A Mid-Cretaceous Origin of Sociality in Xylocopine Bees
with Only Two Origins of True Worker Castes Indicates
Severe Barriers to Eusociality
Sandra M. Rehan1,2*¤, Remko Leys1,3, Michael P. Schwarz1
1School of Biological Sciences, Flinders University of South Australia, Adelaide, South Australia, Australia, 2Department of Biological Sciences, Brock University, St.
Catharines, Ontario, Canada, 3Evolutionary Biology Unit, South Australia Museum, Adelaide, South Australia, Australia
The origin of sterile worker castes, resulting in eusociality, represents one of the major evolutionary transitions in the history
of life. Understanding how eusociality has evolved is therefore an important issue for understanding life on earth. Here we
show that in the large bee subfamily Xylocopinae, a simple form of sociality was present in the ancestral lineage and there
have been at least four reversions to purely solitary nesting. The ancestral form of sociality did not involve morphological
worker castes and maximum colony sizes were very small. True worker castes, entailing a life-time commitment to non-
reproductive roles, have evolved only twice, and only one of these resulted in discrete queen-worker morphologies. Our
results indicate extremely high barriers to the evolution of eusociality. Its origins are likely to have required very unusual life-
history and ecological circumstances, rather than the amount of time that selection can operate on more simple forms of
Citation: Rehan SM, Leys R, Schwarz MP (2012) A Mid-Cretaceous Origin of Sociality in Xylocopine Bees with Only Two Origins of True Worker Castes Indicates
Severe Barriers to Eusociality. PLoS ONE 7(4): e34690. doi:10.1371/journal.pone.0034690
Editor: Gro V. Amdam, Arizona State University, United States of America
Received November 25, 2011; Accepted March 6, 2012; Published April 12, 2012
Copyright: ? 2012 Rehan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by Australian Research Council Discovery grants to M. Schwarz and S. Cooper and by an NSERC award (Natural Sciences and
Engineering Research Council) and an Endeavour Fellowship to S. Rehan. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
The evolution of life has been marked by a number of major but
very infrequent transitions, such as the origins of eukaryotes,
multicellularity, and sexual reproduction . One of these key
transitions is the origin of eusociality, where individuals within
groups show life-time specialization in reproductive or non-
reproductive roles. Like the other transitions, eusociality has arisen
very rarely, but where it has arisen it has had a major impact in
shaping biotic and ecosystem diversity.
The question of why eusociality has been so successful, yet its
origins have been so rare, has been a major puzzle in evolutionary
biology. One approach to this issue has involved identifying so-
called ‘pre-adaptations’ or ‘conditions’ for eusociality – combina-
tions of genetic, life-history and ecological features that facilitate
the evolution of strong forms of altruism [2–5]. We would expect, a
priori, that such conditions should be very restrictive, otherwise
transitions to eusociality would be common. Past attempts at
identifying these conditions have looked for common factors
underlying eusocial origins, but this approach runs the risk of
casting a net too broadly. Common factors might be identifiable,
but whether or not they are sufficient is less straightforward.
One way to address this problem of identifying sufficient factors
is to examine clades where putative pre-adaptations have been
in place for long periods of evolutionary time and ask whether
they have facilitated transitions to eusociality. For this, we require
taxa where the history of conditions or pre-adaptations for
eusociality are known, and where the origins of eusociality can
The bees contain multiple origins of eusociality, but most bee
species are solitary or only weakly social. Molecular studies have
indicated three origins of eusociality in halictine bees with up to 12
subsequent losses , and a single origin in the corbiculate bees,
comprising the tribes Bombini, Euglossini, Meliponini and the
Apini, with one subsequent loss . Although tribal relationships
for the corbiculates have not been firmly resolved for a long time
, two recent molecular studies [9,10] using seven and 12 nuclear
genes respectively both recover the same (Bombini+Meliponi-
ni)+(Apini+Euglossini) phylogeny that was used to infer the single
origin of sociality by .
