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Article history:
and has over 50 described species. Melipona, like Apis, possesses the remarkable ability to use represen-
tational communication to indicate the location of foraging patches. Although Melipona has been the sub-
(von Frisch, 1967; Michener, 1974; Dyer, 2002; Nieh, 2004). All
Apis use a form of referential communication known as the waggle
dance, whereby returning foragers inform colony members about
newly discovered resource sites (von Frisch, 1967; Seeley, 1995;
Dyer, 2002). The waggle dance communicates distance and direc-
tion (von Frisch, 1967; Gould, 1976; Michelsen et al., 1992; Esch
et al., 2001; Dyer, 2002; Sherman and Visscher, 2002). The commu-
ences in the ability to communicate different spatial dimensions
among species of Melipona, which correlate well with spatial distri-
bution of ?oral resources in their current environment (Nieh et al.,
2003). Whether and how this information is actually utilized by
nest mates is still a subject of intense investigation, as was the case
for decades in Apis.
Although the genus has been the focus of behavioral, genetic,
ecological, and pollination studies (Roubik, 2006), only partial phy-
logenetic analyses have been carried out to date (Rego, 1990; Costa
et al., 2003; Fernandes-Salom?o et al., 2005; Rasmussen and Cam-
eron, 2010). The stingless bee genus Melipona is clustered within
* Corresponding author. Present address: University of California, Berkeley, 137
Mulford Hall #3114, Berkeley, CA 94720-3114, USA.
Molecular Phylogenetics and Evolution xxx (2010) xxx?xxx
Contents lists availab
ne
.e l
ARTICLE IN PRESSE-mail address: sramirez@post.harvard.edu (S.R. Ram?rez).1. Introduction
The stingless bee genus Melipona contains at least 50 species of
medium-sized (8?15 mm), robust, and often hirsute bees inhabit-
ing forests of tropical America, from Mexico to Argentina (Schwarz,
1932; Michener, 2007). Most species of Melipona inhabit lowland
wet forests, with the greatest species diversity concentrated in
the Amazon Basin (Moure and Kerr, 1950). These bees are highly
eusocial, which means they exhibit reproductive division of labor,
cooperative brood care, and overlap of generations (Wilson, 1971).
Similar to honey bees (Apis), Melipona are remarkable for in-
sects, in their ability to recruit nest mates to speci?c foraging sites
nication mechanisms of Melipona are less studied, but experimen-
tal evidence indicates functional referential communication in
some species (Esch, 1967; Aguilar and Brice?o, 2002; Nieh,
2004), but not in others (Hrncir et al., 2006). Upon returning to
the nest, successful M. panamica and M. seminigra foragers may
perform short piloting ?ights outside of the nest in the direction
of the resource (Nieh, 1998; Nieh and Roubik, 1998), while inside
the nest, they produce sound pulses while distributing food sam-
ples to potential recruits (Esch, 1967; Nieh, 2004). The average
duration of sound pulses correlates with, and thus potentially en-
codes, distance to food sources relative to the location of the nest
(Esch, 1967; Nieh and Roubik, 1998). Additionally, there are differ-Accepted 20 April 2010
Available online xxxx
Keywords:
Relaxed molecular clock
Penalized likelihood
Eusociality
Stingless bees
Honey bees
Melipona
Apis
Referential communication1055-7903/$ - see front matter 2010 Elsevier Inc. A
doi:10.1016/j.ympev.2010.04.026
Please cite this article in press as: Ram?rez, S.R.,
Evol. (2010), doi:10.1016/j.ympev.2010.04.026ject of numerous behavioral, ecological, and genetic studies, the evolutionary history of this genus
remains largely unexplored. Here, we implement a multigene phylogenetic approach based on nuclear,
mitochondrial, and ribosomal loci, coupled with molecular clock methods, to elucidate the phylogenetic
relationships and antiquity of subgenera and species of Melipona. Our phylogenetic analysis resolves the
relationship among subgenera and tends to agree with morphology-based classi?cation hypotheses. Our
molecular clock analysis indicates that the genus Melipona shared a most recent common ancestor at
least 14?17 million years (My) ago. These results provide the groundwork for future comparative anal-
yses aimed at understanding the evolution of complex communication mechanisms in eusocial Apidae.
2010 Elsevier Inc. All rights reserved.Received 30 March 2009
Revised 2 April 2010
Stingless bees (Meliponini) constitute a diverse group of highly eusocial insects that occur throughout
tropical regions around the world. The meliponine genus Melipona is restricted to the New World tropicsA molecular phylogeny of the stingless b
Santiago R. Ram?rez a,*, James C. Nieh b, Tiago B. Quen
Naomi E. Pierce a
aMuseum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, M
bDivision of Biological Sciences, Section of Ecology, Behavior, and Evolution, University
c Smithsonian Tropical Research Institute, MRC 0580-12, Unit 9100 Box 0948, DPO AA 3
d Faculdade de Filoso?a, Ci?ncias e Letras de Ribeir?o Preto, Universidade de S?o Paulo,
a r t i c l e i n f o a b s t r a c t
Molecular Phyloge
journal homepage: wwwll rights reserved.
