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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 The Linnean Society of London,
Biological Journal of the Linnean Society,
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 The Linnean Society of London,
Biological Journal of the Linnean Society,
2007,
91
, 161–188
Tab
le 1
.
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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
Cu
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ahlb
omii
Gu
érin
-Mén
evil
leA
rau
co P
rov.
, Ch
ile
C. C
arlt
on01
6D
Q78
7996
AF
4929
31K
AF
4929
98K
AF
4928
64K
EF
0509
40d
igre
ssu
s (M
illi
ron
)R
. Gra
nde
de
Oro
sí, M
exic
oR
. Del
gado
307
AY
2684
03C
DQ
7881
78A
Y26
8381
CD
Q78
8418
EF
0509
59d
ilig
ens
Sm
ith
Jali
sco,
Mex
ico
F. N
ogu
era
171
AY
2684
04C
DQ
7881
79A
Y26
8382
CD
Q78
8419
EF
0509
55ex
cell
ens
Sm
ith
Ara
gua,
Ven
ezu
ela
J. G
arc’
a30
8A
Y26
8405
CD
Q78
8184
AY
2683
83C
DQ
7884
24E
F05
0942
ferv
idu
s (F
abri
ciu
s)M
isso
uri
, US
AS.
Cam
eron
309
AY
2684
06C
AF
4929
30K
AF
4929
97K
AF
4928
63K
EF
0509
58m
ediu
s C
ress
onC
hia
pas,
Mex
ico
RA
, RB
222
AY
2684
07C
DQ
7882
28A
Y26
8385
CD
Q78
8459
EF
0509
51m
exic
anu
s C
ress
onC
hia
pas,
Mex
ico
RA
, RB
220
AY
2684
08C
DQ
7882
32A
Y26
8386
CD
Q78
8461
EF
0509
52m
orio
(S
wed
eru
s)P
orto
Ale
gre,
Arg
enti
na
D. W
ittm
an31
0A
Y26
8409
CD
Q78
8239
AY
2683
87C
DQ
7884
67E
F05
0941
opif
ex S
mit
hP
un
o, P
eru
J. A
rcas
175
DQ
7880
75D
Q78
8249
DQ
7883
56D
Q78
8477
EF
0509
54pe
nsy
lvan
icu
s (D
eGee
r)M
isso
uri
, US
AS.
Cam
eron
317
EF
0323
52E
F03
2374
EF
0323
92E
F03
2411
EF
0509
48M
isso
uri
, US
AS.
Cam
eron
311
EF
0323
51E
F03
2375
EF
0323
93E
F03
2412
EF
0509
49pu
llat
us
Fra
nkl
inP
itil
la, C
osta
Ric
aS.
Cam
eron
312
AY
2684
11C
DQ
7882
61A
Y26
8389
CD
Q78
8486
EF
0509
45so
nor
us
Say
*A
rizo
na,
US
AT.
Gri
swol
d05
1D
Q82
2475
DQ
7882
77E
F03
2394
DQ
7885
00E
F05
0946
Mex
ico
S. C
amer
on31
8A
Y26
8412
CE
F03
2376
AY
2683
90C
EF
0324
10E
F05
0947
stei
nd
ach
ner
i H
andl
irsc
hM
orel
os, M
exic
oR
. Aya
la31
3A
Y26
8413
CD
Q78
8280
AY
2683
91C
DQ
7885
02E
F05
0953
tran
sver
sali
s (O
livi
er)
Mad
re d
e D
ios,
Per
uS.
Cam
eron
314
AY
2684
14C
DQ
7882
91A
Y26
8392
CD
Q78
8510
EF
0509
44tr
inom
inat
us
Dal
la T
orre
Oax
aca,
Mex
ico
H. H
ines
229
DQ
7881
23D
Q78
8292
DQ
7883
79D
Q78
8511
EF
0509
60w
eisi
Fri
ese
Jali
sco,
Mex
ico
F. N
ogu
era
315
AY
2684
15C
DQ
7883
03A
Y26
8393
CD
Q78
8520
EF
0509
61
Fs
Fes
tivo
bom
bus
Tka
lck
(1
/1)
fest
ivu
s S
mit
hG
uiz
hou
Pro
v., C
hin
aM
. Yu
n03
2D
Q78
8007
DQ
7881
87Q
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 10
4A
Y73
9465
HD
Q78
8427
EF
0509
10
Su
bgen
us
Spe
cies
Col
lect
ion
loc
alit
yC
olle
ctor
V.#
16S
EF
-1α
Ops
inA
rgK
PE
PC
K
Tab
le 1
.C
onti
nu
ed
BUMBLE BEE PHYLOGENY 165
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
Fr
Fra
tern
obom
bus
Sko
riko
v (1
/1)
frat
ern
us
(Sm
ith
)Il
lin
ois,
US
AH
. Hin
es18
3D
Q78
8013
AF
4929
74K
AF
4930
41K
AF
4929
07K
EF
0508
84
Fn
Fu
neb
ribo
mbu
s S
kori
kov
(1/1
)fu
neb
ris
Sm
ith
Aba
nca
y, P
eru
C. R
asm
uss
en12
8D
Q78
8016
DQ
7881
94D
Q78
8334
DQ
7884
33E
F05
0883
Kl
Kal
lobo
mbu
s D
alla
T
orre
(1/
1)so
roee
nsi
s (F
abri
ciu
s)E
. Pyr
enee
s, F
ran
ceH
H, P
R13
6D
Q78
8107
DQ
7882
78A
F49
3008
KA
F49
2874
KE
F05
1013
Ls
Lae
sobo
mbu
s K
rüge
r (1
/1)
laes
us
Mor
awit
zK
ars
Pro
v., T
urk
eyP
R e
t al
. 05
2D
Q78
8044
DQ
7882
18D
Q78
8344
DQ
7884
51E
F05
0936
Mg
Meg
abom
bus
Dal
la
Tor
re (
14/1
2)ar
gill
aceu
s (S
copo
li)
Kay
seri
Pro
v., T
urk
eyP
R e
t al
. 05
8D
Q78
7967
DQ
7881
50A
Y73
9453
HD
Q78
8396
EF
0510
01co
nso
brin
us
Dah
lbom
�A
ltai
Mts
, Kaz
akh
stan
P. M
ardu
lyn
261
EF
0323
54E
F03
2379
AY
2671
50K
AY
2671
66K
EF
0509
94ge
rsta
ecke
ri M
oraw
itz
E. P
yren
ees,
Fra
nce
HH
, PR
065
DQ
7880
17D
Q78
8195
DQ
7883
35D
Q78
8434
EF
0510
03h
orto
rum
(L
inn
aeu
s)T
osca
na,
Ita
lyP
R e
t al
. 00
5D
Q78
8024
DQ
7882
00A
F49
2987
KA
F49
2853
KE
F05
0999
kore
anu
s (S
kori
kov)
Kan
gwon
do, S
. Kor
eaP.
