A comprehensive phylogeny of the bumble bees ( Bombus

Biological Journal of the Linnean Society

, 2007,

91

, 161–188. With 4 figures

© 2007 The Linnean Society of London,

Biological Journal of the Linnean Society,

2007,

91

, 161–188

161

Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066© 2007 The Linnean Society of London? 200791?161188Original Articles

BUMBLE BEE PHYLOGENYS. A. CAMERON

ET AL

.

*Corresponding author. E-mail: [email protected]

A comprehensive phylogeny of the bumble bees (

Bombus

)

S. A. CAMERON

1

*, H. M. HINES

1

and P. H. WILLIAMS

2

1

Department of Entomology, 320 Morrill Hall, 505 S. Goodwin Avenue, University of Illinois, Urbana, IL 61801, USA

2

Department of Entomology, Natural History Museum, South Kensington, London SW7 5BD, UK

Received 24 March 2006; accepted for publication 10 August 2006

Bumble bees (

Bombus

Latreille) occupy a wide diversity of habitats, from alpine meadows to lowland tropical forest,yet they appear to be similar in morphology throughout their range, suggesting that behavioural adaptations playa more important role in colonizing diverse habitats. Notwithstanding their structural homogeneity, bumble beesexhibit striking inter- and intraspecific variation in colour pattern, purportedly the outcome of mimetic evolution. Arobust phylogeny of

Bombus

would provide the framework for elucidating the history of their wide biogeographicaldistribution and the evolution of behavioural and morphological adaptations, including colour pattern. However,morphological studies of bumble bees have discovered too few phylogenetically informative characters to reconstructa robust phylogeny. Using DNA sequence data, we report the first nearly complete species phylogeny of bumble bees,including most of the 250 known species from the 38 currently recognized subgenera. Bayesian analysis of nuclear(opsin, EF-1

α

, arginine kinase, PEPCK) and mitochondrial (16S) sequences results in a highly resolved and stronglysupported phylogeny from base to tips, with clear-cut support for monophyly of most of the conventional morphology-based subgenera. Most subgenera fall into two distinct clades (

short-faced

and

long-faced

) associated broadly withdifferences in head morphology. Within the

short-faced

clade is a diverse

New World

clade, which includes nearly one-quarter of the currently recognized subgenera, many of which are restricted to higher elevations of Central andSouth America. The comprehensive phylogeny provides a firm foundation for reclassification and for evaluatingcharacter evolution in the bumble bees. © 2007 The Linnean Society of London,

Biological Journal of the LinneanSociety

, 2007,

91

, 161–188.

ADDITIONAL KEYWORDS:

classification – corbiculate bees – nuclear genes.

INTRODUCTION

Bumble bees (

Bombus

Latreille) are among the morefamiliar creatures inhabiting meadows, gardensand grasslands of the temperate world (Darwin, 1859:notes 1854–1861, translated by Freeman, 1968;Sladen, 1912). Robust and vividly coloured, theyemerge from hibernation in early Spring in farnortherly latitudes, feeding from the earliest willowblossoms before the snows have fully melted. Ther-moregulatory mechanisms maintain their body tem-perature high above ambient in cold to freezingweather (Heinrich, 2004), allowing them full activityunder conditions too extreme for other bees. Althoughthey are most abundant in alpine and high-elevation

grassland habitats of the northern temperate zone,they range widely from Greenland to the AmazonBasin, from sea level to altitudes of 5800 m in theHimalayas (Williams, 1985). Thus, bumble beesexploit a wide diversity of habitats, from alpinemeadows to lowland tropical forest (Sakagami, 1976).Nonetheless, they appear similar in morphologythroughout their range (Michener, 1990; Williams,1998), suggesting that ecological opportunity andbehavioural adaptations play an important role in col-onizing diverse habitats (Sakagami, 1976; Cameron &Whitfield, 1996; Dornhaus & Cameron, 2003; Dorn-haus & Chittka, 2004). For example, temperate spe-cies initiate colonies annually from a single queen,whereas the tropical species appear at least faculta-tively perennial (Sakagami, Akahira & Zucchi, 1967;Olesen, 1989) and even exhibit polygyny (Zucchi,1973; Cameron & Jost, 1998).

162

S. A. CAMERON

ET AL

.



2007,

91

, 161–188

Despite the apparent constraint on structural diver-sity, bumble bees display striking inter- and intraspe-cific variation in colour pattern across their range(Sladen, 1912; Skorikov, 1931; Reinig, 1935, 1939;Tkalc , 1968, 1974, 1989; Williams, 1991, 1998), pur-portedly the outcome of mimetic evolution (Plowright& Owen, 1980). Not only are

Bombus

predisposed toconverge sympatrically on similar colour patterns(Williams, 2007), but other insects mimic the

Bombus

patterns (Evans & Waldbauer, 1982), thus providingexcellent opportunities to examine the influence ofnatural selection on the generation of variation amongMüllerian (Plowright & Owen, 1980) and Batesian(Waldbauer, Sternberg & Maier, 1977; Evans & Wald-bauer, 1982) mimics. Elucidating the evolution ofdiversity requires knowledge of the historical patternof variation, which, in turn, provides unique insightsinto evolutionary processes underlying the pattern ofvariation. A robust phylogeny of

Bombus

, the goal ofour research, is essential for inferring the history oftheir wide geographical distribution and for testinghypotheses of behavioural and mimetic adaptations.

C

LASSIFICATION

AND

CURRENT

STATUS

OF

B

OMBUS

PHYLOGENY

Bombus

comprises the monotypic tribe Bombini,which together with Apini (honey bees), Meliponini(stingless bees), and Euglossini (orchid bees), consti-tutes the corbiculate bees, distinguished by the pollen-carrying structure (corbicula) on the hindleg. Theirclosest relatives, estimated from DNA sequences ofmultiple genes, are the stingless bees (Cameron, 1993,2003; Mardulyn & Cameron, 1999; Cameron &Mardulyn, 2001, 2003; Lockhart & Cameron, 2001;Thompson & Oldroyd, 2004), although some morpho-logical data (Roig-Alsina & Michener, 1993; Schultz,Engel & Ascher, 2001) place them as sister to honeybees

+

stingless bees.Subsequent to Linnaeus (1758), approximately

2800 formal names, including synonyms, have beenassigned to

Bombus

species and lower-ranking taxa(Williams, 1998). Today, we recognize approximately250 species (Williams, 1998) assigned to 38 subgenera,primarily on the basis of male genitalia (Radosz-kowski, 1884; Krüger, 1917; Skorikov, 1922; Richards,1968). Many of the subgenera are monotypic (Rich-ards, 1968; Williams, 1998) as a result of the distinct-ness of the male genitalia. The inflated number ofspecies names was often the result of distinguishingspecies (and later, subspecies) by colour pattern.Numerous alternative classifications have been pro-posed (Milliron, 1961, 1971, 1973a, b; Tkalc , 1972)but largely disregarded.

Previous research on bumble bee phylogeny wasrestricted in scope and utility by either: (1) the appli-

uo

uo

cation of phenetic methods to infer phylogenetic rela-tionships; (2) relatively sparse species sampling; or (3)insufficient numbers of informative characters. Exam-ples of phenetic applications include the investigationby Ito (1985) of male genitalia and analyses of allo-zymes in regional taxa (Pekkarinen, 1979; Pekkarinen,Varvio-Aho & Pamilo, 1979; Obrecht & Scholl, 1981;Pamilo, Pekkarinen & Varvio, 1987). Williams (1985,1994) applied parsimony to morphological data toaddress the higher-level relationships among subgen-era, but the available characters led to considerablelack of subgeneric resolution. DNA analyses of rela-tionships have been restricted to regional fauna (anal-yses of European taxa: Pedersen, 1996, 2002) or haveincluded few species (assessment of

Pyrobombus

monophyly, 10–12 species overall: Koulianos, 1999;analysis of subgenera based on 19 species: Koulianos& Schmid-Hempel, 2000). Kawakita

et al

. (2003, 2004)estimated relationships among many of the subgenerausing sequence data, but their sampling of only 76species limits their ability to draw robust conclusionsabout the monophyly of groups and the evolution oftraits.

Comprehensive molecular studies include examina-tion of relationships within the large New Worldsubgenus

Fervidobombus

, including DNA sequencesand morphology, by Cameron & Williams (2003) andsequence analysis of 37 of the 43 recognized

Pyrobom-bus

species by Hines, Cameron & Williams (2006). Inthe present study, we report on the full hierarchy of

Bombus

relationships for most of the world fauna,including rare and potentially endangered speciessuch as

Bombus franklini

and

Bombus occidentalis

(Thorp, 2005), exploiting the large number of charac-ters available from DNA sequences collected frommultiple genes.