et al. A molecular phylogeny ofgenus Melipona (Hymenoptera: Apidae)
l a, David W. Roubik c,d, Vera L. Imperatriz-Fonseca d,
138, USA
lifornia, San Diego, 0116, La Jolla, CA 92093, USA
-9998, USA
ir?o Preto, 140400-901 S?o Paulo, Brazil
le at ScienceDirect
tics and Evolution
sevier .com/ locate /ympevthe stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.
nuclear EF1-a (1.2 kb), ArgK (0.7 kb), and Pol-II (0.8 kb). DNA
tions assumed unordered. We performed 100 random addition se-
2000), this fossil exhibits synapomorphic characters that unambig-
uously place it within crown Meliponini. Thus, we used its age as a
neti
ARTICLE IN PRESSquences using the TBR algorithm, and estimated node support via
non-parametric bootstrapping (100 replicates). A Maximum Likeli-
hood (ML) phylogenetic analysis was performed in the software
package GARLI (Zwickl, 2006) with model parameters estimated
over the speci?ed number of runs. Bootstrap support values were
estimated in GARLI with 100 heuristic tree searches using the same
parameters as those implemented during tree searches. Addition-
ally, Bayesian analyses were implemented in the software package
MrBayes v3.1.1 (Ronquist and Huelsenbeck, 2003). Bayesian treewas extracted from individual bee specimens from either leg or
thoracic muscle tissue using Qiagen DNA Extraction Kits (Qiagen
Inc., Valencia, California). Polymerase Chain Reactions (PCRs) were
carried on a Bio-Rad DNA Engine Dyad Peltier thermal cycler (Bio-
Rad Laboratories Inc., Hercules, California) in 25 lL reactions with
2.5 mmol/L MgCl2, 2.5 mmol/L PCR buffer, and Taq polymerase
(Qiagen Inc., Valencia, California) using various primer pairs (Dan-
forth et al., 2004; Supplementary Table 1). We puri?ed PCR prod-
ucts by incubating samples at 37 C for 35 min using Escherichia
coli Exonuclease I enzyme (New England Biolabs, Hanover, Mary-
land) and subsequently raising the temperature to 80 C for
20 min. Puri?ed products were cycle-sequenced using BigDye?
Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Bio-
systems Inc., Foster City, California). Samples were directly se-
quenced on an Applied Biosystems Inc., 3100 Genetic Analyzer
(Applied Biosystems Inc., Foster City, CA). Both forward and reverse
strands were sequenced for each of the ?ve markers; complemen-
tary strands were assembled using the software Sequencher? v4.2
(Gene Codes Corp., Ann Arbor, MI).
All major lineages within Melipona and Apis were sampled for
this study, including four subgenera, 35 species, and 51 individuals
of approximately 50 described species of Melipona representing all
main species groups, and three subgenera, six species, and 10 indi-
viduals of Apis. Additionally, we sampled 30 taxa within the corbi-
culate bees, including bumble bees, stingless bees, and orchid bees,
and two outgroups (Epicharis and Centris). We include a total of 88
terminals. GenBank accession numbers are provided in Supple-
mentary Table 3.
2.2. Phylogenetic analyses
A single DNA matrix containing ?ve loci was assembled using
MacClade v4.06 (Maddison and Maddison, 2003). Parsimony anal-
yses were implemented in the software package Paup v4.0b
(Swofford, 2003) with all characters weighted equally and transi-the Neotropical Meliponini (Rasmussen and Cameron, 2010), and
its monophyly is well supported (Rego, 1990; Costa et al., 2003;
Fernandes-Salom?o et al., 2005). A recent global phylogenetic anal-
ysis of the entire tribe Meliponini supported a Miocene (24 My)
origin for Melipona, but only 20 of the 50 described species were
sampled and the internal relationships were not well resolved
(Rasmussen and Cameron, 2010). Here, we present the ?rst com-
prehensive species-level phylogenetic analysis of Melipona coupled
with a molecular clock analysis.
2. Materials and methods
2.1. DNA sequencing and taxonomic sampling
We sequenced 4.5 kb of DNA from ?ve different fragments
including mitochondrial CO1 (1.2 kb), ribosomal 16S (0.6 kb),
2 S.R. Ram?rez et al. /Molecular Phylogesearches were made assuming both single (GTR+C+I) and multiple
models of sequence evolution for each locus (see Supplementary
Table 2). In addition, we ran a tree search where models of se-
Please cite this article in press as: Ram?rez, S.R., et al. A molecular phylogeny of
Evol. (2010), doi:10.1016/j.ympev.2010.04.026minimum age calibration for all Meliponini. The placement of E.
moronei within extant (crown) Euglossa is justi?ed by the presence
of elongated mouthparts, labrum shape, and pubescence (Engel,
1999b). The phylogenetic position of A. lithohermaea within extant
Apis is justi?ed by the enlarged body size, elongated metabasitar-
sus, wing venation, and infuscated wing membrane (Engel,
2006). The placement of P. dominicana within extant Neotropical
Meliponini is justi?ed by the short trapezoidal clypeus, triangular
shape of forewing medial cell, and shape of tibiae and basitarsi
(Camargo et al., 2000). The concordance among calibration points
was assessed with the cross-validation method (Near et al., 2005;
Supplementary Fig. 3). Since our phylogenetic sampling included
divergent extant lineages within Apis (Raf?udin and Crozier,
2007), Euglossa (Ram?rez et al., in press) and Neotropical Melipo-quence evolution were partitioned by codon positions, with
parameters estimated separately for ?rst, second, and third codon
positions of nuclear coding genes. Markov chain Monte Carlo
(MCMC) searches were run for 10,000,000 generations, sampling
every 1000 generations for a total of 10,000 trees; model parame-
ters were estimated during the run. Three parallel runs were car-
ried, and for each run one unheated and three incrementally
heated chains were used. We checked for convergence within tree
searches by plotting tree likelihood values against the number of
generations, and among searches by comparing resulting topolo-
gies. Bayesian posterior probabilities were estimated as the pro-
portion of trees containing each node over the trees sampled
during runs. The trees corresponding to the ?rst 1000 generations
were discarded (??burn-in?).