Tri
poti
n27
7E
F03
2355
AF
4929
69K
AF
4930
36K
AF
4929
02K
EF
0509
95po
rtch
insk
y R
ados
zkow
ski
Art
vin
Pro
v., T
urk
eyP
R e
t al
. 07
2D
Q78
8085
DQ
7882
59D
Q78
8361
DQ
7884
84E
F05
1000
reli
gios
us
(Fri
son
)Q
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 14
1D
Q78
8091
DQ
7882
64D
Q78
8364
DQ
7884
89E
F05
0998
rud
erat
us
(Fab
rici
us)
Val
divi
a P
rov.
, Ch
ile
C. C
arlt
on01
8D
Q78
8096
AF
4929
77K
AF
4930
44K
AF
4929
10K
EF
0510
02se
curu
s (F
riso
n)
Qio
ngl
ai S
h, S
ich
uan
, Ch
ina
SC
et
al.
142
DQ
7881
00D
Q78
8270
DQ
7883
68D
Q78
8493
EF
0509
97su
prem
us
Mor
awit
zQ
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 10
1D
Q78
8112
DQ
7882
84D
Q78
8375
DQ
7885
05E
F05
1004
sush
kin
i (S
kori
kov)
Hon
gyu
an, S
ich
uan
, Ch
ina
SC
et
al.
143
DQ
7881
13A
F49
2921
KA
F49
2988
KA
F49
2854
KE
F05
0996
tich
enko
i (S
kori
kov)
AF
4929
22K
AF
4929
89K
AF
4928
55K
Ml
Mel
anob
ombu
s D
alla
T
orre
(14
/10)
alag
esia
nu
s R
ein
ig*
Art
vin
Pro
v., T
urk
eyP
R e
t al
. 08
5D
Q78
7962
DQ
7881
45D
Q78
8312
DQ
7883
92E
F05
0903
erzu
rum
ensi
s (Ö
zbek
)*A
rtvi
n P
rov.
, Tu
rkey
PR
et
al.
126
DQ
7880
03D
Q78
8183
AY
7394
63H
DQ
7884
23E
F05
0899
form
osel
lus
(Fri
son
)A
F49
2939
KA
F49
3006
KA
F49
2872
K
fris
ean
us
Sko
riko
vQ
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 10
5D
Q78
8015
DQ
7881
93A
Y73
9467
HD
Q78
8432
EF
0509
06in
cert
us
Mor
awit
zE
rzu
rum
Pro
v., T
urk
eyP
R e
t al
. 08
6D
Q78
8035
DQ
7882
11D
Q78
8341
DQ
7884
43E
F05
0901
keri
ensi
s M
oraw
itz
Hon
gyu
an, S
ich
uan
, Ch
ina
SC
et
al.
114
DQ
7880
41D
Q78
8216
AY
7394
74H
DQ
7884
48E
F05
0904
lad
akh
ensi
s R
ich
ards
Aba
, Sic
hu
an, C
hin
aS
C e
t al
. 15
8D
Q78
8043
AY
7395
75H
AY
7394
75H
DQ
7884
50E
F05
0905
lapi
dar
ius
(Lin
nae
us)
�S
an Q
uír
ico,
Ita
lyS
C, J
W00
6D
Q78
8045
DQ
7882
19A
F49
3005
KA
F49
2871
KE
F05
0902
min
iatu
s B
ingh
amH
imac
hal
Pra
desh
, In
dia
M. S
ain
i24
4D
Q78
8059
DQ
7882
33D
Q78
8348
DQ
7884
62E
F05
0908
rufo
fasc
iatu
s S
mit
hH
ongy
uan
, Sic
hu
an, C
hin
aS
C e
t al
. 13
3D
Q78
8098
DQ
7882
69A
Y73
9489
HD
Q78
8492
EF
0509
07si
chel
ii R
ados
zkow
ski
Obe
rgu
rgl,
Au
stri
aS
C, J
W03
4D
Q78
8103
DQ
7882
73D
Q78
8371
DQ
7884
96E
F05
0900
sim
illi
mu
s S
mit
h �
Him
ach
al P
rade
sh, I
ndi
aM
. Sai
ni
243
DQ
7881
04D
Q78
8274
DQ
7883
72D
Q78
8497
EF
0509
09
Md
Men
dac
ibom
bus
Sko
riko
v (1
2/7)
avin
ovie
llu
s (S
kori
kov)
�H
imac
hal
Pra
desh
, In
dia
M. S
ain
i24
2A
Y26
8416
CD
Q78
8155
AY
2684
16C
DQ
7884
00E
F05
1020
con
vexu
s W
ang
Qio
ngl
ai S
h, S
ich
uan
, Ch
ina
SC
et
al.