MATERIAL AND METHODS

T

AXA

EXAMINED

A total of 218

Bombus

taxa, representing most of thespecies from all 38 subgenera listed by Williams(1998), was sampled for analysis and their locality,collector, and voucher numbers recorded (Table 1).Intraspecific variants and taxa of controversial speciesstatus (Williams, 1998) were included when possible(Table 1). Outgroups were represented by exemplarsselected from each of the other tribes of corbiculatebees (Table 1). Identification of taxa received fromother collectors was verified by the authors beforesequencing. Voucher specimens for all taxa used in themolecular investigation are deposited at the IllinoisNatural History Survey, Urbana, Illinois. Additionalvouchers from the morphological character examina-tion are maintained by PHW at the Natural HistoryMuseum, London.

BUMBLE BEE PHYLOGENY

163



2007,

91

, 161–188

Tab

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164 S. A. CAMERON ET AL.

© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188

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et

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nae

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(19

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hn

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t �

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DQ

7880

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129

DQ

7880

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187

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da, M

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Nu

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ames

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um

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peci

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niz

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y W

illi

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(199

8) (

excl

udi

ng

exti

nct

spe

cies

) fo

llow

ed b

y th

e n

um

ber

repr

esen

ted

in t

he

pres

ent

stu

dy. A

ster

isks

in

dica

te t

axa

that

wer

ere

cogn

ized

as

con

spec

ific

wit

h o

ther

spe

cies

by

Wil

liam

s (1

998)

. Su

pers

crip

t le

tter

s fo

llow

ing

acce

ssio

n n

um

bers

indi

cate

seq

uen

ces

obta

ined

from

pre

viou

s st

udi

es.

BB

ae e

t al

., u

npu

bl. G

enB

ank

refe

ren

ce, 2

001;

CC

amer

on &

Wil

liam

s (2

003)

; C2 C

amer

on (

1993

); DD

anfo

rth

& J

i (2

001)

; HH

ines

et

al. (

2006

); KK

awak

ita

et a

l. (2

003,

2004

); M

Mar

duly

n &

Cam

eron

(19

99);

Mi M

ich

el-S

alza

t, C

amer

on &

Oli

veir

a (2

004)

; RR

asm

uss

en &

Cam

eron

, 200

7).

HH

, P

R =

H.

Hin

es,

P. R

asm

ont;

SC

et

al. =

S.

Cam

eron

, P.

Wil

liam

s, J

. Wh

itfi

eld;

PR

et

al. =

P.

Ras

mon

t, M

. Ayt

ekin

, H

. H

ines

, M

. Ter

zo,

I. B

arbi

er;

SC

, JW

= S

.C

amer

on, J

. Wh

itfi

eld;

RA

, RB

= R

. Aya

la, R

. Bro

oks.



GENES

Five genes were selected for analysis based on priortesting for useful signal: mitochondrial 16S rRNA, andthe nuclear protein-encoding genes long-wavelengthrhodopsin copy 1 (opsin), elongation factor-1 alphaF2 copy (EF-1α), arginine kinase (ArgK) and phos-phoenolpyruvate carboxykinase (PEPCK). 16S wasselected for its high evolutionary rate, useful forresolving tip relationships; the nuclear genes providebetter phylogenetic signal at deeper hierarchical lev-els in the phylogeny. A full discussion of the first fourgenes, primer sequences, and rationale underlyingtheir selection is provided elsewhere (Hines et al.,2006). PEPCK was selected because it has recoveredrelationships within Lepidoptera at similar taxonomiclevels to those in Bombus (Friedlander et al., 1996) andcontains both highly variable intron and conservedexon regions. PEPCK primers used for most taxa wereFHv4-5′-TGTATRATAATTCGCAAYTTCAC-3′ andRHv4-5′-CTGCTGGRGTYCTAGATCC-3′. Some taxarequired an alternative forward primer, either FH2-5′-GTSTCTTATGGGAGSGGTTACGG-3′ or FHv5-5′-AGAACAATTATCTYAAATRCTAARCTTC-3′. Theforward primer sequences began within an intronand were unable to amplify some of the outgrouptaxa. For these and two problematic Bombus taxa(Bombus haematurus and Bombus pressus), pri-mers were designed to amplify only the exon region:PEPCKexF-5′-GATAYTGGCTATCACARATCC-3′ and21dNrc from Friedlander et al. (1996). Some previ-ously reported sequences were obtained from Gen-Bank (Table 1). Two of these were renamed to conformto current species names, deduced from their collec-tion locality: Bombus parthenius from Taiwan is rec-ognized as Bombus sonani and Bombus nevadensisfrom the eastern USA is recognized as Bombusauricomus.

MORPHOLOGY

We analysed an updated version of the morphologicaldata set of Williams (1994), incorporating 47 bumblebee taxa scored for 53 morphological characters,including character states of the male genitalia (30characters), male soma (seven characters) and femalesoma (16 characters) (Appendices 1 and 2). Each sub-genus is represented by one or two exemplars, whichexpress the character state for the remainder of thesubgenus; variable subgenera were represented bymore than one species. Because genitalic characters ofApini and Meliponini used in the molecular analyseswere difficult to homologize with Bombini, the nextclosest outgroup, Euglossini (Eufriesea), was selected,along with the slightly more distant Xylocopinae(Lestis).

POLYMERASE CHAIN REACTION (PCR) AND DNA SEQUENCING

Detailed extraction, PCR and sequencing protocols arereported in Hines et al. (2006). PCR amplification ofPEPCK was conducted at annealing temperatures of49–50 °C and extension at 72 °C. Gene fragments ofthe following sizes were amplified: (1) ∼530 nucleotides(bp) of 16S; (2) ∼680 bp of opsin, including two intronscomprising 178 bp (Mardulyn & Cameron, 1999); (3)∼725 bp of EF-1α F2 copy, containing an intron of∼200 bp; (4) ∼910 bp of ArgK containing an intron of∼325 bp; and (5) ∼900 bp of PEPCK containing twointrons of ∼525 bp. Sequences obtained from thesefragments, using the Applied Biosystems BigDyeTerminator kit version 3.0 or 3.1, were visualized withan ABI 3730XL automated sequencer at the W. M.Keck Center for Comparative and Functional Genom-ics, University of Illinois. Both strands were sequencedfor all taxa and consensus sequences were deposited inGenBank (accession numbers are shown in Table 1).

ALIGNMENT

Sequences were edited and aligned (default parame-ters, CLUSTAL W) in BioEdit (version 5.0.9) (Hall,1999), with some manual adjustment if identical orhighly similar regions were aligned differently acrosstaxa. A comparative check on the 16S alignment wasmade using CLUSTAL X, version 1.81 (Jeanmouginet al., 1998), with gap opening of 10 and gap extensionof 0.10. Nucleotides of uncertain alignment for 16S(∼50 bp from four hypervariable AT-rich regions) wereexcluded from the data matrices. ArgK and PEPCKcontained a few indels that were problematic to alignfor a few taxa; the ambiguous regions for these weretreated as missing data, as in Kawakita et al. (2003).Nucleotides of EF-1α that varied between our taxaand those of Kawakita et al. (2003) were confirmedby reinspection of chromatograms. Gap regions ofunambiguous alignment were coded as separatebinary characters following the methods of Simmons& Ochoterena (2000) and Simmons, Ochoterena &Carr (2001), incorporating only the regions that wereparsimony informative in collective analyses. With theintroduction of gaps in the alignments, individualgene fragments had the following numbers of aligned/gap-coded sites: 16S, 573/8; opsin, 751/5; EF-1α, 906/13; ArgK, 1108/43; and PEPCK, 1425/57. The com-bined (five-genes) data set included 4763 bp and 126gap-coded characters.

PHYLOGENETIC ANALYSES

DNA sequencesInter- and intrasubgeneric relationships of Bombuswere inferred largely from Bayesian analyses,



implemented in MrBayes, version 3.1.2 (Ronquist &Huelsenbeck, 2003) on an IBM p-series 690 supercom-puter operated by the National Center for Super-computing Applications (University of Illinois atUrbana-Champaign, Champaign, IL). Genes wereanalysed individually and collectively, each parti-tioned into exon, intron and gap regions (when appli-cable) to account for variation in evolutionary ratesamong gene regions. Four taxa sequenced only for the16S fragment (Bombus tunicatus, Bombus wilemani,Bombus coreanus, and B. luteipes) were excluded fromcollective analyses.