The only other bee group where eusociality has been reported is
the subfamily Xylocopinae, which is in the same family Apidae as
the corbiculates. The Xylocopinae comprises four extant tribes.
The relictual tribe Manueliini contains three species and two of
these are known to be solitary [11,12]. The tribes Ceratinini and
Xylocopini contain both solitary and social species, but sociality in
these groups never entails lifelong castes. Instead, it involves
reproductive hierarchies among totipotent females, where the
reproductive status of nestmates can change over time and where
sterile worker castes are not evident [13–20]. Lastly, all species in
the tribe Allodapini exhibit at least weak forms of sociality ,
but many species have well-defined behavioural castes  and in
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two species, Exoneurella tridentata and Hasinamelissa minuta, queen
and worker castes are discrete and life-long [23,24].
The widespread distribution of sociality in the Xylocopinae
could reflect multiple origins of sociality, or else a single and older
origin followed by both reversions to solitary behaviour and
elaborations into complex sociality. Both possibilities have major
consequence for understanding social evolution. Repeated origins
of sociality would suggest the presence of some facilitating factor or
pre-adaptation common to the subfamily, whereas a single origin
would entail a longer history of social behaviour, but one where
there have been very few elaborations into complex sociality. An
ancient origin of weak sociality in the Xylocopinae followed by
very few origins of complex sociality would indicate very
formidable barriers to highly eusocial behaviour even when simple
forms of sociality are in place.
Distinguishing between a single and multiple origins of sociality
in the Xylocopinae requires that phylogenetic relationships of the
constituent tribes are well resolved. Previous studies [7,25–28]
have all recovered Allodapini and Ceratinini as distally-placed
sister tribes, but disagreed on whether the most basal tribe is
Manueliini or Xylocopini. The phylogenetic positions of these two
latter tribes is critical because the two Manueliini species that have
been studied in detail are solitary [11,12,29], whereas more than
half of the studied Xylocopini species are social [19,30]. A basal
position for Manueliini is therefore likely to decrease the likelihood
that sociality is ancestral for the Xylocopinae as a whole (Figure 1).
Here we use phylogenetic analyses based on two mitochondrial
and two nuclear genes from 70 Xylocopinae species to produce a
well resolved and strongly supported phylogeny of the xylocopine
tribes. We then use social data from studies of 47 Xylocopinae
species to infer a single origin of sociality in this subfamily and
some traits of the ancestral social lineage.
We used three different Bayesian approaches and a maximum
parsimony analysis to recover the phylogeny of the Xylocopinae.
All analyses indicated that the Xylocopini comprise the most basal
tribe and Manueliini was the next-most basal tribe (Figure 2 and
Figures S1, S2, S3 electronic supplementary material). Bayesian
posterior probability support for these bifurcations, from each of
the three analytic approaches, was $98% for all of the tribal and
supra-tribal nodes, though MP bootstrap support was lower. A
Bayes Factor test, comparing log likelihoods for an unconstrained
analysis with those for an analysis where Manueliini was
constrained to a basal position in the subfamily, indicated very
strong support (BF=24.170) for Xylocopini as the most basal
We developed chronograms for the Xylocopinae using an
uncorrelated log normal relaxed clock model, setting the root node
uniting the corbiculate apid bees with the Xylocopinae at
107 Mya, based on a recent molecular phylogenetic analysis of
the Apidae . Using this calibration date we found the crown age
of the Xylcopinae to be in the mid-Cretaceous, about 98 Mya,
with the crown ages of Manueliini and Xylocopini dated at ca. 46
and 50 My, and Allodapini and Ceratinini at ca. 53 and 51 Mya
respectively (Table 1). We also varied the set age of the root node
to 120, 100 and 90 Mya to explore the effect of uncertainty in age
of this node (Table 1). The lower value of 90 Mya gave a crown
age for the Xylocopinae of ca. 83 Mya, still well in the Cretaceous.