109
DQ
7879
93D
Q78
8174
DQ
7883
25D
Q78
8415
EF
0510
21d
efec
tor
Sko
riko
vA
F49
2958
KA
F49
3025
KA
F49
2891
K
han
dli
rsch
ian
us
Vog
tA
rtvi
n P
rov.
, Tu
rkey
PR
et
al.
087
DQ
7880
22D
Q78
8198
DQ
7883
37D
Q78
8436
EF
0510
17m
end
ax G
erst
aeck
erG
urg
ltal
, Au
stri
aS
C, J
W01
9D
Q78
8057
AY
7395
84H
AF
4930
24K
AF
4928
90K
EF
0510
19sh
apos
hn
ikov
i S
kori
kov
Art
vin
Pro
v., T
urk
eyP
R e
t al
. 09
9D
Q78
8102
DQ
7882
72D
Q78
8370
DQ
7884
95E
F05
1018
wal
ton
i C
ocke
rell
Qio
ngl
ai S
h, S
ich
uan
, Ch
ina
SC
et
al.
102
DQ
7881
34D
Q78
8302
DQ
7883
85D
Q78
8519
EF
0510
22
Mc
Mu
cid
obom
bus
Krü
ger
(1/1
)m
uci
du
s K
rüge
rE
. Pyr
enee
s, F
ran
ceP
R e
t al
. 05
9D
Q78
8066
AF
4929
35K
AF
4930
02K
AF
4928
68K
EF
0509
34
Su
bgen
us
Spe
cies
Col
lect
ion
loc
alit
yC
olle
ctor
V.#
16S
EF
-1α
Ops
inA
rgK
PE
PC
K
166 S. A. CAMERON ET AL.
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
Ob
Obe
rtob
ombu
s R
ein
ig
(2/1
)ob
erti
Mor
awit
zM
t. K
aila
s, T
ibet
T. B
arde
ll23
4D
Q78
8073
DQ
7882
47D
Q78
8355
DQ
7884
75E
F05
0898
Or
Ori
enta
libo
mbu
s R
ich
ards
(3/
2)fu
ner
ariu
s S
mit
hL
uoj
ish
an, S
ich
uan
, Ch
ina
P. W
illi
ams
270
EF
0323
56E
F03
2378
EF
0323
96E
F03
2415
EF
0510
12h
aem
orrh
oid
alis
Sm
ith
Ch
ian
g M
ai P
rov.
, Th
aila
nd
N. W
arri
t19
1D
Q78
8020
AF
4929
35K
AF
4930
02K
AF
4928
68K
EF
0510
11
Pe
Pre
ssib
ombu
s F
riso
n
(1/1
)pr
essu
s (F
riso
n)
Utt
aran
chal
, In
dia
M. S
ain
i23
9D
Q78
8088
EF
0323
68E
F05
0837
Ps
Psi
thyr
us
Lep
elet
ier
(29/
21)
ash
ton
i (C
ress
on)
�O
ttaw
a, C
anad
aJ.
Wh
itfi
eld
164
DQ
7879
69A
F49
2983
KA
F49
3050
KA
F49
2916
KE
F05
0978
barb
ute
llu
s (K
irby
) �
Upp
lan
d C
o., S
wed
enB
. Ced
erbe
rg07
3D
Q78
7975
DQ
7881
58D
Q78
8318
DQ
7884
03E
F05
0972
boh
emic
us
(Sei
dl)
�E
. Pyr
enee
s, F
ran
ceP
R e
t al
. 05
5D
Q78
7980
AF
4929
25K
AF
4929
92K
AF
4928
58K
EF
0509
79ca
mpe
stri
s (P
anze
r)D
alar
na
Co.
, Sw
eden
B. C
eder
berg
040
DQ
7879
86A
F49
2927
KA
F49
2994
KA
F49
2860
KE
F05
0974
chin
ensi
s (M
oraw
itz)
�Q
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 15
2D
Q78
7988
DQ
7881
70D
Q78
8321
DQ
7884
12E
F05
0982
citr
inu
s (S
mit
h)
�V
irgi
nia
, US
AA
. Dea
ns
170
DQ
7879
90D
Q78
8171
DQ
7883
22A
Y26
7169
KE
F05
0975
core
anu
s (Y
asu
mat
su)
AF
3648
30B
corn
utu
s (F
riso
n)
�N
ingn
an, S
ich
uan
, Ch
ina
T. Y
a27
1E
F03
2353
EF
0323
77E
F03
2395
EF
0324
13E
F05
0984
fern
ald
ae (
Fra
nkl
in)
�N
ew M
exic
o, U
SA
SC
, JW
088
DQ
7880
06A
F49
2926
KA
F49
2993
KA
F49
2859
KE
F05
0969
flav
idu
s E
vers
man
n �
Lap
plan
d C
o., S
wed
enB
. Ced
erbe
rg04
1D
Q78
8010
DQ
7881
89D
Q78
8332
DQ
7884
29E
F05
0970
insu
lari
s (S
mit
h)
Cal
ifor
nia
, US
AH
. Hin
es16
2D
Q78
8038
AF
4929
75K
AF
4930
42K
AF
4929
08K
EF
0509
76m
axil
losu
s K
lug
�K
ayse
ri P
rov.
, Tu
rkey
PR
et
al.
074
DQ
7880
54D
Q78
8227
AY
7394
80H
DQ
7884
58E
F05
0973
nor
vegi
cus
(Spa
rre-
Sch
nei
der)
Dal
arn
a C
o., S
wed
enB
. Ced
erbe
rg08
9D
Q78
8072
DQ
7882
46D
Q78
8354
DQ
7884
74E
F05
0971
quad
rico
lor
(Lep
elet
ier)
�U
ppla
nd
Co.