Model selection for each gene was based on Akaikeinformation criteria in Modeltest, version 3.7 (Posada& Crandall, 1998) and MrModeltest (Nylander, 2004).The model parameters used for each gene parti-tion were: 16S (GTR + I + G), 16S gap characters(standard + G); EF-1α intron (GTR + I + G), EF-1αexon (GTR + I + G), EF-1α gap characters (standard +G); opsin intron (GTR + I + G), opsin exon (HKY + G),opsin gap characters (standard + G); ArgK intron(GTR + G), ArgK exon (GTR + I + G), ArgK gap char-acters (standard + G); PEPCK intron (GTR + G),PEPCK exon (HKY + G) and PEPCK gap characters(standard + G). In combined analyses, gap-coded char-acters were treated as a single partition (standard + G).

Four to six independent analyses were carried outfor each gene fragment and for the combined data(eight million generations for individual genes, 12million for combined analyses, four chains withmixed-models, flat priors, saving trees every 1000generations). Consensus trees were estimated from atleast three independent analyses, with convergentlog-likelihood plots examined in Tracer 1.2 (Rambaut& Drummond, 2003). All trees estimated prior to sta-tionarity were discarded. Trees remaining after con-vergence from replicate runs were combined to createa single majority rule consensus tree. Posterior prob-ability (PP) values represent the proportion of allMarkov chain samples that contain a given node, andare interpreted as the probability that a node ormonophyletic group is correct given the model and thedata.

To assess the influence of the gap regions on treetopology, additional parallel analyses were conductedwith gap-coded characters excluded.

Maximum parsimony nonparametric bootstrapanalysis of the combined five-genes data [heuristicsearch, 300 replicates, ten random additions perreplicate, retaining a maximum of 300 trees in eachreplicate, tree-bisection-reconnection (TBR) branchswapping] was implemented in PAUP*4.0 (Swofford,2001) to compare parsimony bootstrap values withBayesian posterior probabilities. Correspondinglyhigh values for these two different measures of sup-port provide good confidence for testing the monophyly

of the conventional Bombus subgenera and acceptingother monophyletic groups.

Morphological dataMorphological characters were analysed under parsi-mony in PAUP* (heuristic search, 100 replicates witha maximum of 500 trees saved per replicate, TBRbranch-swapping, all characters of equal weight).Because male genitalic characters have been the focusin prior examinations of Bombus phylogeny (Williams,1985, 1994), we separated the morphological charac-ters into two partitions, one with the full set ofcharacters, the other with only genitalic characters.Replicate analyses established that tree topology andtree statistics were stable. Nonparametric bootstrap-ping (heuristic search: 100 replicates, ten randomadditions per replicate, maximum of 500 trees savedper replicate) provided measures of relative supportfor each node in the parsimony tree.

Morphological characters were also combined witha reduced five-genes data set of the 47 Bombus taxaanalysed under parsimony, implemented in MrBayes(two independent runs, three million generations,four chains, saving trees every 1000 generations, flatpriors, mixed models using the same parameters asthose applied to the total sequence data set, standardmodel applied to the morphological characters).Results were compared with those from a Bayesiananalysis of the reduced five-genes data set alone(same model parameters and conditions as applied togenes + morphology). Outgroups for this analysiswere selected from the full-scale molecular data set(Plebeia, Trigona, Heterotrigona, Apis, and Eulaema;Table 1), coded as missing data for the morphologicalcharacters.

RESULTS

RESOLUTION AND SUPPORT FOR THE PHYLOGENY

Independent Bayesian analyses of each nucleargene data set resulted in highly resolved and wellsupported phylogenies overall (supplementary treesonline). On the whole, opsin was less useful near thetips of the phylogeny but resolved many of the deeperclades, whereas EF-1α, ArgK, and PEPCK providedgood resolution and support for relationships withinand between clades throughout the trees. By contrast,mitochondrial 16S (supplementary tree online) wasespecially useful in resolving the tip clades within con-ventional groupings (subgenera) but generally poor atresolving intersubgeneric and deeper relationships(Table 2).

Simultaneous Bayesian analysis of the combinedsequence data produced a tree with nearly completeresolution and high branch support at all levels



(Fig. 1, Table 2). Of the 204 Bombus nodes in the tree,all but 12 were resolved and 158 (77%) were supportedby posterior probability values > 0.95. Relation-ships among subgenera indicated by high support(PP > 0.95) are summarized in Fig. 2.

SUBGENERIC RELATIONSHIPS

The Bayesian combined (Fig. 1) and individual nuc-lear gene phylogenies, and to some extent the 16Sphylogeny, indicated strong support for all but the fol-lowing five conventional Bombus subgenera (Table 2):Separatobombus, Dasybombus, Cullumanobombus,Fervidobombus, and Tricornibombus. Of the threelarger nonmonophyletic subgenera, Cullumanobom-bus was paraphyletic as Bombus rufocinctus fell out assister group to the remaining Cullumanobombus spe-cies (Bombus apollineus + Bombus semenoviellus) + aNew World clade (Fig. 1). Fervidobombus emerged as

polyphyletic as the group (Bombus morio + Bombusexcellens + Bombus dahlbomii) separated and at-tached to the Thoracobombus–Rhodobombus clade(PP = 0.81), leaving the remaining Fervidobombusstrongly supported (PP = 1.0). In turn, a polyphyleticTricornibombus, which excluded Bombus (Tricorni-bombus) imitator, and included Bombus (Exilobom-bus) exil (PP = 1.0), attached as sister group toFervidobombus (minus the dahlbomii-group) withstrong support (PP = 1.0). Of the remaining two non-monophyletic subgenera (both ditypic), Separatobom-bus was unresolved due to poor support (Fig. 1), andDasybombus was polyphyletic as Bombus (Dasybom-bus) macgregori and Bombus (Dasybombus) handlirs-chi fell into different well supported clades (both withPP = 1.0). Two other ditypic subgenera represented inthis study by both species (Coccineobombus and Bom-bias) were resolved as strongly monophyletic(PP = 1.0).

Table 2. The left half of the table indicates resolution (number of internal nodes resolved) and node support (PP = posteriorprobability) for the conventional Bombus subgenera from Bayesian analyses of the combined sequences from five genes.In the right half of the table, posterior probability values are reported for subgeneric monophyly based on individual genesequences. Abbreviations of subgeneric names are given in Table 1. The 12 monotypic subgenera (Table 1) are not listed,nor are subgenera comprising two species for which we have only one representative (Br, Fn, Ob, Rf). u, unresolved; pa,paraphyletic; po, polyphyletic

Subgenus

Numberof taxain study

Number of internalnodes resolvedin combinedgene tree

Number of nodes PP ≥ 0.95in combinedgene tree

PP support for monophyly of subgenera

16S Opsin ArgK Ef-1α PEPCK

Pr 46 42 28 u 1.0 1.0 1.0 1.0Bo 13 10 4 1.0 1.0 1.0 1.0 1.0Al 5 4 4 u 1.0 1.0 1.0 1.0Rb 7 6 5 1.0 1.0 0.86 1.0 1.0Cc 2 1 1 0.87 1.0 1.0 0.98 1.0Sp 2 0 0 u u u u uDs 2 0 0 u po po po poCu 3 1 1 0.99 u pa u uMl 12 11 10 1.0 0.89 1.0 1.0 0.91Ag 5 4 4 u 0.85 1.0 1.0 1.0Sb 5 4 3 u 0.99 0.84 1.0 0.98Fv* 19 17 15 u 1.0 0.97 u 1.0Tr 3 1 1 po u pa u poRh 3 2 2 1.0 1.0 0.88 1.0 1.0Th 18 15 11 1.0 u 1.0 1.0 1.0Ps 20 19 17 1.0 1.0 1.0 1.0 1.0Mg 12 11 9 u 0.94 1.0 1.0 0.78Sx 2 1 1 1.0 0.98 1.0 1.0 1.0Dv 4 2 1 1.0 1.0 1.0 1.0 1.0St 8 7 5 1.0 0.98 1.0 1.0 1.0Or 2 1 1 1.0 1.0 1.0 1.0 1.0Bi 2 1 1 1.0 1.0 1.0 1.0 1.0Md 7 6 5 1.0 1.0 1.0 1.0 1.0

*Fervidobombus minus the dahlbomii + morio + excellens group.

BUMBLE BEE PHYLOGENY

173



2007,

91

, 161–188

Fig

ure

1.

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NW



Figure 2. Bombus phylogeny representing only the subgeneric relationships with strong support (PP = 0.95) from theBayesian tree in Fig. 1. Branches are colour-coded as in Fig. 1. Values on branches are Bayesian posterior probabilityvalues.

SF

LF

1.0

1.0 Melanobombus

Obertobombus

Festivobombus

Exilobombus

Alpigenobombus

Senexibombus

Diversobombus

Megabombus

Orientalibombus

Thoracobombus

Psithyrus

Rhodobombus

Tricornibombus

Kallobombus

Eversmannibombus

Mucidobombus

Subterraneobombus

Fervidobombus

Confusibombus

Bombias

Mendacibombus

Pyrobombus

Bombus

Alpinobombus

NW

Cullumanobombus

Cullumanobombus

Sibericobombus

Rufipedibombus

Fervidobombus

Tricornibombus (B. imitator)

Laesobombus

NW

(dahlbomii gr.)