Our tribal crown ages based on a root node age of 107 Mya
correspond well with other molecular studies that have included
these tribes [7,31–34].
Importantly, our penalized likelihood estimation of divergence
dates, based on a MrBayes phylogram, gave very similar results to
our BEAST analysis (Table S1 and Figure S1). When the root
node was set to 107 Mya for both analyses, age discrepancies for
our key nodes all varied by less than 10%, except for the crown
ages of Xylocopini and Manueliini (Table 1). For these two latter
tribes, the penalized likelihood analysis gave crown ages that were
approximately 10 My older than the BEAST analyses.
Maximum likelihood (ML) and Bayesian MCMC analyses
based on 41 extant Xylocopinae species for which social data are
available (Table S1) and using 2000 post-burnin chronograms
indicated that social nesting was the ancestral trait for the
Xylocopinae, with an estimated mean probability of P=0.997
from ML analyses and P=0.821 from the MCMC analyses (both
averaged over the 2000 chronograms). The MCMC probabilities
for this node as well as the nodes for each of the Xylocopinae
tribes are summarized as pie charts in Figure 1. A Bayes Factor
test, where social and solitary nesting were separately constrained
as ancestral conditions, favoured social nesting as the ancestral
We used maximum likelihood analyses to infer maximum
colony size in the ancestral Xylocopinae lineage, along with l, k
and d values . l is a measure of how well phylogeny explains
variation in a trait, with values close to 1 indicating a strong
phylogenetic signal. k provides a measure of how changes in a trait
vary with branch lengths, and d provides a measure of whether
rates of change in a trait vary with distance from the root .
Our analyses indicated a strong effect of phylogeny on colony size
(mean l=0.99) and an ancestral maximum size of 3 females per
nest (Table S1). Interestingly, the mean k value was 2.29,
indicating relatively greater change with increasing branch
lengths, rather than a model of punctuated evolution, and the
mean d was 0.88, suggesting gradualism without a tendency
for accelerated change either close to the root or close to the
Lastly, our analyses indicated an extremely low probability for
castes being present in the Xylocopinae root node (P=0.023).
There was moderately low probability for true castes being present
in the root nodes of the two genera containing species with true
castes, Exoneurella (P=0.211) and Hasinamelissa (P=0.303), and
extremely low probability for true castes being present in the most
Figure 1. Alternative scenarios of carpenter bee relationships
with likely implications for origins of sociality. Studies conflict
over whether Manueliini or Xylocopini is the most basal tribe the
Xylocopinae. All allodapines are social and most ceratinines and
xylocopines are social, whereas the only two well-studied species of
Manuelia are solitary. A Manueliini-basal phylogeny (left) would make it
more likely that sociality is not the ancestral state for the subfamily as
well as imply a more recent origin of sociality. Arrows contrast the
possible ranges in timings of social origins, but more than one origin is
still possible under both scenarios.
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recent common ancestor (MRCA) of these two clades (P=0.021).
Combined with our chronogram analyses, this indicates two
origins of true castes, both occurring more recently than the
estimated age of 44.3 Mya for the MRCA of Hasinamelissa and
The bee tribe Manueliini contains only three species, and the
two species whose nesting biology is well known are both strictly
solitary. A basal position of Manueliini in the Xylocopinae would
make it more likely that solitary nesting is the ancestral trait for this
subfamily. However, our analyses overwhelmingly indicate that
Xylocopini comprises the basal tribe within the Xylocopinae,
supporting a recent study of the Apidae using five gene fragments
from a wide sample of apid tribes , but contrasting with some
earlier studies that employed much narrower taxon representation
and smaller character sets (e.g. [25,28]). Our phylogenetic results
are therefore important for inferring social evolution in the
Xylocopinae because we can now be certain that the tribe
Manueliini is not basal.