, Sw
eden
B. C
eder
berg
090
DQ
7880
90D
Q78
8263
DQ
7883
63D
Q78
8488
EF
0509
66ru
pest
ris
(Fab
rici
us)
Upp
lan
d C
o., S
wed
enB
. Ced
erbe
rg00
9D
Q78
8099
AF
4929
28K
AF
4929
95K
AF
4928
61K
EF
0509
83sk
orik
ovi
(Pop
ov)
�A
ba, S
ich
uan
, Ch
ina
SC
et
al.
159
DQ
7881
06D
Q78
8276
DQ
7883
73D
Q78
8499
EF
0509
68su
ckle
yi G
reen
eC
olor
ado,
US
AS
C, J
W09
1D
Q78
8110
DQ
7882
82D
Q78
8374
DQ
7885
03E
F05
0981
sylv
estr
is (
Lep
elet
ier)
Upp
lan
d C
o., S
wed
enB
. Ced
erbe
rg02
0D
Q78
8115
DQ
7882
86D
Q78
8377
DQ
7885
07E
F05
0967
tibe
tan
us
(Mor
awit
z) �
Min
Sh
an, S
ich
uan
, Ch
ina
SC
et
al.
134
DQ
7881
20D
Q78
8290
DQ
7883
78D
Q78
8509
EF
0509
85va
riab
ilis
(C
ress
on)
�M
isso
uri
, US
AS.
Cam
eron
316
AY
2684
19C
DQ
7882
95A
Y26
8397
CD
Q78
8513
EF
0509
77ve
stal
is (
Geo
ffro
y) �
Ken
t, E
ngl
and
P. W
illi
ams
169
DQ
7881
28D
Q78
8297
AY
7394
95H
DQ
7885
15E
F05
0980
Pr
Pyr
obom
bus
Dal
laal
boan
alis
Fra
nkl
in*
Ala
ska,
US
AS
AC
, JB
W25
7E
F03
2343
EF
0323
60E
F03
2380
EF
0323
98E
F05
0813
Tor
re (
43/4
0)ar
den
s S
mit
h �
Dae
-Don
g, S
. Kor
eaD
. Kim
131
DQ
7879
66D
Q78
8149
AF
4930
31K
AF
4928
97K
EF
0508
23av
anu
s (S
kori
kov)
*L
uoj
ish
an, S
ich
uan
, Ch
ina
P. W
illi
ams
272
EF
0323
44E
F03
2365
EF
0323
84E
F03
2402
EF
0508
30be
atic
ola
(Tka
lck
)A
F49
2963
KA
F49
3030
KA
F49
2896
K
bifa
riu
s C
ress
onN
ew M
exic
o, U
SA
M. H
ause
r20
8D
Q78
7977
DQ
7881
60A
F49
3010
KA
F49
2876
KE
F05
0838
bim
acu
latu
s C
ress
onA
rkan
sas,
US
AS.
Cam
eron
218
DQ
7879
78D
Q78
8161
AY
7394
56H
DQ
7884
05E
F05
0847
biro
i V
ogt
Ket
men
Mts
., K
azak
hst
anM
. Hau
ser
210
DQ
7879
79D
Q78
8162
AY
7394
57H
DQ
7884
06E
F05
0825
brod
man
nic
us
Vog
tA
rtvi
n P
rov.
, Tu
rkey
PR
et
al.
077
DQ
7879
84D
Q78
8166
AY
7394
58C
DQ
7884
09E
F05
0818
cali
gin
osu
s (F
riso
n)
Cal
ifor
nia
, US
AH
. Hin
es15
0D
Q78
7985
DQ
7881
68A
F49
3035
KA
F49
2901
KE
F05
0853
cen
tral
is C
ress
onW
ash
ingt
on, U
SA
J. W
hit
fiel
d14
6D
Q78
7987
DQ
7881
69A
Y73
9459
CD
Q78
8411
EF
0508
52
Su
bgen
us
Spe
cies
Col
lect
ion
loc
alit
yC
olle
ctor
V.#
16S
EF
-1α
Ops
inA
rgK
PE
PC
K
Tab
le 1
.C
onti
nu
ed
BUMBLE BEE PHYLOGENY 167
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
cin
gula
tus
Wah
lber
gL
appi
, Fin
lan
dP.
Ras
mon
t21
2D
Q78
7989
AF
4929
48K
AF
4930
15K
AF
4928
81K
EF
0508
12ep
hip
piat
us
Say
Oax
aca,
Mex
ico
R. A
yala
042
DQ
7880
02C
hia
pas,
Mex
ico
M. G
uzm
an19
8D
Q78
8182
AY
7394
62H
DQ
7884
22E
F05
0844
flav
esce
ns
(Sm
ith
)M
ei-f
ang,
Tai
wan
H. T
own
es18
1D
Q78
8009
AF
4929
50K
AF
4930
17K
AF
4928
83K
EF
0508
24fl
avif
ron
s C
ress
on �
Cal
ifor
nia
, US
AJ.
Wh
itfi
eld
095
DQ
7880
11D
Q78
8190
AF
4930
16K
AF
4928
82K
EF
0508
50fr
igid
us
Sm
ith
Ala
ska,
US
AA
. Sch
oll
185
DQ
7880
14D
Q78
8192
AY
7394
66H
DQ
7884
31E
F05
0811
hae
mat
uru
s K
riec
hba
um
er �
Tra
bzon
Pro
v., T
urk
eyP.
