of invariant species with heavy reliance on colour-pattern characters; (2) a middle phase (1935–98) showing lumping, associated with a shift to a polytypic concept of species emphasizing morphological characters, often justified with an interbreeding concept of species, but only rarely associated directly with process-related characters; and (3) a recent phase (after 2000), using a concept of species as evolutionarily independent lineages, as evidenced by corroboration from integrative assessment, usually including evidence for coalescents of species in fast-evolving genes compared with morphology. Analysis of splitting or lumping should help to improve biodiversity comparisons and conservation.
... This can have substantial consequences, especially for conservation, because IUCN focuses Red List assessments of threat status, at least initially, primarily on species (IUCN, 2001). The splitting of species into multiple separate species in the sense used here is an issue separate from the subdivision of species into multiple constituent subspecies, which is not supported here because of inconsistencies in concepts and operational practice regarding subspecies (Wilson & Brown, 1953;Barrowclough, 1982;Zink, 2004;Williams et al., 2015). ...
... Accepting polytypic species was part of a move toward thinking of species as populations or metapopulations and, combined with 'the modern synthesis' of evolution with Mendelian genetics (Dobzhansky, 1937;Huxley, 1942), led to the rise in popularity of the interbreeding or 'biological' concept of species (Poulton, 1904;Mayr, 1963). In applying an interbreeding concept (evident in the writings of, for example : Vogt, 1909;Reinig, 1935;Krüger, 1951), increasing emphasis was given to coincident discontinuities in variation Source Smith, 1854von Dalla Torre, 1896Skorikov, 1923Skorikov, 1937Milliron, 1973Williams, 1998 The species listed as 'unnamed' by Williams et al. (2015) was subsequently described under the name Bombus kluanensis by Williams et al. (2016a). CLGS: male cephalic labial gland secretions, compared using data from Martinet et al. (2018). ...
Article
Splitting or lumping of species is a concern because of its potential confounding effect on comparisons of biodiversity and on conservation assessments. By comparing global lists of species reported by previous authors to lists of the presently recognized species that were known to those authors, a simple ratio can be used to describe their relative splitting or lumping of species. One group of 'model' organisms claimed for the study of what species are and how to recognize them is bumblebees. A comparison of four bumblebee subgenera shows: (1) an early phase (up to and including 1931) showing splitting, in which taxonomy was dominated by a typological concept of invariant species with heavy reliance on colour-pattern characters; (2) a middle phase (1935-98) showing lumping, associated with a shift to a polytypic concept of species emphasizing morphological characters, often justified with an interbreeding concept of species, but only rarely associated directly with process-related characters; and (3) a recent phase (after 2000), using a concept of species as evolutionarily independent lineages, as evidenced by corroboration from integrative assessment, usually including evidence for coalescents of species in fast-evolving genes compared with morphology. Analysis of splitting or lumping should help to improve biodiversity comparisons and conservation.
... динавского полуострова (Williams et al., 2015). Палеарктический вид. ...
... Источники информации. Elfving, 1960;Løken, 1973;Радченко, Песенко, 1994;Потапов и др., 2013;Williams et al., 2015;Potapov et al., 2017;Lhomme, Hines, 2018 (Elfving, 1960;Løken, 1973 Татаринов, Долгин, 1999Коршунов, 2002;Gorbunov, Kosterin, 2003;Львовский, Моргун, 2007;Tshikolovets, 2011;Татаринов, Кулакова, 2013б, Татаринов, 2016 Татаринов, Долгин, 1999Коршунов, 2002;Болотов, Семушин, 2003;Львовский, Моргун, 2007;Tshikolovets, 2011;Татаринов, 2016 Описание взрослой стадии. Длина переднего крыла бабочки -18-28 мм. ...