Our maximum likelihood analyses indicated an extremely high
probability (P=0.997) that social living is ancestral for the
Xylocopinae. Our MCMC analyses indicated a lower, but still
substantial, probability (P=0.821) for sociality being ancestral,
and a Bayes Factor test indicated this was positive, verging on
strong, support. We note that that these probabilities are
predicated on an absence of social/solitary nesting for many
species in our analyses, particularly in the tribes Xylocopini and
Ceratinini. However, there are reasons to believe that many of the
species for which we have missing data may be social. For
example, in a review that covered evidence for solitary or
cooperative nesting in Xylocopa, Vicidomini  listed 13 social
nesters from nine subgenera, and 12 solitary nesters also from nine
subgenera, but two of these subgenera (Koptortosoma and Neox-
ylocopa) contained both solitary and social species. In addition to
species covered in Vicidomini’s study, social nesting has been
reported in another four Xylocopa species, X. (Koptortosoma) pubescens
, X. (Ctenoxylocopa) sulcatipes , X. (Lestis) bombylans and X. (L.)
aeratus . Likewise, Ceratina species have long been considered
solitary with few studies reporting social traits [13–15,39].
However, recent studies have shown sociality in C. (Pithitis)
smaragdula, C. (Ceratinidia) nigrolateralis, C. (C.) accusator and C.
(Neoceratina) australensis [40–44]. When combined, multiple studies
therefore indicate that social nesting in Xylocopa and Ceratina is both
frequent and taxonomically widespread.
Given the above considerations, our results have important
implications for understanding social evolution in bees. Cardinal
and Danforth  showed that eusociality evolved once in the
corbiculate bees, but it is not known if this origin was preceded by
a period of less complex sociality in an ancestral lineage and, if so,
what kind of social structure that ancestor may have had.
However, the Xylocopinae, which is nested within the same
subfamily Apinae as the corbiculates, provides numerous examples
of simple forms of sociality as well as some socially complex species
. Our analyses show that a simple form of sociality is ancestral
for the Xylocopinae and that sociality has been in place for about
100 My. A number of important consequences flow from this
conclusion, which we now discuss.
Eusociality in the corbiculates evolved some 87 Mya, but that
was only 10 My after divergence of that group from its sister clade,
the solitary-nesting tribe Centridini . This means that
eusociality in the corbiculates would have been preceded by, at
most, 10 My of evolution involving simple forms of sociality. In
contrast, for the Xylocopinae the lag time from simple ancestral
sociality (about 100 Mya) to eusociality involving true worker
castes (less than the most recent common ancestor of Exoneurella
and Hasinamelissa, ca. 45 Mya) was about 55 My and potentially
much longer, depending on when worker castes evolved in the
lineages leading to E. tridentata and H. minuta. Combined, these
findings indicate that complex eusociality can evolve from a non-
social ancestor comparatively quickly, as in the corbiculates, or
very slowly despite a very long period of simple sociality, as in the
Xylocopinae. This disparity in lag times from solitary to eusocial
suggests that origins of eusociality cannot be simply explained by
the length of time that evolution can operate on primitively social
Attempts to understand how eusociality has evolved, or why it
has evolved so few times, have often involved identifying so-called
pre-adaptations or conditions for eusociality (e.g. [3–5]). These all
involve features that are present in social species of Xylocopinae,
such as overlap of generations, use of a defensible nest with
resources concentrated in that nest, opportunities for kin to
Figure 2. Evolution of sociality in the Xylocopinae. Chronogram of the Xylocopinae based on 70 species from all extant xylocopine tribes. The
chronogram was derived from a log normal relaxed clock model in BEAST and posterior probability support for each node is indicated by numbers
above branches. Social species are coloured red, solitary species are blue and species where social status are unknown are black. Outgroup clades are
indicated by grey branches and the root node, uniting the outgroup and the Xylocopinae, was set at 107 Mya. The two Xylocopinae species known
to have true worker castes are indicated by black rectangles. The relative probabilities of social and solitary as states for key internal nodes were
estimated using a Bayesian analysis and are summarized as pie charts with the probability of being social (red slices) indicated by italic numbers. For
Xylocopini and Ceratinini, which are both monogeneric, we have used the subgeneric rather than generic names.