Ras
mon
t21
1D
Q78
8019
EF
0323
64E
F05
0829
hu
nti
i G
reen
eW
ash
ingt
on, U
SA
J. W
hit
fiel
d15
1D
Q78
8027
DQ
7882
03A
F49
3045
KA
F49
2911
KE
F05
0840
hyp
nor
um
(L
inn
aeu
s)K
löst
erle
, Au
stri
aS
C, J
W07
8D
Q78
8029
DQ
7882
05A
F49
3013
KA
F49
2879
KE
F05
0826
Qio
ngl
ai S
h, S
ich
uan
,Ch
ina
SC
et
al.
207
EF
0323
59E
F03
2363
EF
0323
83E
F03
2401
EF
0508
27im
pati
ens
Cre
sson
Illi
noi
s, U
SA
H. H
ines
060
DQ
7880
33D
Q78
8209
AF
4930
09K
AF
4928
75K
EF
0508
42in
firm
us
(Tka
lck
) �
Qio
ngl
ai S
h, S
ich
uan
, Ch
ina
SC
et
al.
157
DQ
7880
36D
Q78
8212
AY
7394
71H
DQ
7884
44E
F05
0836
infr
equ
ens
(Tka
lck
)*Q
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 14
0D
Q78
8037
DQ
7882
13A
Y73
9472
HD
Q78
8445
EF
0508
31jo
nel
lus
(Kir
by)
Lap
plan
d C
o., S
wed
enB
. Ced
erbe
rg07
9D
Q78
8039
DQ
7882
14A
Y73
9473
HD
Q78
8446
EF
0508
14la
ppon
icu
s (F
abri
ciu
s)L
appl
and
Co.
, Sw
eden
B. C
eder
berg
103
DQ
7880
46D
Q78
8220
DQ
7883
45D
Q78
8452
EF
0508
45le
mn
isca
tus
Sko
riko
vQ
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 16
1D
Q78
8047
DQ
7882
21A
Y73
9477
HD
Q78
8453
EF
0508
34le
pid
us
Sko
riko
vQ
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 15
5D
Q78
8048
DQ
7882
22A
Y73
9478
HD
Q78
8454
EF
0508
35lu
teip
es R
ich
ards
Pok
har
a, N
epal
S. M
arti
n19
5D
Q78
8052
mel
anop
ygu
s N
ylan
der
Cal
ifor
nia
, US
AH
. Hin
es21
5D
Q78
8055
DQ
7882
29A
F49
3011
KA
F49
2877
KE
F05
0849
mix
tus
Cre
sson
Was
hin
gton
, US
AS
C, J
W02
4D
Q78
8060
DQ
7882
34A
F49
3014
KA
F49
2880
KE
F05
0816
mod
estu
s E
vers
man
nA
ba, S
ich
uan
, Ch
ina
SC
et
al.
160
DQ
7880
63D
Q78
8237
AY
7394
82H
DQ
7884
65E
F05
0820
�S.
Alt
ai, K
azak
hst
anP.
Mar
duly
n23
8E
F03
2358
EF
0323
62E
F03
2382
EF
0324
00E
F05
0821
mon
tico
la S
mit
hE
. Pyr
enee
s, F
ran
ceP
R e
t al
. 17
6D
Q78
8064
DQ
7882
38A
Y73
9483
HD
Q78
8466
EF
0508
48pa
rth
eniu
s R
ich
ards
Him
ach
al P
rade
sh, I
ndi
aM
. Sai
ni
241
DQ
7880
76D
Q78
8250
DQ
7883
57D
Q78
8478
EF
0508
32pe
rple
xus
Cre
sson
Ott
awa,
Can
ada
J. W
hit
fiel
d16
6D
Q78
8079
DQ
7882
54A
F49
3012
KA
F49
2878
KE
F05
0828
pici
pes
Ric
har
dsQ
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 18
0D
Q78
8082
DQ
7882
57A
Y73
9487
HD
Q78
8482
EF
0508
33pr
ator
um
(L
inn
aeu
s)K
löst
erle
, Au
stri
aS
C, J
W07
5D
Q78
8087
AF
4929
66K
AF
4930
33K
AF
4928
99K
EF
0508
19py
ren
aeu
s P
érez
Gu
rglt
al, A
ust
ria
SC
, JW
035
DQ
7880
89D
Q78
8262
AY
7394
88H
DQ
7884
87E
F05
0822
san
der
son
i F
ran
klin
Isle
au
Hau
t, M
ain
e, U
SA
SC
, JW
255
EF
0323
46E
F03
2361
EF
0323
81E
F03
2399
EF
0508
15si
tken
sis
Nyl
ande
rC
alif
orn
ia, U
SA
H. H
ines
144
DQ
7881
05D
Q78
8275
AY
7394
90H
DQ
7884
98E
F05
0817
son
ani
(Fri
son
)*A
F49
2951
KA
F49
3018
KA
F49
2884
K
sylv
icol
a K
irby
�*
New
Mex
ico,
US
AS
C, J
W10
8D
Q78
8116
DQ
7882
87A
Y73
9493
HD
Q78
8508
EF
0508
47te
rnar
ius
Say
Nov
a S
coti
a, C
anad
aS
C, J
W11
6D
Q78
8117
AF
4929
79K
AF
4930
46K
AF
4929
12K
EF
0508
39va
gan
s S
mit
hW
isco
nsi
n, U
SA
J. W
hit
fiel
d04
4D
Q78
8125
DQ
7882
93D
Q78
8380
DQ
7885
12E
F05
0854
van
dyk
ei (
Fri
son
)W
ash
ingt
on, U
SA
J. W
hit
fiel
d14
9D
Q78
8126
DQ
7882
94A
F49
3049
KA
F49
2915
KE
F05
0851
vosn
esen
skii
Rad
oszk
owsk
i �
Was
hin
gton
, US
AJ.