2.3. Divergence time estimation
Divergence times were estimated using a fully resolved topol-
ogy obtained by applying a 50% Majority-Rule (MR) consensus to
all the trees obtained from a Bayesian phylogenetic search; the
remaining polytomies (six) were resolved randomly using the R
software package APE v2.3. Using a Likelihood Ratio Test (LRT)
we estimated this tree had a signi?cantly lower score value (nL
39222.08) when a molecular clock was enforced than when the
assumption was relaxed (nL 38987.29). We calculated branch
lengths on the 50% MR consensus tree via maximum likelihood
in the software package Paup, optimized under the model of se-
quence evolution GTR+C+I (molecular clock not enforced). Node
divergence times were estimated with Penalized Likelihood (PL)
using the Truncated-Newton algorithm in the software package
r8s v1.71 (Sanderson, 1997). Mean ages ? SD were calculated using
non-parametric bootstrapping.
We used two sets of calibration ages, corresponding to the
youngest and oldest estimates of the ages of the fossils used as
node age constraints. A total of ?ve different ages were used to cal-
ibrate our molecular clock trees (indicated by letters in Fig. 1): A,
maximum root age (80?100 My, based on oldest stem bee fossil
(Poinar and Danforth, 2006) and molecular clock analysis done
by Hines (2008)); B, Cretotrigona prisca (65?70 My, Michener and
Grimaldi, 1988; Engel, 2000) used as a minimum age calibration;
C, Euglossa moronei (15?20 My, Engel, 1999b) used as a minimum
age calibration; D Apis lithohermaea (14?16 My, Engel, 2006) used
as a minimum age calibration; and E, Proplebeia dominicana (15?
20 My, Wille and Chandler, 1964; Camargo et al., 2000) used as a
minimum age calibration. Although the age of C. prisca has been
the subject of controversy (Michener and Grimaldi, 1988; Engel,
cs and Evolution xxx (2010) xxx?xxxnini (Rasmussen and Cameron, 2010) we used fossil ages as mini-
mum age constraints, even though in some cases lineage sampling
was incomplete (e.g. Euglossa).
the stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.
neti
ARTICLE IN PRESSS.R. Ram?rez et al. /Molecular Phyloge3. Results and discussion
3.1. Phylogenetic relationships
Our maximum parsimony, maximum likelihood, and Bayesian
phylogenetic analyses, based on ?ve loci, resolved relationships
91
88
96
100
100
100
67
73
100
100
61
62
100
100
100
Parsimony bootstrap
100
100
76
94
100
100
94
100
99
92
*
100
100
Likelihood bootstrap *
*
B
A
Fig. 1. Strict consensus cladogram of 32 equally short maximum parsimony trees showin
asterisks denote unavailable values. Five different ages were used to calibrate a molecula
Hines (2008) and Poinar and Danforth (2006)); B, minimum age constraint, Cretotrigon
constraint, Euglossa moronei (15?20 My, Engel, 1999b); D, minimum age constraint, Apis
dominicana (15?20 My, Wille and Chandler, 1964; Camargo et al., 2000).
Please cite this article in press as: Ram?rez, S.R., et al. A molecular phylogeny of
Evol. (2010), doi:10.1016/j.ympev.2010.04.02675
76
M. panamica MP1
M. costaricensis MP14
Melipona melanopleura MP12
M. sp. (male) MP127
M. solani MP86
cs and Evolution xxx (2010) xxx?xxx 3within and between Melipona, Apis, and related clades of corbicu-
late bees. We obtained well-resolved and supported phylogenetic
trees (Figs. 1 and 2) that are congruent with each other under dif-
ferent optimization schemes and model parameters (Figs. 1, 2 and
Supplementary Figs. 1, 2). Parsimony analyses yielded 32 shortest
trees (TL = 6344), with a strict consensus almost identical to a max-
58
100 81
76 87
97 86
95
100
100
100
59
57
93 87
100
100
99
97
52 100
50
98
100 99
73
97
53
95
100
100 100
100
100
67 95
80
99
93
97
92
51
51 70
69
*
100
100
94
95 91
100
93
100
71
92
100
100 100
100
67
100
76
96
99
100
100
100
68
76
100
100
*
*
*
*
*
*
*
Nannotrigona perilampoides
Euglossa asarophora
M. quadrifasciata anth. MP23
Euglossa villosa
M. crinita MP92
Trigona sp.