Table 1. Age estimates of Xylocopinae root age and tribe
origins obtained from a relaxed clock model.
Root node set to:90 My100 My 107 My120 My
Xylcopinae82.9192.12 98.57 (103.30)110.54
73.23 81.36 87.06 (90.70)97.63
68.5276.14 81.47 (85.46) 91.36
Manueliini (M)38.6542.9445.95 (54.42)51.53
Xylocopini (X)42.0146.6849.95 (61.90) 56.02
Allodapini (A) 44.4649.4052.86 (49.18) 59.28
Ceratinini (C)42.68 47.4250.74 (50.11) 56.91
MRCA 37.3041.4444.34 (41.59)49.73
The root node, connecting the corbiculate outgroup with the Xylocopinae, was
set at four different values, ranging from 90 Mya to 120 Mya to explore the
effects on internal node estimates. The root node age set to 107 Mya
corresponds to the estimate by Cardinal and Danforth . Age estimates are
also given for the node uniting Manueliini, Ceratinini and Allodapini (M+C+A),
the node uniting the Ceratinini and Allodapini (C+A), and the most recent
common ancestor (MRCA) for the two allodapine species (Exoneurella tridentata
and Hasinamelissa minuta) that have true worker castes. Bayesian analyses
indicate that this MRCA did not have true castes, so that the age of this node
predates the two origins of true workers. Node age estimates are also given in
parentheses for a penalized likelihood transformation of the consensus
phylogram obtained from a MrBayes analysis where the root node was set to
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cooperate in nest use, and opportunities for such cooperation to
enhance nest defence or resource acquisition. For example,
cooperative nesting leads to almost ubiquitous decreases in rates
of brood loss in allodapines . Increased defence of brood is also
widely reported for social species of Xylocopa and Ceratina
[13,25,40,42,45–47]. Overlap of generations has also been
reported in all social species in the Xylocopinae studied to date
[17–19,32,48,49], and the only studies of Xylocopinae to report
cooperative nesting among unrelated females involve artificially
forced associations in Ceratina (e.g. [15,25,43,50]). Consequently,
these conditions for eusociality, and the length of time that simple
sociality has been in place, are not sufficient to explain why
eusociality has evolved so infrequently in the Xylocopinae. It
therefore seems likely that much more stringent conditions are
Schwarz et al.  argued that, among allodapines, the
evolution of sterile workers in Exoneurella tridentata and Hasinamelissa
minuta is linked to harsh environmental conditions that simulta-
neously limit opportunities for dispersal and opportunities for
subordinate females to survive long enough to assume a position of
reproductive dominance. This argument falls broadly into an
approach that posits ‘causal mosaics’ (sensu Crespi ) – specific
combinations of selective factors and life history traits that may
be able to explain individual origins of eusociality where more
general hypotheses have little or no predictive value. Dew et al.
 have shown that within the allodapines, large colony size in E.
tridentata represents a threshold event rather than the result of
gradual evolutionary change within that tribe. This is also
concordant with the idea that some rare coincidence of selective
factors is required for eusociality to evolve, rather than eusociality
representing an outcome from a gradual and long-term evolu-
The notion that the evolution of true worker castes requires a
highly unusual mosaic of selective and life history conditions could
potentially explain why its origins have been so few and yet so
widely distributed over time. If eusociality requires a gradual
accumulation of more and more complex traits we might expect to
see origins becoming more frequent closer to the present, but this
does not seem to be the case. If causal mosaics have a different
composition of facilitating factors for each inferred origin, they will
not permit straightforward statistical assessment, and recent
molecular studies on at least bees [7,53], indicate that the number
of eusocial origins now known is much smaller than earlier studies
Whilst recent molecular phylogenetic studies have decreased the
number of eusocial origins available for comparative studies, they
have increased the number of known reversions to solitary living.