Wh
itfi
eld
112
DQ
7881
33D
Q78
8301
AF
4930
47K
AF
4929
13K
EF
0508
41w
ilm
atta
e C
ocke
rell
*C
hia
pas,
Mex
ico
R. V
anda
me
199
DQ
7881
36D
Q78
8304
AY
7394
96H
DQ
7885
21E
F05
0843
Su
bgen
us
Spe
cies
Col
lect
ion
loc
alit
yC
olle
ctor
V.#
16S
EF
-1α
Ops
inA
rgK
PE
PC
K
168 S. A. CAMERON ET AL.
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
Rh
Rh
odob
ombu
s D
alla
T
orre
(3/
3)ar
men
iacu
s R
ados
zkow
ski
Kay
seri
Pro
v., T
urk
eyP
R e
t al
. 08
0D
Q78
7968
DQ
7881
51D
Q78
8315
DQ
7883
97E
F05
0937
mes
omel
as (
Ger
stae
cker
)S
wit
zerl
and
C. G
erla
ch03
7D
Q78
8058
DQ
7882
31E
F03
2390
EF
0324
14E
F05
0938
pom
oru
m (
Pan
zer)
�E
rzin
can
Pro
v., T
urk
eyP
R e
t al
. 05
3D
Q78
8084
DQ
7882
58D
Q78
8360
DQ
7884
83E
F05
0939
Rb
Rob
ust
obom
bus
Sko
riko
v (7
/7)
ecu
ador
ius
Meu
nie
r �
Aba
nca
y, P
eru
C. R
asm
uss
en13
5D
Q78
8001
DQ
7881
81D
Q78
8329
DQ
7884
21E
F05
0874
hor
tula
nu
s F
ries
e �
Mag
dale
na,
Col
ombi
aJ.
Can
till
o20
0D
Q78
8025
DQ
7882
01A
Y73
9468
HD
Q78
8438
EF
0508
75m
elal
eucu
s H
andl
irsc
hO
xapa
mpa
, Per
uC
. Ras
mu
ssen
173
DQ
7879
60D
Q78
8143
DQ
7883
11D
Q78
8460
EF
0508
76ro
bust
us
Sm
ith
Boy
acá,
Col
ombi
aS.
Cam
eron
050
DQ
7880
93D
Q78
8266
DQ
7883
66E
F03
2405
EF
0508
77tu
cum
anu
s V
ach
alT
ucu
mán
, Arg
enti
na
M. L
uci
a27
6E
F03
2349
EF
0323
67E
F03
2386
EF
0324
04E
F05
0873
vogt
i F
ries
e* �
Aba
nca
y, P
eru
C. R
asm
uss
en17
2D
Q78
8130
DQ
7882
99D
Q78
8383
DQ
7885
17E
F05
0878
volu
cell
oid
es G
riba
do*
San
Jos
e, C
osta
Ric
aP.
Han
son
122
DQ
7881
31A
Y26
7133
KA
Y26
7149
KA
Y26
7165
K
Rc
Ru
bicu
nd
obom
bus
Sko
riko
v (1
/1)
rubi
cun
du
s S
mit
hC
un
din
amar
ca, C
olom
bia
A. P
erez
021
DQ
7880
94B
oyac
á, C
olom
bia
S. C
amer
on20
2D
Q78
8267
DQ
7883
67D
Q78
8491
EF
0508
88
Rf
Ru
fipe
dib
ombu
s S
kori
kov
(2/1
)ex
imiu
s S
mit
hA
lish
an, T
aiw
anJ.
de
Boe
r04
9D
Q78
8005
DQ
7881
86A
Y73
9464
CD
Q78
8426
EF
0509
11
Sx
Sen
exib
ombu
s F
riso
n
(4/2
)bi
colo
ratu
s S
mit
hN
anto
u, T
aiw
anC
. Lin
225
DQ
7879
76A
F49
2971
KA
F49
3038
KA
F49
2904
KE
F05
1005
kuli
nge
nsi
s C
ocke
rell
Zh
ejia
ng
Pro
v., C
hin
aS
C e
t al
. 09
7D
Q78
8042
DQ
7882
17D
Q78
8343
DQ
7884
49E
F05
1006
Sp
Sep
arat
obom
bus
Fri
son
(2/
2)gr
iseo
coll
is (
DeG
eer)
Illi
noi
s, U
SA
H. H
ines
082
DQ
7880
18D
Q78
8196
AF
4930
39K
AF
4929
05K
EF
0508
79m
orri
son
i C
ress
onA
rizo
na,
US
AT.
Sil
lett
014
DQ
7880
65U
tah
, US
AT.
Gri
swol
d19
6D
Q78
8240
DQ
7883
50D
Q78
8468
EF
0508
80
Sb
Sib
iric
obom
bus
Vog
t (5
/4)
asia
ticu
s M
oraw
itz
�H
imac
hal
Pra
desh
, In
dia
M. S
ain
i24
9D
Q78
7970
EF
0323
69E
F03
2387
EF
0324
06E
F05
0896
niv
eatu
s K
riec
hba
um
erK
ayse
ri P
rov.
, Tu
rkey
PR
et
al.
093
DQ
7880
70D
Q78
8244
DQ
7883
53D
Q78
8472
EF
0508
93si
biri
cus
(Fab
rici
us)
Kh
övsg
öl N
uu
r, M
ongo
lia
D S
hep
pard
274
EF
0323
48E
F03
2370
EF
0323
88E
F03
2407
EF
0508
97su
lfu
reu
s F
ries
eK
ayse
ri P
rov.
, Tu
rkey
PR
et
al.
064
DQ
7881
11D
Q78
8283
AY
7394
92H
DQ
7885
04E
F05
0895
vort
icos
us
Ger
stae
cker
*A
ksar
ay P
rov.
, Tu
rkey
PR
et
al.