M. aff. costaricaensis MP21
Geotrigona kraussi
M. manda?aia MP81
M. quinquefasciata MP80
Bombus vagans
A. dorsata
Friesella schrotkyii
A. cerana
M. captiosa MP43
Eulaema peruviana
M. favosa favosa MP19
M. compressipes MP37
M. sp (eburnea group) MP85
M. quadrifasciata anth. MP76
M. grandis MP128
M. grandis MP97
M. amazonica MP94
Cephalotrigona zexmeniae
Apis cerana
M. interrupta MP44
M. crinita MP32
A. cerana
Euglossa decorata
M. sp. (eburnea group) MP93
Nogueirapis mirandula
Meliponula bocandei
A. florea
M. beecheii MP18
M. triplaridis MP8.1
A. koschevnikovi
M. seminigra atrofulva MP38
Scaptotrigona barrocoloradensis
Exaerete azteca
Plebeia franki
M. illustris MP46a
Frieseomelitta silvestrii
Bombus transversalis
M. aff. costaricensis MP33
M. grandis MP22
Eufriesea caerulescens
Cephalotrigona sp.
Centris sp.
M. sp (M de Dios) MP70
Scaptotrigona barrocoloradensis
M. sp. (male) MP96
M. flavolineata MP49
M. sp. (queen) MP91
M. scutellaris MP78
M. asilvai MP82
Scaptotrigona polysticta
M. lateralis MP45
Tetragonisca angustula
M. rufiventris MP24
M. melanoventer MP34
M. bicolor MP83
Epicharis sp.
A. andreniformis
Aglae caerulea
M. nebulosa MP48
M. marginata MP79
M. fuscopilosa MP35
M. n. sp. MP98
M. sp. (crinita group) MP90
M. ogliviei MP41
M. fuliginosa MP42
A. mellifera
Euglossa mixta
M. micheneri MP39
A. koschevnikovi
Meliwillea bivea
M. fulva MP15
M. fallax MP13
Lestrimelitta sp.
M. illota MP95
M. rufiventris rufiventris MP77
M. interrupta MP20
M. flavolineata MP72
Apini
Euglossini
Bom
bini
outgroups
M
eliponini
E
D
C
g both maximum likelihood and maximum parsimony parametric bootstrap values;
r clock (indicated by letters): A, maximum root age (80?100 My, estimated based on
a prisca (65?70 My, Michener and Grimaldi, 1988; Engel, 2000); C, minimum age
lithohermaea (14?16 My, Engel, 2006); and E, minimum age constraint, Proplebeia
the stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.
neti
ARTICLE IN PRESS4 S.R. Ram?rez et al. /Molecular Phylogeimum likelihood analysis (Fig. 1). Bayesian phylogenetic analyses
also yielded well-supported trees that varied little whether using
single (GTR+C+I), or partitioned (locus-speci?c and codon-speci?c)
models of sequence evolution (Supplementary Figs. 1 and 2). All
phylogenetic methods returned (i) Melipona as sister to the other
Fig. 2. Relaxed-clock chronogram of Melipona and related groups; the tree correspon
Divergence times were calculated (in millions of years, My) using the software package r8
single model of sequence evolution was ?tted to all loci (GTR+C+I) to estimate branch
Please cite this article in press as: Ram?rez, S.R., et al. A molecular phylogeny of
Evol. (2010), doi:10.1016/j.ympev.2010.04.026cs and Evolution xxx (2010) xxx?xxxNeotropical meliponine taxa included in our study (14 genera);
(ii) Melipona + Neotropical Meliponini as sister to the African
stingless bee genus Meliponula; and (iii) all stingless bees (Melipo-
nini) + bumble bees (Bombini) as a monophyletic clade. These re-
sults concord with previous molecular studies (Cameron and
ds to the maximum a posteriori topology resulting from a Bayesian tree search.
s v1.71. Node bars represent the 95% con?dence intervals of the age of each node. A
lengths. Statistics for numbered nodes are indicated in Table 1.
the stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.
quent molecular clock analyses, we used these two alternative tree
topologies. The results from our PL analysis suggest that whereas
extant Apis shared a recent common ancestor during the Oligocene,
29 ? 2 to 34 ? 2 My ago, Melipona shared a most recent common
ancestor during the Miocene, 14 ? 1 to 17 ? 1 My ago, depending
on whether we use the oldest or youngest ages of the fossil calibra-
tions (Table 1). These time estimates varied little when using either
of the two alternative models of sequence evolution (Table 1) or
the two alternative topologies (data not shown).
Melipona is one of the two largest (species rich) genera in the
highly eusocial stingless bees (Plebeia is the other genus). Melipona
is also exclusively Neotropical, and its origin is not known (Ras-
mussen and Cameron, 2010). The few endemic species on islands
in both the Lesser Antilles and Paci?c Panama (Camargo and Pedro,
2007) indicate relicts of past mainland connections during Mio-
cene times (Roubik and Camargo, unpublished data). Our molecu-
lar clock analysis coincides with this scenario.
Our fossil-calibrated molecular clock provides an age estimate
for the origin of Apis, Melipona, and the main clades of corbiculate
Apidae. This provides a temporal framework with which to esti-
mate the antiquity of referential communication. Although the
genus Apis has an extensive fossil record, with the oldest fossil dat-
ing to the Oligocene (25 My old) (Engel, 1998, 1999a, 2006; Engel
et al., 2009), all honey bee fossils known to date (with the excep-
tion of A. lithohermaea) are stem relatives of extant Apis (Engel,
netics and Evolution xxx (2010) xxx?xxx 5
ARTICLE IN PRESSMardulyn, 2001; Rasmussen and Cameron, 2007, 2010; Kawakita
et al., 2008). In a recent study by Rasmussen and Cameron
(2010), Melipona was recovered as sister to most Neotropical gen-
era, except Celetrigona, Dolichotrigona, Trigonisca, and Leurotrigona.