There are 12 inferred losses of sociality in halictines , one loss
in the corbiculate bees , and our study indicates at least four
losses in the Xylocopinae. Because the large majority of
Xylocopini and Ceratinini species have received no detailed
studies of nesting biology, it is likely that there are more than
four losses of sociality in the Xylocopinae. In the Xylocopini
reversals to solitary nesting (as in X. violacea  and X. caffra )
occur in species that are found in more temperate climates
and coincide with transitions from multivoltine to univoltine,
indicating that climate may play a role here. Likewise, reversals to
solitary nesting in Ceratina (Zadontomerus) occur in species that do
not reuse nesting substrates [20,49]. Life history traits such as
obligate nest dispersal and univoltine colony cycles limit
opportunities for overlapping generations and cooperative brood
care. Paradoxically, comparative studies focussed on losses of
sociality may be our best strategy for understanding the origin of
Taxa and sampling localities along with NCBI accession
numbers are listed in Table S1. All new data have been deposited
in Genbank accession numbers JQ230006–JQ230057. Our
ingroup comprised 70 species sampled from all four tribes of the
Xylocopinae : Allodapini (22 species), Ceratinini (31 species),
Manueliini (3 species), and Xylocopini (11 species). Our taxa
covered all species of Manueliini, and choice of species in the three
other tribes was based on availability of sequence data and the
desirability of representing as wide a range as possible of the major
intra-tribal clades identified by previous studies [31–33].
Four gene fragments were used for phylogenetic analyses: two
mitochondrial genes cytochrome oxidase subunit 1 (COI - 1279
base pairs) and cytochrome b (cytb - 428 base pairs), and two
nuclear genes,the F1 and F2 copies of Elongation Factor-1a (EF-
1a), with 460 and 772 base pairs respectively. DNA extraction,
PCR amplification and sequencing were performed as described in
Leys et al. , Schwarz et al.  and Rehan et al. . Most
sequences were from previous studies and references for their
sources, along with accession numbers for newly sequenced
species, are listed in the supplementary material (Table S1). The
intron region of the F2 copy of EF-1a was largely unalignable and
was not included in the analyses.
We used three methods to explore tribal relationships in the
Xylocopinae. Firstly we used a Bayesian Monte Carlo Markov
Chain (MCMC) approach, implemented in BEAST 1.6.2 
with a relaxed log-normal clock model. For this analysis we
separately combined the two mitochondrial genes and the two
nuclear genes and then each group was partitioned into 1stplus
2nd, and 3rdcodon positions, producing a total of six partitions. A
GTR+I+C model was fitted to each partition because this is the
most general model available and effectively allows more
restrictive models when some model parameters converge to
similar values. For tree construction we used a Yule process with
the prior for birthrate drawn from a uniform distribution bounded
by 0 and infinity. We used a total of 20 million generations,
sampling every 1000thgeneration and with a burnin of 10 million.
Stationarity in models was assessed by plotting parameter values in
the program Tracer 1.4.1 .
As a check that the phylogeny produced from our BEAST
analysis was robust to different analytical approaches, we also
carried out Bayesian analyses in MrBayes version 3.1.2 ,
BayesPhylogenies 1.1 , and a maximum parsimony analysis in
PAUP* v4.0b10 .
For MrBayes analyses we used the same gene partitioning
scheme as for our BEAST analyses, leading to six partitions . We
used default MrBayes priors, with a GTR+I+C model for each
partition, and partitions were unlinked for all substitution model
parameters. Two analyses were run in parallel, each for 20 million
generations with 16 chains, sampling every 1000thgeneration.
Stationarity in model parameters was assessed by examining the
average standard deviation of split frequencies (ASDSF), along
with trace plots of log likelihood (LnL) values along with other
parameters, such as transition rates and base composition
frequencies for each partition, again using Tracer 1.4.1 . We
chose a burnin of 15 million, well after stationarity was reached, so
that the consensus phylogram and posterior probabilities were
based on 10,000 post-burnin trees.