(B. rufocinctus)

1.0

1.01.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

0.99

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

0.98

1.0

0.99

1.0

Rb

Ds

Fn

Sp

Fr

Cc

Ds

Rc

Br

Sp

Cr

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0



Of the 11 recognized monotypic subgenera (Table 1),most of their relationships to other taxa were wellresolved with good support (Figs 1, 2). Notably, theattachment of Exilobombus (B. exil) within Tricorni-bombus was strongly supported (PP = 1.0), as was theplacement of Pressibombus (= B. pressus) as sister toBombus infirmus within Pyrobombus (PP = 0.99;Fig. 1). Sister group relationships of the monotypicsubgenera are summarized in Table 3.

The four species sequenced only for the 16S frag-ment were resolved in the 16S tree within their ex-pected subgenera. In particular, B. luteipes fell withinthe Pyrobombus clade (Bombus avanus + B. sonani+ Bombus infrequens + B. parthenius) (PP = 0.90),attaching to B. infrequens + B. parthenius as an unre-solved trichotomy (PP = 0.97); B. tunicatus attachedas sister to Bombus terricola (PP = 0.74); B. coreanuswas sister to Bombus ashtoni + Bombus bohemicus(PP = 0.85); and B. wilemani was sister to Bombus tri-fasciatus (PP = 1.0).

Parsimony nonparametric bootstrap analysis of thecombined sequences resulted in a tree nearly identicalto but somewhat less resolved than the Bayesian tree[Fig. 1, bootstrap values (BV) shown below branches].The only substantive contradiction was Cullumano-bombus, which was monophyletic in the parsimonytree (BV = 87%) but paraphyletic in the Bayesian tree(PP = 0.99). Parsimony resulted in decreased resolu-tion within portions of Pyrobombus, Bombus s.s.,Psithyrus, and Fervidobombus, principally for rela-tionships that were poorly supported with Bayesiananalysis. Inconsistencies between parsimony andBayesian support values (Fig. 1) also occurred near afew of the tip taxa within several subgenera, whereposterior probability values were generally low(PP = 0.65). Parsimony gave stronger support for the

hypothesis that Mendacibombus falls nearest the root(BV = 86%).

DEEP RELATIONSHIPS

The individual nuclear- and combined-genes phyloge-nies resolved nearly all subgenera into two large andstrongly supported sister clades (Figs 1, 2), short-faced(SF) and long-faced (LF), which broadly correspondto differences in facial morphology and ecologicallyimportant differences in tongue-length, with someexceptions. ArgK (Fig. 3D) gave somewhat weakersupport for SF (PP = 0.83) and opsin (Fig. 3C) did notfully resolve it, whereas PEPCK did not fully resolvethe LF clade (Fig. 3E). However, the sister group rela-tionship between SF + LF, with or without Kallobom-bus, was supported in all nuclear gene analyses(Fig. 3B–E).

Nested within the short-faced clade was a large, wellsupported group consisting of taxa found exclusivelyin the New World, including Robustobombus andnumerous mono- and ditypic subgenera (Figs 1, 2).Eight of the 19 mono- and ditypic subgenera fell intothis New World (NW) clade, which was also stronglysupported in the individual gene trees, except 16S.The subgeneric tree (Fig. 2, inset) shows clearly thatthe New World clade has poorer resolution among sub-genera than any other part of the tree.

Although the nuclear gene trees were generallycompatible, uncertainty surrounded both the root andthe placement of the monotypic Kallobombus (Kl) withrespect to the short- and long-faced clades. A consen-sus of the basal relationships for the five genes(Fig. 3A) summarizes the uncertainty. Kallobombus inthe EF-1α tree (Fig. 3B) formed a trichotomy with theshort- and long-faced clades whereas, in the opsin tree,

Table 3. Synopsis of the closest relatives of each of the 11 conventional monotypic subgenera of Bombus

Subgenus Sister group relationship PP value/BV

Pressibombus Pe + Bombus (Pr) infirmus 0.99/94Fraternobombus Fr + Rb 0.99/–Crotchiibombus Cr + Bombus (Sp) griseocollis 0.89/–Rubicundobombus Rc + Cc + Bombus (Ds) handlirschi 1.0/99Festivobombus Fs + Ml 0.94/–Mucidobombus/Eversmannibombus/ (Mc + Ev) + Ls 1.0/88Laesobombus ((Mc + Ev) + Ls) + Th 1.0/98Exilobombus Ex + (Bombus (Tr) atripes + Bombus tricornis) 1.0/99Kallobombus Kl + LF + SF 1.0/98Confusibombus Cf + Bi 1.0/100

The sister group relationships are inferred from combined DNA sequences of 16S, EF-1α, opsin, and ArgK.Abbreviated subgeneric names in column 2 are given in Table 1.PP, posterior probability; BV, parsimony bootstrap value; –, no support.



it was more closely related to the long-faced clade(Fig. 3C) and, with ArgK and PEPCK, it was sister toSF + LF (Fig. 3D, E). In the combined-genes analysis,Kallobombus was resolved as sister to SF + LF withmoderate support (PP = 0.93, BV = 63%). With respectto rooting the Bombus tree, opsin and PEPCK sug-gested a rooting along the Mendacibombus branch

(Fig. 3C, E), although support from opsin was poor(PP = 0.63), and EF-1α and ArgK indicated a rootingwith Confusibombus + Bombias (PP = 0.99 and 0.74,respectively). EF-1α did not resolve the sister grouprelationship between Bombias + Confusibombus,which was strongly supported by all the other genes(PP = 0.98–1.0). The combined analysis (Fig. 1) sug-

Figure 3. Bombus trees summarizing the basal relationships for each of four nuclear genes. A, summary consensus ofthe four individual gene trees: EF-1α (B), opsin (C), ArgK (D), and PEPCK (E). Kl, Kallobombus; Bi, Bombias; Cf,Confusibombus; Md, Mendacibombus. SF and LF refer to the short-faced and long-faced clades, respectively. Proportionsshown on the branches of individual gene trees are Bayesian posterior probabilities.

SF

LF

Kl

Md

Bi

Cf

SFSF

LF

Kl

Md

Bi

Cf

SF

LF

Kl

Md

Bi

Cf

SF

LF

Kl

Md

BiCf

LF

SF

LF

Kl

Bi

Cf

Md

C. opsinB. EF-1a

D. ArgK E. PEPCK

A. Consensus

0.81

1.0

0.85

0.970.780.63

0.801.0

0.670.99

0.99

1.0

0.83

1.00.90

1.0

0.74

1.0

0.99

1.0

0.91

0.86

1.0

1.0

1.0 1.0

0.99

0.96



gested Mendacibombus as sister group to the remain-ing bumble bees, with weak to moderate support(PP = 0.77, BV = 86%).

EFFECTS OF GAP-CODED CHARACTERS

Excluding the gap-coded characters from the five-genes data set had minor influences on tree topologyoverall and support values were highly similar to thecomplete data set. In a few cases, gap characterssubstantially increased support for a clade, includ-ing (1) the sister group relationship betweenB. rufocinctus and members of the New World clade +(B. semenoviellus + B. apollineus) (PP = 0.65 withoutgaps, 0.99 with gaps); (2) the sister group relationshipbetween Bombus citrinus and Bombus insulariswithin Psithyrus (from 0.90 to 1.0); and (3) thesister group relationship between (Megabombus +Senexibombus + Diversobombus) and Thoracobom-bus–Psithyrus (from 0.91 to 0.98). By contrast, gapcharacters substantially decreased support for therelationship between Bombus velox and Bombusruderarius (within Thoracobombus) from 0.99 to0.52. Regarding topological changes, excluding thegap characters caused the Bombus hypnorum–B. haematurus group within Pyrobombus (Fig. 1) toattach as sister to the clade Bombus cingulatus–B. biroi (PP = 0.62), and within Alpinobombus (Fig. 1),Bombus balteatus attached as sister to Bombus hyper-boreus in place of Bombus neoboreus (PP = 0.94). Mostimportantly, excluding gap characters shifted the posi-tion of the root from Mendacibombus to Bombias +Confusibombus (PP = 0.61).