Although our study did not include these taxa, our results are con-
gruent with their hypothesis about the placement of Melipona. The
placement of both honey bees (Apini) and orchid bees (Euglossini)
were inconsistent in our study, depending on methodology (Sup-
plementary Figs. 1 and 2). The parsimony and codon-partitioned
Bayesian analyses supported the topology of (Euglossini (Apini,
(Bombini, Meliponini))), whereas the Likelihood, Bayesian (opti-
mized with both single and gene-partitioned models) supported
the topology of ((Euglossini, Apini), (Bombini, Meliponini)).
Although our analyses recovered the clade Apini + Euglossini as
monophyletic only in some analyses, we obtained strong support
for the clade Bombini + Meliponini, a grouping that has been con-
troversial (Kawakita et al., 2008). Bombini is primarily boreal and
temperate in distribution and Meliponini is restricted to tropical
latitudes. Overall, our results differ from earlier morphology-based
hypothesis about the relationships of corbiculate bees (Michener,
1944; Engel, 2001; Schultz et al., 2001) and, perhaps not surpris-
ingly, support hypotheses based on molecular data (Cameron and
Mardulyn, 2001; Kawakita et al., 2008).
Our phylogenetic analyses indicate that three of the four sub-
genera recognized by morphology within Melipona (Melikerria,
Melipona s. str., and Michmelia) are monophyletic, but one (Eome-
lipona) is polyphyletic (Fig. 2). All Melipona species currently unas-
signed to a speci?c subgenus (Incertae sedis)?except M. fuliginosa
(Camargo and Pedro, 2007, 2008)?form a monophyletic clade, sis-
ter to the subgenus Michmelia (Fig. 2). In our MP analysis, M.
amazonica was sister to the rest of species in the genus, but this
placement was supported by a low bootstrap value (67). On the
other hand, our Bayesian analysis supported the placement of M.
amazonica as sister to M. marginata and M. bicolor, which was sup-
ported by a high Bayesian posterior probability (98). M. amazonica
constitutes a problematic taxon and additional gene fragments
may be required to resolve its placement. Additionally, some of
the internal branches within Melipona were not resolved or were
not well supported. We note that multiple branches within Melip-
ona are relatively short, particularly in the subgenus Michmelia and
thus a rapid lineage diversi?cation may explain the observed low
support values. The results from our phylogenetic analysis may
guide future taxonomic studies, particularly on the delineation of
subgenera and assignment of unplaced species.
Our phylogenetic analyses agree with the proposed and widely
accepted hypothesis of the internal relationships of honey bees:
(Micrapis, (Megapis, Apis s. str.)) (Arias and Sheppard, 1996, 2005;
Oldroyd and Wongsiri, 2006; Raf?udin and Crozier, 2007) and con-
cur with molecular phylogenetic studies of the corbiculate bee
tribes based on molecular data (Cameron and Mardulyn, 2001;
Thompson and Oldroyd, 2004; Kawakita et al., 2008).
3.2. Molecular clock analysis
We performed molecular clock analyses calibrated with ?ve dif-
ferent fossil ages using Penalized Likelihood (PL). Because previous
phylogenetic analyses (and our own results) have produced uncer-
tainty in the placement of Apini and Euglossini, we used two alter-
native tree topologies that resulted from our Bayesian analyses
(Supplementary Fig. 1) that were produced by applying both single
and partitioned models of sequence evolution. The main difference
between these two alternative topologies was in the relative posi-
tion of Apis and Euglossini, where Apis was recovered sister to
S.R. Ram?rez et al. /Molecular PhylogeEuglossini (single model of sequence evolution), and Euglossini
was recovered sister to the remaining corbiculate bees (gene and
codon partitions). To account for this uncertainty in our subse-
Please cite this article in press as: Ram?rez, S.R., et al. A molecular phylogeny of
Evol. (2010), doi:10.1016/j.ympev.2010.04.0262006). By implementing molecular clocks, we show that extant
Apis likely shared a most recent ancestor during the Eocene?Oligo-
cene (29?33 My ago), whereas Melipona appears to have shared a
common ancestor more recently, during the Miocene (14?17 My
ago). Our age estimates for the most recent common ancestor of
Melipona differ from those obtained by Rasmussen and Cameron
(2010), which suggested that living members of genus shared an
ancestor 25 My ago. In their analysis, Rasmussen and Cameron
(2010) speci?ed a maximum age for the root node (Meliponini)
of 125 My based on the oldest fossil angiosperms (most bees, ex-
cept roughly 20 meliponine species (Lestrimelitta, Cleptotrigona,
and the Trigona hypogea group) depend on ?owering plants for
feeding. We used a different age estimate (80?100 My) for the
Table 1
Mean age estimates (in millions of years, My) of major clades of corbiculate bees
calculated via Penalized Likelihood (PL) optimized with a single model of sequence
evolution (GTR+C+I) and a partitioned (locus-speci?c) model of sequence evolution.