124
DQ
7881
32D
Q78
8300
DQ
7883
84D
Q78
8518
EF
0508
94
St
Su
bter
ran
eobo
mbu
s V
ogt
(9/7
)ap
posi
tus
Cre
sson
�U
tah
, US
AT.
Gri
swol
d14
5D
Q78
7965
DQ
7881
48D
Q78
8314
DQ
7883
95E
F05
0986
bore
alis
Kir
byM
ain
e, U
SA
SC
, JW
250
DQ
7879
81A
F49
2976
KA
F49
3043
KA
F49
2909
KE
F05
0987
dif
fici
llim
us
Sko
riko
v*H
ongy
uan
, Sic
hu
an, C
hin
aS
C e
t al
. 15
4D
Q78
7998
DQ
7881
77D
Q78
8327
DQ
7884
17E
F05
0990
dis
tin
guen
du
s M
oraw
itz
�E
desb
ecka
, Fin
lan
dC
. Gri
nte
r19
7D
Q78
7999
DQ
7881
80D
Q78
8328
DQ
7884
20E
F05
0988
frag
ran
s (P
alla
s)K
ayse
ri P
rov.
, Tu
rkey
PR
et
al.
061
DQ
7880
12D
Q78
8191
DQ
7883
33D
Q78
8430
EF
0509
92m
elan
uru
s L
epel
etie
rD
zun
gars
kij A
lata
u, K
azak
h.
M. H
ause
r02
2D
Q78
8056
DQ
7882
30A
F49
2990
KA
F49
2856
KE
F05
0991
pers
onat
us
Sm
ith
Hon
gyu
an, S
ich
uan
, Ch
ina
SC
et
al.
138
DQ
7880
81D
Q78
8256
DQ
7883
59D
Q78
8481
EF
0509
93su
bter
ran
eus
(Lin
nae
us)
Upp
lan
d C
o., S
wed
enB
. Ced
erbe
rg04
6D
Q78
8109
DQ
7882
81A
F49
3027
KA
F49
2893
KE
F05
0989
Th
Th
orac
obom
bus
Dal
lad
eute
ron
ymu
s S
chu
lzP
rim
orsk
iy K
rai,
Ru
ssia
K. H
olst
on14
7D
Q78
7997
DQ
7881
76D
Q78
8326
AY
2671
70K
EF
0509
22T
orre
(19
/16)
filc
hn
erae
Vog
t �
Hon
gyu
an, S
ich
uan
, Ch
ina
SC
et
al.
206
DQ
7880
08D
Q78
8188
DQ
7883
31D
Q78
8428
EF
0509
33h
edin
i B
isch
off
Qio
ngl
ai S
h, S
ich
uan
, Ch
ina
SC
et
al.
129
DQ
7880
23D
Q78
8199
DQ
7883
38D
Q78
8437
EF
0509
30h
onsh
uen
sis
(Tka
lck
)A
F49
3029
KA
F49
2962
KA
F49
2895
K
hu
mil
is I
llig
erE
. Pyr
enee
s, F
ran
ceH
H, P
R05
6D
Q78
8026
DQ
7882
02A
Y73
9469
HD
Q78
8439
EF
0509
24im
petu
osu
s S
mit
hX
inm
ian
, Sic
hu
an, C
hin
aP.
Wil
liam
s.28
4E
F03
2350
EF
0323
73E
F03
2391
EF
0324
09E
F05
0927
mlo
kosi
evit
zii
Rad
oszk
owsk
iA
rtvi
n P
rov.
, Tu
rkey
PR
et
al.
081
DQ
7880
61D
Q78
8235
DQ
7883
49D
Q78
8463
EF
0509
17
Su
bgen
us
Spe
cies
Col
lect
ion
loc
alit
yC
olle
ctor
V.#
16S
EF
-1α
Ops
inA
rgK
PE
PC
K
Tab
le 1
.C
onti
nu
ed
BUMBLE BEE PHYLOGENY 169
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
mu
scor
um
(L
inn
aeu
s)Ö
lan
d C
o., S
wed
enB
. Ced
erbe
rg03
3D
Q78
8067
DQ
7882
41D
Q78
8351
DQ
7884
69E
F05
0925
pasc
uor
um
(S
copo
li)
Tos
can
a, I
taly
SC
, JW
023
DQ
7880
77D
Q78
8251
AF
4930
01K
AY
4928
67K
EF
0509
32po
tan
ini
Mor
awit
z*H
ongy
uan
, Sic
hu
an, C
hin
aS
C e
t al
. 11
3D
Q78
8086
DQ
7882
60D
Q78
8362
DQ
7884
85E
F05
0928
pseu
dob
aica
len
sis
Vog
tP
rim
orsk
iy K
ray,
Ru
ssia
K. H
olst
on25
3E
F03
2357
EF
0323
72A
Y26
7155
KA
Y26
7171
KE
F05
0923
rem
otu
s T
kalck
�Q
ion
glai
Sh
, Sic
hu
an, C
hin
aS
C e
t al
. 19
2D
Q78
8092
DQ
7882
65D
Q78
8365
DQ
7884
90E
F05
0929
rud
erar
ius
(Mü
ller
)O
berg
urg
l, A
ust
ria
SC
, JW
047
DQ
7880
95A
F49
2932
KA
F49
2999
KA
F49
2865
KE
F05
0920
sch
ren
cki
Mor
awit
z �
W. K
hen
tey,
N. M
ongo
lia
M. M
üh
len
berg
298
AF
3648
28B
AF
4929
33K
AF
4930
00K
AF
4928
66K
EF
0509
31sy
lvar
um
(L
inn
aeu
s)U
ppsa
la C
o., S
wed
enS
C, J
W11
0D
Q78
8114
DQ
7882
85D
Q78
8376
DQ
7885
06E
F05
0918
velo
x (S
kori
kov)
*A
rtvi
n P
rov.