MORPHOLOGY

Figure 4 illustrates comparative results of parsimonybootstrap analyses based on the complete set of mor-phological characters (Fig. 4A) and on the male geni-talia alone (Fig. 4B), with bootstrap values indicatingsupport for nodes > 50%. Genitalic characters aloneresulted in higher tree resolution than when somaticcharacters were included, although support for theadditional resolution was weak. Both data sets recov-ered a New World clade + Sibiricobombus but boot-strap support was < 50% with the inclusion of somaticcharacters. The genitalic data resolved a short-facedclade, with weak support (BV = 59%), and therewas no resolution of a long-faced clade in eithertree. The trichotomy Senexibombus + Diversobombus+ Megabombus was well supported in both trees(BV = 83% and 78%). Five of the nine subgenera rep-resented by two exemplars in the complete data setwere recovered with good support (BV > 70%), onlythree were supported by genitalia alone. The basaldivergences resembled the comprehensive DNA tree

(Figs 1, 2), except that Mendacibombus was paraphyl-etic and Bombias and Confusibombus did not form aclade.

Bayesian analysis of the sequence data for these 47taxa recovered a tree (not shown) entirely consistentwith the subgeneric relationships from the all-taxonBayesian analysis (Fig. 2), with nearly identical highsupport values (all but a few PP = 0.98). Adding themorphological characters to the 47-taxon sequencedata resulted in decreased support for Subterrane-obombus + Orientalibombus (from PP = 0.92 to 0.85)and for the placement Kallobombus as sister to theshort-faced + long-faced clades (from PP = 0.94 to0.68). Also, Alpigenobombus moved from its positionas an unresolved polytomy with the clades circum-scribing the New World taxa–Sibiricobombus andMelanobombus–Rufipedibombus to that of sister tothese two clades, although support was weak(PP = 0.85).

DISCUSSION

MONOPHYLY OF CONVENTIONAL SUBGENERA

An unequivocal result of this molecular phylogeneticexamination is the strong support that it provides forgroups of species represented by the conventional sub-generic system, as developed over many years frommorphological taxonomy (Radoszkowski, 1884; Vogt,1911; Krüger, 1917; Skorikov, 1922; Frison, 1927;Richards, 1968; summarized in Williams, 1998). Thisis consistent with the more limited molecular analysesof Pedersen (2002) and Kawakita et al. (2004). In re-trospect, the long stability of the Bombus subgenera isnotable given the intense controversies over theirhigher-level classification (for a review, see Ito, 1985).Clearly, characters of the male genitalia (Radosz-kowski, 1884; Vogt, 1911; Richards, 1968; Williams,1985, 1994) have proven reliable indicators of species-group membership (see also Hines et al., 2006).

The problems generally arise when these samecharacters are used to assess relationships amongthe subgenera. On the whole, morphological charac-ters, whether analysed phenetically (Plowright &Stephen, 1973; Ito, 1985) or cladistically (Williams,1985, 1994; Ito & Sakagami, 1985), have been unableto fully resolve the intersubgeneric relationships or toreconstruct higher-level clades that are congruentwith the DNA-based phylogeny. Although finding mor-phological characters that can serve as synapomor-phies for higher-level groupings of Bombus could beuseful, another approach to the study of morphologywould make use of the well-resolved DNA phylogenyto elucidate the evolution of functionally interestingmorphological traits, such as features of the male gen-italia, the tongue, and colour pattern.



In a few cases, our results lead to the splitting ofconventional subgenera. The large subgenus Fervi-dobombus is a conspicuous example, as the dahl-bomii-group (Fig. 1) falls outside this clade, leavingbehind a strongly supported clade attached to a poly-phyletic Tricornibombus. The polyphyly of Fervi-dobombus was suggested in earlier phenetic (Ito,1985) and cladistic analyses of subgeneric relation-ships (Koulianos & Schmid-Hempel, 2000; Kawakitaet al., 2003). The report by Cameron & Williams(2003), rooting Fervidobombus with Thoracobombus(thought to be among its closest relatives), producedan apparently monophyletic clade because other sub-genera were not represented in the analysis. More

complete taxon sampling outside Fervidobombusupdates this.

Williams (1998) assigned B. handlirschi to themonotypic Dasybombus (Labougle & Ayala, 1985)based on similarities of the male genitalia, but theDNA data show that B. handlirschi is closely relatedto Rubicundobombus (sensu Franklin, 1912, 1913)and Coccineobombus (Fig. 1). The two remainingoccurrences of nonmonophyly of subgenera (Cullu-manobombus and Separatobombus) are less clear cutin terms of support, and would benefit from tar-geted studies of those groups.

In one of the more surprising results, it appears thatPressibombus is a part of Pyrobombus. Morphologi-

Figure 4. Nonparametric parsimony bootstrap trees estimated from morphological characters for 47 Bombus subgenericexemplars. A, analysis of the total morphology data set. B, analysis of genitalic characters alone. Values above branchesindicate bootstrap support (BV) > 50%. Subgeneric taxon labels are given in Table 1.

87

54

100

72

100

Ds

CcBrRcSbFrCrRbSpSpFn

SxDvMgOr

Bo

Al

Ml

Ob

Ps

Th

Ag

Kl

St

RhTrFv

EvMcExLs

PrPrFs

Rf

Cu

CfBi

MdMd

macgregorihandlirschicoccineus

brachycephalusrubicundusniveatusfraternuscrotchii

volucelloidesmorrisonigriseocollisfunebris

kulingensistrifasciatussupremus

haemorrhoidalis

sporadicusterrestris

hyperboreus

insularissylvestris

filchneraesylvarum

nobilisbreviceps

soroeensis

melanurus

armeniacustricornisfervidus

persicusmucidus

exillaesus

hypnorumlapponicusfestivus

eximius

rufocinctus

confususnevadensisconvexus

avinoviellus

9456 62

63

simillimusladakhensis

oberti

65 67

59

pressusPe

65

95

83 90

89 96

68 57

91 69

86

76 6456

83 78

51

86 79

Ds

Ml

Bo

Ag

Th

Ps

51

54

56

B. GenitaliaA. All Morphology

Ds

CcBrRcSbFrCrRbSpSpFn

SxDvMgOr

Bo

Al

Ml

Ob

Ps

Th

Ag

Kl

St

RhTrFv

EvMcExLs

PrPrFs

Rf

Cu

CfBi

MdMd

Pe

Ds

Ml

Bo

Ag

Th

Ps

SF

NW



cally, B. pressus is characterized by distinctive malegenitalia, particularly by a strong reduction of thegonostylus, which appears to a lesser extent in othermembers of the parthenius-group, and by a uniqueventrolateral production of the head of the penis valve.Apart from this new addition to the group, Pyrobom-bus, the largest subgenus, is decisively monophyletic(for additional discussion, see Hines et al., 2006). Incontrast to earlier reports by Williams (1991, 1994,1998) suggesting that Mendacibombus is paraphyleticand Sibiricobombus sensu Richards (1968) (whichincluded Obertobombus) is polyphyletic, both groupsare monophyletic and, from the single species studiedhere, Obertobombus indeed forms a monophyleticgroup with Sibiricobombus. The monotypic Festivo-bombus and Rufipedibombus form a strongly sup-ported group with the larger Melanobombus.

The socially parasitic subgenus Psithyrus has pro-voked a great of deal of taxonomic controversy. Histor-ically, Psithyrus was considered a separate genus ofbumble bees (Lepeletier, 1832) because of the diver-gent biology and morphology associated with itsinquiline mode of life. Our results strongly supportPsithyrus as a monophyletic group within Bombus,which is consistent with the majority of findings overthe last few decades (Plowright & Stephen, 1973;Pekkarinen et al., 1979; Ito, 1985; Williams, 1985,1991, 1994; Pamilo et al., 1987; Pedersen, 2002; Kawa-kita et al., 2004). Furthermore, Psithyrus divides intothe same clades (Fig. 1) established as subgenera inearlier taxonomic studies (Franklin, 1912, 1913; Fri-son, 1927; Popov, 1931; Pittioni, 1949). Williams (1991,1998) treated these as species groups, synonymizingthe subgeneric names; Rasmont et al. (1995) consid-ered them as separate subgenera within Bombus. Therelationships among these species groups receiveweaker support, highlighting the consistency in rela-tive degree of support between the molecular and mor-phological data.

Of potential interest to Psithyrus evolution is thefinding by Bromham & Leys (2005) that rates ofmolecular evolution in social parasites are fasterthan those of their social relatives. If this conclu-sion is accepted, and if substitution rates areexpected to evolve in a lineage-dependent fashion, itfollows that the socially parasitic Psithyrus shouldexhibit faster rates than most of their social rela-tives. There may be a hint of this in the greater com-bined branch lengths of Psithyrus relative to theother subgenera, but testing this requires sister-group comparison of substitution rates in their non-parasitic sister taxa.