Tree branch lengths were calculated with maximum likelihood under the substitution
model GTR+C+I using a 50% majority-rule consensus tree obtained in the Bayesian
tree searches; SD were calculated via non-parametric bootstrapping. Divergence
times for nodes that collapsed in the 50% majority-rule consensus are denoted by
??NA?.
Node Younger calibrations Older calibrations
GTR+C+I
(l ? SD)
Locus-
speci?c
(l ? SD)
GTR+C+I
(l ? SD)
Locus-
speci?c
(l ? SD)
Apis 29.92 ? 1.51 30.57 ? 1.57 33.18 ? 1.67 33.84 ? 1.72
Melipona 15.43 ? 0.89 14.56 ? 0.82 17.21 ? 0.98 16.21 ? 0.91
Euglossini 20.25 ? 1.27 20.89 ? 1.34 22.49 ? 1.33 23.16 ? 1.45
Neotropical
Meliponini
32.92 ? 1.82 33.29 ? 1.79 36.73 ? 1.91 37.05 ? 1.91
1 6.34 ? 0.48 6.32 ? 0.44 7.07 ? 0.52 7.03 ? 0.48
2 8.49 ? 0.65 8.73 ? 0.65 9.47 ? 0.71 9.72 ? 0.72
3 11.66 ? 0.73 12.23 ? 0.78 13.00 ? 0.80 13.62 ? 0.86
4 8.29 ? 0.82 8.22 ? 0.88 9.25 ? 0.90 9.15 ? 0.98
5 NA 10.60 ? 0.83 NA 11.80 ? 0.92
6 32.04 ? 2.51 31.69 ? 2.44 35.37 ? 2.77 34.91 ? 2.69
7 17.27 ? 1.42 17.51 ? 1.49 19.16 ? 1.58 19.40 ? 1.65
8 22.32 ? 1.48 22.68 ? 1.54 24.76 ? 1.65 25.12 ? 1.72
9 18.56 ? 1.80 18.89 ? 1.79 20.59 ? 1.99 20.93 ? 1.98
10 77.62 ? 1.14 75.89 ? 0.98 85.24 ? 1.37 83.14 ? 1.17
11 83.87 ? 1.36 81.76 ? 1.23 92.76 ? 1.62 90.20 ? 1.47
the stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.
in Apis. This study should guide future comparative analyses of ref-
neti
ARTICLE IN PRESSerential communication and of other aspects of life history evolu-
tion in Melipona and other eusocial bees.
Acknowledgments
We thank Paulo Nogueira-Neto and Jay Evans for providing cru-
cial samples used in this study, Jennifer Davis and Jessica Girard for
assistance in the laboratory, and Jo?o M.F. Camargo and Claus Ras-
mussen for their assistance in the identi?cation of bee specimens.
Beth Pringle provided useful comments on the manuscript. This
project was supported by grants from ORBS (Opportunities for Re-
search in the Behavioral Sciences Program), the National Science
Foundation (NSF-DDIG, DEB 0608409, NSF-IBN 0316697, NSF DEB
0447242, and NSF-IBN 0545856) and grants from the Putnam
Expedition Fund to J.C.N., S.R.R., and N.E.P. Samples were collected
in Brazil under the Permit No. 057/2003 (IBAMA) and Peru under
Permit No. 005141 (AG-INRENA).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2010.04.026.
References
Aguilar, I., Brice?o, D., 2002. Sounds in M. costaricensis (Apidae: Meliponini): effect
of sugar concentration and nectar source distance. Apidologie 33, 375?388.
Arias, M.C., Sheppard, W.S., 1996. Molecular phylogenetics of honey bee species
(Apis mellifera L.) inferred from mitochondrial DNA sequence. Mol. Phylogenet.
Evol. 5, 557?566.
Arias, M.C., Sheppard, W.S., 2005. Phylogenetic relationships of honey bees
(Hymenoptera: Apinae: Apini) inferred from nuclear and mitochondrial DNA
sequence data. Mol. Phylogenet. Evol. 37, 25?35.
Camargo, J.M.F., Pedro, S.R.M., 2007. Meliponini Lepeletier, 1836. In: Moure, J.S.,
Urban, D., Melo, G.A.R. (Eds.), Catalogue of Bees (Hymenoptera: Apoidea) in the
Neotropical Region. (accessed
30.10.08).
Camargo, J.M.F., Pedro, S.R.M., 2008. Revis?o das esp?cies de Melipona do grupo
fuliginosa (Hymenoptera: Apoidea: Apidae: Meliponini). Rev. Bras. Entomol. 52,
411?427.
Camargo, J.M.F., Grimaldi, D., Pedro, S.R.M., 2000. The extinct fauna of stingless bees
(Hymenoptera: Apidae: Meliponini) in Dominican amber: two new species and
redescription of the male of Proplebeia dominicana (Wille and Chandler). Am.
Mus. Novit. 3293, 1?24.divergence between corbiculate bees and the outgroup (Centridi-
ni). We suspect that the difference between both studies stems
from applying different ages to the root node. The discovery of
new fossils may shed new light on the time of origin of apid
lineages.
Because no morphological characters have been associated with
the use of recruitment communication in bees, we cannot infer
whether stem fossil Apis, or any other extinct stingless bee lineages
(Engel, 2001), exhibited this form of communication behavior.