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BayesPhylogenies  implements an MCMC method allowing
a mixture model where multiple models of sequence evolution can
be applied to nucleotides without having to partition data a priori.
In our analyses, this served to check whether tribal relationships
inferred from the BEAST and MrBayes analyses were dependent
on the partitions that we set prior to analysis. For our
BayesPhylogenies analysis we chose to use four patterns of
sequence evolution, each with a separately estimated GTR model,
with gamma rate heterogeneity and base frequencies estimated
separately for each of the four models. We used three chains run
for five million iterations sampling every 500thgeneration.
Stationarity in the models was assessed using Tracer to examine
parameters across sampled generations, as was done for the
MrBayes analysis, and we used a burnin of 4 million iterations.
Our maximum parsimony (MP) analysis was implemented in
PAUP*, as a further check for robustness of tribal relation-
ships. MP analysis employed 50 random sequence addition
heuristic searches, holding 10 trees at each step. Node support
was assessed using bootstrap analysis with the same heuristic
search procedure, with 1000 bootstrap pseudoreplicates.
Estimating Divergence Ages
Chronograms in BEAST were produced using a log normal
relaxed clock model and default priors. For the MrBayes analysis,
we produced chronograms using Sanderson’s penalised likelihood
(PL) transformation of phylograms, enabled in the program r8s
, and this was applied to both the consensus phylogram from
our MrBayes analysis, as well as 300 random postburnin
phylograms from the same analysis.
The only reliable internal calibration point available for the
Xylocopinae is the presence of Boreallodapini (extinct sister clade
to Allodapini with Ceratinini representing the next-most basal
divergence) fossils from Baltic amber dated at 45.1 Mya . In
initial analyses we therefore explored the effect of setting a
minimum divergence age of 45 Mya between Ceratinini and
Allodapini and using a variety of root node ages (uniting the
corbiculates with the Xylocopinae) ranging from 90 Mya to
120 Mya. Our chronograms from both the BEAST and
MrBayes+r8s analyses were subsequently calibrated by setting a
fixed date for the root node and we varied this from 90 Mya to
120 Mya and also included a set date of 107 Mya corresponding
to the date estimated by Cardinal and Danforth .
Social evolution analysis
Each species was coded as either social, solitary or as unknown
based on a review of the current literature (ESM Table 1). We use
the term ‘social’ here in a broad sense to include species where two
or more adult females are present in a nest while eggs are being
laid and brood are being actively provisioned. Our use of the term
sociality therefore covers all forms of sociality that have previously
been designated as eusocial, semisocial and quasisocial [2,39,62]
but does not include subsocial colonies with only a single adult
female, or communal colonies. We do not use the term ‘sociality’
here to imply that all such nestmates are actively involved in
rearing brood, but rather that reproductive females tolerate the
presence of other adult females whether the latter help in brood
rearing or not. Such cases comprise a clear ‘pre-adaptation’ to
sociality involving worker-like behaviour, since selection is able to
operate on already-present associations to produce a division of
labour. We coded the two Inquilina social parasites as social, rather
than solitary, because this genus is derived from a social ancestor
(its host clade) and because their mode of living entails important
social traits, including nestmate recognition, integrating with the
host social hierarchy, soliciting trophallaxis via social communi-
cation, and having their brood reared by alloparental care rather
than kleptoparastism . We coded the social status of the rare
Middle Eastern allodapine Exoneuridia hakkariensis as missing
because the nesting biology of this species has never been
We used both maximum likelihood (ML) and MCMC methods,
both implemented in BayesTraits [35,64] to infer the ancestral
states (solitary or social) for each tribe and for the root node of the
Xylocopinae. Species for which we had sequence data but not
social data were included in phylogenetic trees but their social
states were treated as missing. To account for the effects of
phylogenetic uncertainty, analyses were applied to both the
consensus phylogram from the BEAST analysis, as well as 2000
For ML analyses we calculated the probability of any one state
being ancestral for the Xylocopinae root node as well as the root
nodes for each of the four tribes. Support for any one state can be
gauged usinga likelihoodratio
LR=2(LnL(better fitting model)2LnL(worse fitting model)) .