DEEP DIVISIONS WITHIN BOMBUS

Krüger (1917) recognized a division of the bumblebees into two larger sections, Odontobombus (‘with

spine’) and Anodontobombus (‘without spine’), basedon several diagnostic morphological characters,including head shape and presence/absence of a mid-basitarsal spine. His Odontobombus (Thoracobombus,Laesobombus, Mucidobombus, Rhodobombus, Sub-terraneobombus, and Megabombus) and Anodonto-bombus (Pyrobombus, Bombus s.s., Alpinobombus,Cullumanobombus, Melanobombus, Kallobombus,Confusibombus, and Mendacibombus) correspondapproximately to our long-faced and short-facedclades. In detail, the long-faced clade includes anadditional eight subgenera (Fig. 1) not included inKrüger’s more restricted investigation of CentralEuropean fauna, and the short-faced clade includes 13subgenera omitted by Krüger. Krüger’s characters ledhim to group Kallobombus, Confusibombus, and Men-dacibombus within the equivalent of the short-facedclade, whereas our results reveal that these taxa falloutside both divisions, representing more basal diver-gences in the phylogeny. Kawakita et al. (2004) dis-cussed two higher-level Bombus groupings (clades Aand B), which are consistent with our long-faced andshort-faced divisions, but with many fewer taxa andweak support for their A clade. Interestingly, withBayesian analysis of sequences from only 47 (∼20%) ofthe 218 taxa collected for this study, we recovered theidentical structure to that of the comprehensive tree,with well supported intersubgeneric relationships,including the short-faced, long-faced, and New Worldsubdivisions. This indicates that taxon sampling isless important in recovering higher level phylogeneticstructure of Bombus than is the availability of a largenumber of informative characters.

RELATIONSHIPS NEAR THE RANK OF SPECIES

In several cases, sister taxa share such a remarkabledegree of sequence similarity that we might askwhether they are conspecific. Given the 1–2 bp differ-ences we commonly see within a gene for many taxonsamples, for example, within nuclear genes of het-erozygous females or from comparisons of conspecifictaxa sequenced both by us and by Kawakita et al.(2003), we consider it likely that two nominal taxa areconspecific if they differ by only 1–2 bp across all genesequences (equivalent to only 0.03–0.05% sequencedivergence). Of course, final decisions about speciesstatus will require examining the patterns of variationamong a broad sampling of individuals.

Several sympatric species pairs exhibit discordantcolour patterns yet have virtually identical sequences.For example, Bombus (Sibiricobombus) niveatus(Fig. 1), which is white-banded, and Bombus vortico-sus, which is yellow-banded, differed by a single nucle-otide (ArgK) across all five genes, supporting theirstatus as dimorphic colour forms of the same species.



Williams (1991, 1998) concluded this from morphologyand Rasmont et al. (2005) from mandibular glandsecretions. Bombus (Mendacibombus) handlirshianus(white-band) and Bombus shaposhnikovi (yellow-banded) differ by two bases (1 bp 16S, 1 bp PEPCK),and individuals of these two colour forms were evenobserved to share a common nest entrance at a site innorth-east Turkey (H. M. Hines, pers. observ.). Some ofthese colour dimorphisms may be the result ofintraspecific diallelic differences (Owen & Plowright,1980). The lack of correspondence between colour pat-tern variation and degree of nucleotide differentiationshows that much remains to be learned about colourpattern evolution, and sets the stage for examiningpossible adaptive causes, including mimicry.

Some putative allopatric species pairs, also describedfrom distinct colour morphs, may be conspecific. Oneexample involves the two Senexibombus species,Bombus bicoloratus (Taiwan) and Bombus kulingensis(mainland China), which differ by only a single base inboth the 16S and PEPCK sequences. Another exampleincludes Bombus pensylvanicus from eastern NorthAmerica and Bombus sonorus from western NorthAmerica: both taxa, which are similar in morphologybut variable in colour pattern (Williams, 1998), exhibitsigns of introgression as B. sonorus from San LuisPotosí, Mexico (an area of sympatry with B. pensyl-vanicus) and B. sonorus from Arizona are not mono-phyletic relative to two B. pensylvanicus sequencesfrom Missouri (Fig. 1). Other likely conspecific taxa,questioned by Williams (1998), include Bombuserzurumensis/Bombus sichelii (3 bp difference for 16S,3 bp for EF1α, 1 bp for PEPCK) and Bombus volucel-loides/Bombus melaleucus (2 bp 16S, 3 bp ArgK).

In two cases, allopatric pairs of Psithyrus taxaexhibit nearly identical sequences and could be con-specific: Bombus fernaldae/Bombus flavidus (1 bp dif-ference EF1a, 1 bp ArgK) and B. ashtoni/B. bohemicus(1 bp ArgK). It is unusual that one member of eachpair occurs only in the New World and the other onlyin the Old World, respectively. If conspecific, each pairwould encompass a nearly Holarctic temperate distri-bution, thus far found only in the lucorum complexand some Alpinobombus species. The possible conspec-ificity of B. fernaldae and B. flavidus has not been con-sidered until now, but Williams (1991) suggested thatB. ashtoni and B. bohemicus could be parts of thesame species. Both taxa specialize on hosts of the sub-genus Bombus s.s.: B. ashtoni on Bombus affinis andB. terricola (Plath, 1934; Fisher, 1984), B. bohemicuson Bombus lucorum (Alford, 1975)

Other species pairs considered potentially con-specific (Williams, 1998) are somewhat geneticallydivergent based on 16S, including B. occidentalis/B. terricola (7 bp, 1.3%); Bombus fervidus/Bombuscalifornicus (8 bp, 1.6%); B. velox/Bombus deuter-

onymus and Bombus sylvicola/Bombus lapponicus(10 bp, 1.9%); B. moderatus + B. cryptarum/B. lucorum(11 bp, 2.1%, 14 bp, 2.6%; neither taxon is monophy-letic with B. lucorum); Bombus alagesianus/Bombuskeriensis (14 bp, 2.6%) and Bombus difficillimus/Bombus melanurus (20 bp, 3.8%). The separation ofB. auricomus and B. nevadensis on the basis of mor-phology, colour pattern (Williams, 1998), and alloz-ymes (Scholl et al., 1992) is also supported by oursequence data (22 bp, 4.2%). Interestingly, Bombusmodestus from Kazakhstan is genetically distinct fromB. modestus from Sichuan, China for 16S, exhibitinggreater genetic distance (24 bp, 4.5%) than many pairsof sister species.

IMPLICATIONS FOR BOMBUS CLASSIFICATION

Several authors have suggested that the subgenericsystem for bumble bees would benefit from simplifica-tion (Menke & Carpenter, 1984; Williams, 1998). Thechallenge is to recognize monophyletic groups that aremorphologically and behaviourally meaningful, whilereducing the number of small subgenera. Our Bombusphylogeny raises the possibility of revising the classi-fication with more confidence by providing a frame-work to ensure monophyletic subgenera and reducethe proliferation of formal group names. Here weintend no nomenclatural action as specific recommend-ations concerning recognition of groups will be madeelsewhere, based in part on some of the following rel-evant details.

Concerning the New World clade, rather than recog-nizing even more monotypic subgenera, it might bemore practical to recognize the entire New World cladeas a single subgenus consisting of several speciesgroups, including Robustobombus. Further reductionsin numbers of subgenera could be achieved by placingObertobombus (Bombus oberti) into Sibiricobombus,and by including Festivobobombus (Bombus festivus)and Rufipedibombus in Melanobombus. Whether toconsolidate these taxa into one group is complicatedby the fact that many of the monotypic subgeneraare sister to clades subtended by conspicuously longbranches, suggesting long separation/distinctness(Fig. 1). In view of these apparently large intersubge-neric genetic distances, it might be prudent to con-sider additional biological characters in decisions as towhether to combine the taxa.

Given the strong support for Pressibombus fallingwithin Pyrobombus, it is reasonable to synonymizePressibombus under Pyrobombus. The clade com-prising the monotypic subgenera Mucidobombus,Eversmannibombus, and Laesobombus could be con-sidered as members of Thoracobombus or as a sepa-rate three-species subgenus. Exilobombus attaches toTricornibombus (excluding B. imitator) and should be



considered a part of Tricornibombus. Megabombuscould include Senexibombus. Bombias and Confusi-bombus might be combined into a single subgenus,although the branches appear long and the respectivetaxa distinct.

New names might be required to accommodate thesplitting of a few subgenera. The obvious division ofFervidobombus into two monophyletic groups couldrequire a new species group or subgenus designationfor B. dahlbomii + B. morio + B. excellens. Other clas-sification decisions may involve Cullumanobombus,Separatobombus, Dasybombus, and Tricornibombus.

FUTURE RESEARCH

PhylogenyWithin some clades (e.g., Bombus s.s. and Fervidobom-bus), a few of the internodes are short but have highposterior probabilities (Fig. 1). We interpret theserelationships with caution because Bayesian inferenceis highly sensitive to phylogenetic signal (indicatinghigh posterior probability values with relatively fewcharacters) and has been shown (with simulated data)to occasionally attach high support values to incorrectshort internodes (Alfaro, Zoller & Lutzoni, 2003).Gathering additional characters should resolve theuncertainties associated with these short branches.Importantly, new characters from other genes shouldincrease resolution and support for relationshipswithin the New World clade and strengthen the place-ment of Kallobombus and assignment of the root.