However, since all extant members of Apis use recruitment com-
munication, it is likely that the most recent common ancestor of
extant Apis had a form of recruitment communication similar to
that exhibited by modern species. If true, this would suggest that
recruitment communication in honey bees has been stable since
the Eocene?Oligocene. On the other hand, our results suggest that
the genus Melipona shared a most recent common ancestor more
recently, during the Miocene. Although detailed behavioral obser-
vations are available for less than 10 of the 50 Melipona species in
our phylogeny (Nieh, 2004), available data suggest that communi-
cation abilities in Melipona are more variable (Nieh, 2004). Thus,
our study suggests that the traits associated with communication
behavior are perhaps younger and more ?exible in Melipona than
6 S.R. Ram?rez et al. /Molecular PhylogeCameron, S.A., Mardulyn, P., 2001. Multiple molecular data sets suggest
independent origins of highly eusocial behavior in bees (Hymenoptera:
Apinae). Syst. Biol. 50, 194?214.
Please cite this article in press as: Ram?rez, S.R., et al. A molecular phylogeny of
Evol. (2010), doi:10.1016/j.ympev.2010.04.026Costa, M.A., Del Lama, M.A., Melo, G.A.R., Sheppard, W.S., 2003. Molecular
phylogeny of the stingless bees (Apidae: Apinae: Meliponini) inferred from
mitochondrial 16S rDNA sequences. Apidologie 34, 73?84.
Danforth, B.N., Fang, J., Sipes, S., Brady, S.G., Almeida, E., 2004. Phylogeny and
Molecular Systematics of Bees (Hymenoptera: Apoidea). Cornell University,
Ithaca, NY. Available from: .
Dyer, F.C., 2002. The biology of the dance language. Annu. Rev. Entomol. 47, 917?
949.
Engel, M.S., 1998. Fossil honey bees and evolution in the genus Apis (Hymenoptera:
Apidae). J. Hymenopt. Res. 29, 265?281.
Engel, M.S., 1999a. The taxonomy of recent and fossil honey bees (Hymenoptera:
Apidae: Apis). J. Hymenopt. Res. 8, 165?196.
Engel, M.S., 1999b. The ?rst fossil Euglossa and phylogeny of the orchid Bees
(Hymenoptera: Apidae: Euglossini). Am. Mus. Novit. 3272, 1?14.
Engel, M.S., 2000. A new interpretation of the oldest fossil bee (Hymenoptera:
Apidae). Am. Mus. Novit. 3296, 1?11.
Engel, M.S., 2001. Monophyly and extensive extinction of advanced eusocial bees:
insights from an unexpected Eocene diversity. Proc. Natl. Acad. Sci. USA 98,
1661?1664.
Engel, M.S., 2006. A giant honey bee from the middle Miocene of Japan
(Hymenoptera: Apidae). Am. Mus. Novit. 3504, 1?12.
Engel, M.S., Hinojosa-D?az, I.A., Rasnitsyn, A.P., 2009. A honey bee from the Miocene
of Nevada and the biogeography of Apis (Hymenoptera: Apidae: Apini). Proc.
Cal. Acad. Sci. 6, 23?38.
Esch, H., 1967. Die Bedeutung der Lauterzeugung f?r die Verst?ndigung der
stachellosen Bienen. Z. Vergl. Physiol. 56, 408?411.
Esch, H.E., Zhang, S., Srinivasan, M.V., Tautz, J., 2001. Honeybee dances
communicate distances measured by optic ?ow. Nature 411, 58.1?58.3.
Fernandes-Salom?o, T.M., Rocha, R.B., Campos, L.A.O., Ara?jo, E., 2005. The ?rst
internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera: Apidae:
Meliponini): characterization and phylogenetic analysis. Insectes Soc. 52, 11?18.
Gould, J.L., 1976. The dance-language controversy. Q. Rev. Biol. 51, 211?244.
Hines, H.M., 2008. Historical biogeography, divergence times, and diversi?cation
patterns of bumble bees (Hymenoptera: Apidae: Bombus). Syst. Biol. 57, 58?75.
Hrncir, M., Barth, F.G., Tautz, J., 2006. Vibratory and airborne-sound communication
in bees (Hymenoptera). In: Drosopoulos, S., Claridge, M.F. (Eds.), Insect Sound
and Communication: Physiology, Behaviour, Ecology and Evolution. Taylor &
Francis Group, Boca Raton, FL, pp. 421?436.
Kawakita, A., Ascher, J.S., Sota, T., Kato, M., Roubik, D.W., 2008. Phylogenetic analysis
of the corbiculate bee tribes based on 12 nuclear protein-coding genes
(Hymenoptera: Apoidea: Apidae). Apidologie 39, 163?175.
Maddison, D.R., Maddison, W.P., 2003. MacClade v4.06 (Computer Program).
Sinauer Associates, Sunderland, MA.
Michelsen, A., Andersen, B.B., Storm, J., Kirchner, W.H., Lindauer, M., 1992. How
honeybees perceive communication dances, studied by means of a mechanical
model. Behav. Ecol. Sociobiol. 30, 143?150.
Michener, C.D., 1944. Comparative external morphology, phylogeny, and a
classi?cation of the bees. Bull. Am. Mus. Nat. Hist. 82, 151?326.
Michener, C.D., 1974. The Social Behavior of the Bees: A Comparative Study.
Harvard University Press, Cambridge, MA.