There is no natural way to combine ML ancestral state analyses
from multiple chronograms into a single assessment , so we
examined results from 2000 post-burnin chronograms as well as
the average of these results.
For MCMC analyses, multiple post-burnin chronograms can be
combined in a single analysis so that phylogenetic uncertainty is
taken into account when estimating posterior probabilities over the
tree samples , and for this we used the same 2000 post-burnin
chronograms as for the ML analysis. Priors for character state
transition rates were based on the distribution of rates from the
ML analyses, which suggested zero-truncated exponential distri-
butions, and these were seeded using a reverse jump hyperprior.
For each analysis we explored a range of rate deviation values with
the criterion that acceptance rates varied between 0.2–0.4.
Harmonic means of the LnL were examined for multiple runs to
determine an appropriate burnin and total number of iterations.
Following these multiple runs, we used a burnin of 100 million
iterations and a total run of 1 billion generations, sampling every
100,000thiteration. Finally we statistically assessed the likelihood
of the root node being social or non-social by fixing (‘fossilising’)
this node for each state and then comparing the model likelihoods
using a Bayes Factor test, where the BF=2(LnL(best fitting
model)2LnL(worse fitting model)), where LnL is calculated as the
harmonic mean of post-burnin log likelihoods, and values of BF.2
indicate support for the better fitting model, and values .5
indicate strong support (e.g. ).
We also examined the evolution of maximum colony size
(maximum number of adult females per nest, excluding callows, in
nests where brood were being actively reared) and the presence of
morphologically-based castes. Maximum colony sizes for included
species are given in table S1 along with references for data sources.
Morphologically-based castes have been reported for only two
species in the Xylocopinae, Hasinamelissa minuta and Exoneuridia
tridentata , so all other species in our data set where nesting
biology has been described were coded as lacking such castes.
Ancestral colony size was inferred using the MCMC option in the
Continuous module of BayesTraits . We used a burnin of 10
million generations and a total of 1 billion iterations, sampling
every 1 millionth iteration to reduce autocorrelation of sampled
values. Stationarity in the model was assessed by plotting the
harmonic mean of the LnL, and the ancestral value was based on
the mean of sampled values after stationarity. The presence of
morphology-based castes at the root and internal nodes was
assessed using the BayesMultiState module in BayesTraits. We
used the same burnin and total iterations as for our Continuous
Ancient Origin of Sociality in Xylocopine Bees
PLoS ONE | www.plosone.org6 April 2012 | Volume 7 | Issue 4 | e34690
analysis, and a rate deviation of 60 to ensure acceptance rates of
between 0.2 and 0.4, as recommended . Probabilities for the
presence/absence of castes at chosen nodes was estimated as
means for sampled iterations after model stationarity, as
determined from plots of the harmonic mean LnL. Both the
Continuous and MultiState analyses were carried out three times
to check for consistency in outcomes.
likelihood transformation of the consensus phylogram
obtained from a MrBayes analysis.
Chronogram obtained from a penalised
BayesPhylogenies analysis, along with posterior proba-
bilities for all nodes.
Consensus phylogeny obtained from the
parsimony analysis implemented in PAUP*.
Bootstrap consensus tree from a maximum
and references for this status for Xylocopinae species in
Genbank accession numbers and social status
Images in Figure 1 are courtesy of Laurence Packer and the Packer
Collection at York University (PCYU). Thanks to Claudia Ratti and Nick
de Silva for taking these images.
Conceived and designed the experiments: SMR RL MPS. Performed the
experiments: SMR RL MPS. Analyzed the data: SMR RL MPS.
Contributed reagents/materials/analysis tools: SMR RL MPS. Wrote
the paper: SMR RL MPS.
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