The missing putative species (Williams, 1998) fromthis study are most notably from Psithyrus (eight spe-cies), Mendacibombus (five species) and Melanobom-bus (four species). Examination of the positions ofthese and remaining species from small subgenera(e.g., Cullumanobombus, Rufipedibombus, Senexi-bombus, and Orientalibombus) would secure ourknowledge of Bombus relationships and subgenericmonophyly.

Diversification in the New WorldThe pattern of diversification of the New World clade,with its numerous distinctive taxa that span NorthAmerica, Mexico, and the Andes, suggests there mayhave been at least two episodes of relatively rapiddivergence after colonization of the New World. Thefirst of these is implied by the very short branchesthat subtend relatively long branches of the morpho-logically distinct monotypic subgenera, which aredifficult to resolve with good support (Fig. 1). Thispattern poses interesting questions concerning theinterplay between colonization of the New World andtrends in bumble bee diversification. Comparativestudies of sister taxa outside this group, which areall Old World (except B. rufocinctus), could reveal

whether any morphological or physiological innova-tions occurred in association with episodes of conti-nental colonization and diversification (Kay et al.,2005). The second episode occurred with Robustobom-bus, the only speciose monophyletic subgenus withinthe New World clade. This group seems to havediverged more recently in upland areas of the north-ern Andes, perhaps in association with new ecologicalopportunities provided after the northern Andeanuplift (3–5 Mya) that involved the colonization bynew plant genera (Hughes & Eastwood, 2006). Ourrobust species phylogeny affords the opportunity totest this hypothesis with estimates of divergencetimes.

The only other large endemic New World subgenus,Fervidobombus, suggests a similar pattern of diversi-fication after colonization from the Old World. How-ever, in contrast to taxa of the New World clade, manyof which inhabit cooler, mountainous habitats, Fervi-dobombus inhabit warmer lowland habitats, includingtropical rain forest (Williams, 1985; Cameron &Williams, 2003). Their predisposition to nest aboveground, a characteristic of many of their closest OldWorld relatives, may underlie their mostly lowlanddistribution, and at least partially explain the innova-tion in nest architecture associated with the atypicalrain forest species, Bombus transversalis (Taylor &Cameron, 2003) and Bombus pullatus (Hines, Cam-eron, & Deans, 2007). Additional research on the biol-ogy of Old World relatives should clarify influences ofecological shifts and phylogeny on life history of thetropical bumble bees.

Colour pattern evolutionColour pattern mimicry in bumble bees has been sug-gested by several authors (Plowright & Owen, 1980;Williams, 1991) to explain many geographical trendsin colour pattern variation. However, conclusions thatcolour patterns do not also evolve along phylogeneticlineages have been limited by incomplete species sam-pling and coding of the full range of patterns andintraspecific polymorphisms. Our comprehensive phy-logenetic results permit the first comparative exami-nation of convergence and mimicry on a worldwidescale.

ACKNOWLEDGEMENTS

First and foremost we thank B. Cederberg andP. Rasmont, without whose collecting and taxonomicassistance this research would have been greatlydiminished. We also thank all of those listed in Table 1for their assistance worldwide in the contribution offresh material for sequencing; most significantly, wethank R. Ayala, A. M. Aytekin, O. Berg, M. Hauser, R.Hepburn, P. Mardulyn, C. Rasmussen, M. Saini, A.



Scholl, R. Thorp, R. Vandame, and J. Whitfield. Weespecially thank Chen Xuexin and Tang Ya for theirinvaluable assistance with organizing the China expe-dition, Pierre Rasmont for organizing the Turkeyexpedition, Ricardo Ayala for organizing collecting inMexico, and Gernot Patzelt for assisting with collect-ing in the Austrian Alps. We thank Sudhakar Pami-dighantam (UIUC NCSA) for invaluable technicalassistance and Ben Grosser and Micah Wolfe for cre-ative assistance with graphics. We are indebted to JimWhitfield for his careful reading and comments on themanuscript. We also thank Sally Corbet, CharlesMichener, and three anonymous reviewers forcomments. Lastly, SAC is especially grateful to JBWfor sharing in the earliest vision of this research. Thisresearch was supported by USDA grants (NRI,CSREES, 2002-35302-11553 and 2004-35302-15077)and NSF grants (DEB 060004) to S.A.C.

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APPENDIX 1

The morphological character states represented in thematrix are listed. Multistate characters analysed asordered are shown with asterisks. Characters of thepenis refer to the penis valves unless otherwisespecified.

MALE PENIS

1. Spatha broader than long (0); spatha longer thanbroad (1).2. Spatha basally broadly rounded (0); spatha basallyacutely pointed (1).3. Spatha laterally continuous with valves (0); spathalaterally overhanging valves (1).4. Dorsal lightly sclerotized channel narrow (0); dor-sal lightly sclerotized channel broad (1).5. Shaft without a distinct ventro-lateral angle (0);shaft with an acute ventro-lateral angle near the mid-point of its length (1); shaft with only a weak trace ofa ventro-lateral angle (2); shaft with ventro-lateralangle broadly rounded as a shallow convexity (3);shaft with ventro-lateral angle pronounced as a verybroadly rounded right angle (4).6. Shaft dorso-ventrally narrow or irregular inbreadth (0); shaft uniformly dorso-ventrally expanded(1).7. Apex straight or curved outwards (0); apex curvedin towards body midline (1).8.* Head strongly laterally compressed (0); headnearly tubular (1); head strongly dorso-ventrally flat-tened (2).(9) Inner basal shelves broad (0); inner basal shelvesnarrow (1).10.* Inner shelves of head absent or weakly defined(0); inner shelves of head strongly marked basally bya pronounced right angle, then running parallel toshaft axis as far as recurved head (1); inner shelvesexpanded basally (2).11.* Head with outer shelf narrow (0); head withouter shelf extended laterally by more than the samebreadth as head (1); head with outer shelf curvedventrally and then twisted towards apex, to form halfof a funnel (2).

12. Head with outer shelf narrow or board (0); headwith outer margin of shaft with straight section nar-row subapically (1).13. Head with inner (median) margin of recurved sec-tion convex (0); head with inner margin of recurvedsection concave.14. Head with or without inwardly recurved hook (0);head straightened with secondary loss of inwardlyrecurved hook (1).15. Head without basal ventral projection (0); headwith basal ventral projection (1).16. Shaft nearly straight in lateral aspect (0); shaftwith a strong ‘S’-shaped dogleg in lateral aspect (1).

MALE VOLSELLA

17.* Small irregular sclerite, not extending apicallyfurther than gonostylus (0); large clasping organ,extending apically further that gonostylus (1); entirevolsella elongated and narrowed (2).18. Outer margin with long setae (0); outer marginwithout long setae (1).19. Lateral margins subapically converging (0); lat-eral margins broadened immediately subapically andthen truncated apically (1).20. Inner margin without a distinct subapical process(0); inner margin with a subapical process, which isusually toothed, arising just before inner margin andprojecting in towards midline of body (1); inner sub-apical process produced distinctly beyond inner mar-gin, often in the form of a broad curved tongue (2);inner subapical process reduced to an indistinct curvein margin, always lacking teeth (3).21. Inner subapical process distinctly separated fromapex, often nearer midpoint of length (0); inner sub-apical process narrowly subapical, at least on long axisof volsella.22.* Inner ventral ridge not swollen or swelling notcurved back proximally towards outer margin (0);inner ventral ridge, near mid-point of volsella length,pronounced at the inner edge of coarsely sculpturedarea in the apical half and curved back proximallytowards the outer margin (1); coarsely sculptured ven-tral area broadened basally before an inner constric-tion to a narrower subapical neck, and pear-shaped (2).23.* Coarsely sculptured ventral area weakly definedor proximal half reaching outer margin adjacent togonocoxite (0); proximal half of coarsely sculpturedventral area separated from outer margin by a con-cave, weakly sculptured area, forming a narrow shin-ing submarginal groove (1); proximal half of coarselysculptured ventral area separated from outer marginby a concave, weakly sculptured area, forming a sub-marginal groove as broad as coarsely sculptured area(2); proximal half of coarsely sculptured ventral areaseparated from margin by broad submarginl groovewith long setae (3).



24.* Inner apical margin without stout setae (0);inner apical margin with stout setae (1); inner apicalmargin with stout setae very long and dense (2).