Michener, C.D., 2007. The Bees of the World. Johns Hopkins University Press,
Baltimore, MD.
Michener, C.D., Grimaldi, D., 1988. The oldest fossil bee: apoid history, evolutionary
stasis, and the antiquity of social behavior. Proc. Natl. Acad. Sci. USA 85, 6424?
6426.
Moure, J.S., Kerr, W.E., 1950. Sugest?es para a modi?ca??o da sistem?tica do g?nero
Melipona (Hymen.-Apoidea). Dusenia 1, 105?129.
Near, T.J., Meylan, P.A., Shaffer, H.B., 2005. Assessing concordance of fossil
calibration points in molecular clock studies: an example using turtles. Am.
Nat. 165, 137?143.
Nieh, J.C., 1998. The food recruitment dance of the stingless bee, Melipona panamica.
Behav. Ecol. Sociobiol. 43, 133?145.
Nieh, J.C., 2004. Recruitment communication in stingless bees (Hymenoptera:
Apidae: Meliponini). Apidologie 35, 159?182.
Nieh, J.C., Roubik, D.W., 1998. Potential mechanisms for the communication of
height and distance by a stingless bee, Melipona panamica. Behav. Ecol.
Sociobiol. 43, 387?399.
Nieh, J.C., Contrera, F.A.L., Ram?rez, S., Imperatriz-Fonseca, V.L., 2003. Variation in
the ability to communicate three-dimensional resource location by stingless
bees from different habitats. Anim. Behav. 66, 1129?1139.
Oldroyd, B.P., Wongsiri, S., 2006. Asian Honey Bees: Biology, Conservation and
Human Interactions. Harvard University Press, Cambridge, MA.
Poinar Jr., G.O., Danforth, B.N., 2006. A fossil bee from Early Cretaceous Burmese
amber. Science 314, 614.
Raf?udin, R., Crozier, R.H., 2007. Phylogenetic analysis of honey bee behavioral
evolution. Mol. Phylogenet. Evol. 43, 543?552.
Ram?rez, S.R., Roubik, D.W., Skov, C., Pierce, N.E., 2010. Phylogeny, biogeography
and diversi?cation of the orchid bees (Hymenoptera: Euglossini). Biol. J. Linn.
Soc., doi:10.1111/j.1095-8312.2010.01440.x.
Rasmussen, C., Cameron, S.A., 2007. A molecular phylogeny of the Old World
stingless bees (Hymenoptera: Apidae: Meliponini) and the non-monophyly of
the large genus. Trigona. Syst. Entomol. 32, 26?39.
cs and Evolution xxx (2010) xxx?xxxRasmussen, C., Cameron, S.A., 2010. Global stingless bee phylogeny supports
ancient divergence, vicariance, and long distance dispersal. Biol. J. Linn. Soc. 99,
206?232.
the stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.
Rego, M.M.C., 1990. Revis?o do genero Melipona Illiger, 1806: Genitalia e esternos
pre-genitais de machos (Meliponinae: Apidae: Hymenoptera). Ribeir?o Preto,
Brazil. M.Sc. Thesis, Universidade de S?o Paulo.
Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572?1574.
Roubik, D.W., 2006. Stingless bee nesting biology. Apidologie 37, 124?143.
Sanderson, M.J., 1997. A nonparametric approach to estimating divergence times in
the absence of rate constancy. Mol. Biol. Evol. 14, 1218?1231.
Schultz, T.R., Engel, M.S., Ascher, J.S., 2001. Evidence for the origin of eusociality in
the corbiculate bees (Hymenoptera: Apidae). J. Kans. Entomol. Soc. 74, 10?16.
Schwarz, H.F., 1932. The genus Melipona: the type genus of the Meliponidae or
stingless bees. Bull. Am. Mus. Nat. Hist. 63, 231?460.
Seeley, T.D., 1995. The Wisdom of the Hive: The Social Physiology of Honey Bee
Colonies. Harvard University Press, Cambridge, MA.
Sherman, G., Visscher, P.K., 2002. Honey bee colonies achieve ?tness through
dancing. Nature 419, 920?922.
Swofford, D.L., 2003. Paup v4.0b (Computer Program). Sinauer Associates,
Sunderland, MA.
Thompson, G.J., Oldroyd, B.P., 2004. Evaluating alternative hypotheses for the origin
of eusociality in corbiculate bees. Mol. Phylogenet. Evol. 33, 452?456.
von Frisch, K., 1967. The Dance Language and Orientation of Bees. The Belknap Press
of Harvard University Press, Cambridge, MA.
Wille, A., Chandler, L.C., 1964. A new stingless bee from the Tertiary of the
Dominican Republic (Hymenoptera: Meliponini). Rev. Biol. Trop. 12, 187?195.
Wilson, E.O., 1971. The Insect Societies. Harvard University Press, Cambridge, MA.
Zwickl, D.J., 2006. GARLI, Genetic Algorithm for Rapid Likelihood Inference 0.94
(Software Program). Available from: .
S.R. Ram?rez et al. /Molecular Phylogenetics and Evolution xxx (2010) xxx?xxx 7
ARTICLE IN PRESSPlease cite this article in press as: Ram?rez, S.R., et al. A molecular phylogeny of
Evol. (2010), doi:10.1016/j.ympev.2010.04.026the stingless bee genus Melipona (Hymenoptera: Apidae). Mol. Phylogenet.