, Tu
rkey
PR
et
al.
094
DQ
7881
27D
Q78
8296
DQ
7883
81D
Q78
8514
EF
0509
21ve
tera
nu
s (F
abri
ciu
s)Ju
ra, F
ran
ceA
. Sch
oll
187
DQ
7881
29D
Q78
8298
DQ
7883
82D
Q78
8516
EF
0509
19zo
nat
us
Sm
ith
Aks
aray
Pro
v., T
urk
eyP
R e
t al
. 06
3D
Q78
8138
DQ
7883
06D
Q78
8386
DQ
7885
22E
F05
0926
Tr
Tri
corn
ibom
bus
Sko
riko
v (3
/3)
atri
pes
Sm
ith
Zh
ejia
ng
Pro
v., C
hin
aP.
Mei
hu
a06
6D
Q78
7971
DQ
7881
53D
Q78
8316
DQ
7883
99E
F05
0964
imit
ator
Pit
tion
i �
Gu
izh
ou P
rov.
, Ch
ina
M. Y
un
028
DQ
7880
32D
Q78
8208
DQ
7883
39D
Q78
8441
EF
0509
65tr
icor
nis
Rad
oszk
owsk
iP
rim
orsk
iy K
ray,
Ru
ssia
K. H
olst
on14
8D
Q78
8121
AF
4929
37K
AF
4930
04K
AF
4928
70K
EF
0509
63
OU
TG
RO
UP
ST
rib
eS
pec
ies
Api
ni
Api
s d
orsa
ta F
abri
ciu
sB
anga
lore
, In
dia
S. C
amer
on32
1L
2289
3C2
AY
2671
46K
AF
0917
33M
AY
2671
78K
EF
0510
28A
pis
mel
life
ra L
inn
aeu
sA
rkan
sas,
US
AS.
Cam
eron
320
L22
891
AF
0152
67D
AF
0917
32M
EF
0323
97E
F05
1030
Eu
glos
sin
iE
ugl
ossa
im
peri
alis
Coc
kere
llS
ão P
aulo
, Bra
zil
S. C
amer
on31
9A
J581
085M
iA
Y26
7144
KA
Y26
7160
KA
Y26
7176
K
Eu
laem
a bo
livi
ensi
s (F
ries
e)L
a P
az P
rov.
, Bol
ivia
M. H
ause
r21
3D
Q78
8139
DQ
7883
07D
Q78
8387
DQ
7885
23E
F05
1029
Mel
ipon
ini
Gen
iotr
igon
a th
orac
ica
Sm
ith
Ku
ala
Mu
da, M
alay
sia
R. H
epbu
rn30
3D
Q78
8140
DQ
7883
08D
Q78
8388
DQ
7885
24E
F05
1023
Het
erot
rigo
na
itam
a C
ocke
rell
Ku
ala
Mu
da, M
alay
sia
R. H
epbu
rn30
4D
Q78
8141
DQ
7883
09D
Q78
8389
DQ
7885
25E
F05
1024
Hyp
otri
gona
gri
bodo
i (M
agre
tti)
Bw
indi
, Uga
nda
R. K
ajob
e32
2D
Q79
0440
RD
Q81
3121
RD
Q81
3199
RD
Q81
3043
RE
F05
1026
Lio
trig
ona
mah
afal
ya
Bro
oks
& M
ich
ener
Mah
ajan
ga P
rov.
, Mad
agas
car
B. F
ish
erD
Q79
0442
RD
Q81
3126
RD
Q81
3204
RD
Q81
3048
R
Ple
beia
fro
nta
lis
Fri
ese
Pu
ebla
, Mex
ico
M. V
illa
mar
323
DQ
7904
59R
DQ
8131
38R
DQ
8132
16R
DQ
8130
59R
EF
0510
27T
rigo
na
amaz
onen
sis
Du
cke
Mad
re d
e D
ios,
Per
uS.
Cam
eron
178
DQ
7881
42D
Q78
8310
DQ
7883
90D
Q78
8526
EF
0510
25
Su
bgen
us
Spe
cies
Col
lect
ion
loc
alit
yC
olle
ctor
V.#
16S
EF
-1α
Ops
inA
rgK
PE
PC
K
Th
e tw
o-le
tter
acr
onym
(in
bol
d)
in c
olu
mn
1 d
enot
es t
he
abbr
evia
ted
subg
enu
s n
ame.
Nu
mbe
rs in
par
enth
eses
fol
low
ing
subg
ener
ic n
ames
ref
er t
o to
tal n
um
ber
of s
peci
es r
ecog
niz
ed b
y W
illi
ams
(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.
170 S. A. CAMERON ET AL.
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
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,
BUMBLE BEE PHYLOGENY 171
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
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
172 S. A. CAMERON ET AL.
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
(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 The Linnean Society of London,
Biological Journal of the Linnean Society,
2007,
91
, 161–188
Fig
ure
1.
Ph
ylog
eny
of
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idobom
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Tric
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lobom
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us
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abom
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igona
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igona
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Psithyr
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LFSF
NW
174 S. A. CAMERON ET AL.
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
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
BUMBLE BEE PHYLOGENY 175
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
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.
176 S. A. CAMERON ET AL.
© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 161–188
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
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Cf
SFSF
LF
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BiCf
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C. opsinB. EF-1a
D. ArgK E. PEPCK
A. Consensus
0.81
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0.85
0.970.780.63
0.801.0
0.670.99
0.99
1.0
0.83
1.00.90
1.0
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1.0
1.0 1.0
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BUMBLE BEE PHYLOGENY 177
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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.
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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
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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.
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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
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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.
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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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u∞∞
u∞∞
u∞∞
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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).
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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).
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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
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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.
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