MALE GONOSTYLUS

25. Inner basal corner without a distinct process (0);inner basal corner with a distinct rounded processprojecting in towards midline of body (1); inner basalcorner with a distinct process; distally narrowed in theform of a single sharp spine (2); inner basal processwith teeth (3).26. Basal inner margin associated with setae (0);basal inner margin without associated setae (1).27. Inner basal process produced (0); inner basal pro-cess reduced to a stub or membranous or both (1).28. Inner apical margin simple (0); inner apical mar-gin double with a submarginal groove (1).29. Apical process present (0); apical process absent(1).30. Apical process rounded or narrowing (0); apicalprocess broadened (1).

MALE HEAD

31. Mandible with sparse long setae from posteriormargin (0); mandible with dense setae from posteriormargin, forming a ‘beard’ (1).32. Antenna short, not reaching back beyond wingbases (0); antenna long, reaching back beyond wingbases (1).33. Flagellar segments nearly straight and cylindrical(0); flagellar segments curved (1).34. Compound eye similar in relative size to femaleeye (0); compound eye distinctly enlarged relative tofemale eye (1).

MALE THORAX

35. Hind tibia with outer surface uniformly convex(0); hind tibia with outer surface partially concavemedially in distal third (1).36. Hind tibia with short or long hairs over entireouter surface (0); hind tibia without even short hairsmedially in distal third (1).

MALE ABDOMEN

37. Gastral sternum VII with posterior margin medi-ally convex or irregular, but not broadly concave (0);gastral sternum VII with posterior margin mediallybroadly concave (1).

FEMALE HEAD

38.* Labrum with median longitudinal ridge (0);labrum with complete transverse ridge between twogrooves (1); labrum with transverse ridge broadly

interrupted medially (2); labrum with median part oftransverse ridge displaced towards apex of labrum toform a projecting lamella, which reaches towards theanterior margin of the labrum (3).39. Labrum broadly rectangular (0); labrum broadlytriangular (1).40. Mandible distally broadly rounded (0); mandibledistally pointed (1).41. Mandible with two to four teeth (0); mandible withsix teeth (1).42. Mandible with basal keel not reaching distal mar-gin (0); mandible with basal keel reaching distal mar-gin (1).43. Oculo-malar distance less than the basal breadthof mandible (0); oculo-malar distance equal to orgreater than the basal breadth of mandible (1).44. Oculo-malar area broadly rounded into the faceanteriorly, the area below the eye uniformly convex(0); oculo-malar area separated from the face anteri-orly by a narrowly rounded angle, the area immedi-ately below the eye partially concave (1).

FEMALE THORAX

45.* Mid-basitarsus with disto-posterior cornerbroadly rounded or forming a right angle (0); mid-basitarsus with acute disto-posterior corner (1);mid-basitarsus with pronounced disto-posteriorspine (2).46. Hind tibia without corbicula (0); hind tibia withcorbicula (1).47. Hind tibia with disto-posterior corner formingright angle (0); hind tibia with disto-posterior corneracute or spinosely produced (1).48. Hind basitarsus with proximo-posterior processno longer broad (0); hind basitarsus with proximo-posterior process longer than broad (1).49. Hind basitarsus with proximal process with fewscattered hairs on outer surface (0); hind basitarsuswith proximal process densely hairy on outer surface(1).

FEMALE ABDOMEN

50.* Gastral sternum II without transverse ridge (0);gastral sternum II with weakly rounded traverseridge (1); gastral sternum II with strongly raisedtransverse ridge (2).51. Gastral sternum VI without subapical swellings,curving gradually dorsally (0); gastral sternum VIwith paired subapical swellings, lateral areasabruptly turned dorsally (1).52. Gastral sternum VI without lateral keels (0); gas-tral sternum VI with lateral keels (1).53. Gastral segments V–VI nearly coaxial with seg-ments I-IV (0); gastral segments V–VI curled ventrallyand back towards anterior (1).



APPENDIX 2

Morphological character matrix. Morphological character states are explained in Appendix 1.

1111111111222222222233333333334444444444555512345678901234567890123456789012345678901234567890123

Lestis 00000-000000-00000-----000000000110000010100001000000Eufriesea 00100-000000-00001-----000000000000100010100010000000avinoviellus 10000-000000-0001000-00001000010011001000010010000000convexus 10010-001000-0001001000001000010011001000010010000000nevadensis 10010-001000-0001102100111000010011012000010111000000confusus 10010-011000-0001102000111000010011003000010111000000soroeensis 111120011000–0001102100101000011100013000100111101000haemorrhoidalis 110120011000–0001112000201100111101103000110011101000kulingensis 110120011000–0001102100231000111101103000100211100000trifasciatus 110120011000–0001102100231000111101103000110211100000supremus 110120011000–0001102100231000111101103000110211100000melanurus 11112011100000001102100111000011101003000110211101000insularis 110120011000–0001103000110100011100003110001200002111sylvestris 110120011000–0001103000110100010000003110001200002111exil 110120011000–0001102000211000011100003000100211101000tricornis 110120011000–0001102100111000011100003000110211101000fervidus 110120011000–0001102000111000011100003000110211101000armeniacus 111120011000–0001102100111000011100003000110211101000persicus 110120011000–0001103000211000011100003000110211101100mucidus 110120011000–0001102000211000011100003000110211101000laesus 110120011000–0011112000211000011101003000100211101000filchnerae 110120011000–0011102000221000011100003000110211101000sylvarum 110120011000–0011102000221000011100003000110211101000hyperboreus 111120121000–1101102100111110010001103000110111101000sporadicus 11114012103001101102100111100010001103000100011100000terrestris 11114012103001101102100111100010001103000100011100000hypnorum 11112012100000001102100111110010001003000100011100000lapponicus 11112012100000001102100111110010001003000100011100000pressus 11114012102000101102100111001010000103000110011100000breviceps 11112012100000001102100111000010000003001100111101000nobilis 11112012100000001102100111000010000103001100111101000eximius 11013012101000001102100111001011001103000110011100000festivus 11111012100000001102100111100010000003000100011100000simillimus 11111012100110001102100111100010011013000110011100000ladakhensis 11011012100110001102100111100010000013000110010100000oberti 11111012100010001102100111000010010003000110011110000niveatus 11111012110000001102110111000011011013000110011110000rufocinctus 11111012100000001102100111000010010113000100011100000brachycephalus 11111012120000001102123111100010010013000100011100000rubicundus 111110121200–0001102123111000010010013000100011100000handlirschi 11111112110010002102123101100010010013000100011100000coccineus 11111112110010001102123111000010010013000100011100000fraternus 11111012100000001102120111000001010013000100011100000crotchii 11111012110000001102121111000011011103000100011100000griseocollis 11111012100000001102122111100010010013000100011100000morrisoni 11111012100000001102122111000010011013000100011100000funebris 111110121000–0001102122111000010010013000100011100000volucelloides 11111012110010001102122111000011010013000100011100000macgregori 11111112100010002102123101100010010013000100011100000



SUPPLEMENTARY MATERIAL

The following supplementary material is available for this article:

Figure S1. 16S. Phylogeny of Bombus estimated from Bayesian analysis of mitochondrial 16S rRNA nucleotidedata. Values above branches are Bayesian posterior probabilities. The left hand portion of the figure connects viathe dashed lines at the bottom to the dotted lines at the top of the right hand portion of the figure.

Figure S2. EF-1∝. Phylogeny of Bombus estimated from Bayesian analysis of elongation factor-1alpha F2 copynucleotide sequences. Values above branches are Bayesian posterior probabilities. The left hand portion of the fig-ure connects via the dashed lines at the bottom to the dotted lines at the top of the right hand portion of the fi gure.

Figure S3. Opsin. Phylogeny of Bombus estimated from Bayesian analysis of long-wavelength rhodopsin copy1 nucleotide sequences. Values above branches are Bayesian posterior probabilities. The left hand portion of thefigure connects via the dashed lines at the bottom to the dotted lines at the top of the right hand portion of thefigure.

Figure S4. ArgK. Phylogeny of Bombus estimated from Bayesian analysis of arginine kinase nucleotidesequences. Values above branches are Bayesian posterior probabilities. The left hand portion of the figure con-nects via the dashed lines at the bottom to the dotted lines at the top of the right hand portion of the figure.

Figure S5. PEPCK. Phylogeny of Bombus estimated from Bayesian analysis of phosphoenolpyruvate carbox-ykinase nucleotide sequences. Values above branches are Bayesian posterior probabilities. The left hand portionof the figure connects via the dashed lines at the bottom to the dotted lines at the top of the right hand portionof the figure.

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1095-8312.2007.00784.x(This link will take you to the article abstract).

Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary mate-rials supplied by the authors. Any queries (other than missing material) should be directed to the correspondingauthor for the article.

http://www.blackwell-synergy.com/doi/abs/10.1111/j.1095-8312.2007.00784.x

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