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Mitochondrial Phylogenomics Resolves the Global Spread of Higher Termites,

Ecosystem Engineers of the Tropics

Article in Molecular Biology and Evolution · March 2017

DOI: 10.1093/molbev/msw253

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Mitochondrial Phylogenomics Resolves the Global Spread
of Higher Termites, Ecosystem Engineers of the Tropics
Thomas Bourguignon,*,1,2,3 Nathan Lo,3 Jan Sobotnık,2 Simon Y.W. Ho,3 Naeem Iqbal,4 Eric Coissac,5,6
Maria Lee,1 Martin M. Jendryka,1 David Sillam-Dussès,7,8 Barbora Krızkova,2 Yves Roisin,9 and
Theodore A. Evans1,10
1
Department of Biological Sciences, National University of Singapore, Singapore, Singapore
2
Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic
3
School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
4
Department of Plant Protection, Faculty of Agricultural Sciences, Ghazi University, Dera Ghazi Khan, Pakistan
5
Centre National de la Recherche Scientifique, Laboratoire d’Ecologie Alpine (LECA), Grenoble, France
6
Laboratoire d’Ecologie Alpine (LECA), Université Grenoble Alpes, Grenoble, France
7
Institut de Recherche pour le Développement, Sorbonne Universités, iEES-Paris, Bondy, France
8
Université Paris 13, Sorbonne Paris Cité, LEEC, Villetaneuse, France
9
Evolutionary Biology and Ecology, Université Libre de Bruxelles, Bruxelles, Belgium
10
School of Animal Biology, University of Western Australia, Perth, WA, Australia
*Corresponding author: E-mail: thomas.bourgui@gmail.com.
Associate editor: Nicolas Vidal

Abstract
The higher termites (Termitidae) are keystone species and ecosystem engineers. They have exceptional biomass and play
important roles in decomposition of dead plant matter, in soil manipulation, and as the primary food for many animals,
especially in the tropics. Higher termites are most diverse in rainforests, with estimated origins in the late Eocene
(54 Ma), postdating the breakup of Pangaea and Gondwana when most continents became separated. Since termites
are poor fliers, their origin and spread across the globe requires alternative explanation. Here, we show that higher
termites originated 42–54 Ma in Africa and subsequently underwent at least 24 dispersal events between the continents
in two main periods. Using phylogenetic analyses of mitochondrial genomes from 415 species, including all higher
termite taxonomic and feeding groups, we inferred 10 dispersal events to South America and Asia 35–23 Ma, coinciding
with the sharp decrease in global temperature, sea level, and rainforest cover in the Oligocene. After global temperatures
increased, 23–5 Ma, there was only one more dispersal to South America but 11 to Asia and Australia, and one
dispersal back to Africa. Most of these dispersal events were transoceanic and might have occurred via floating

Article
logs. The spread of higher termites across oceans was helped by the novel ecological opportunities brought about
by environmental and ecosystem change, and led termites to become one of the few insect groups with specialized
mammal predators. This has parallels with modern invasive species that have been able to thrive in human-
impacted ecosystems.
Key words: historical biogeography, insects, Isoptera.

Introduction some habitats (Bignell 2006; Jouquet et al. 2011, 2016).

Termites have a nearly global distribution and comprise
3,000 described species (Krishna et al. 2013). Despite their
modest levels of diversity, they are among the most successful
terrestrial organisms on the earth in terms of sheer abun-
dance, making up 10–20% of the animal biomass in tropical
ecosystems worldwide (Eggleton et al. 1996; Ellwood and
Foster 2004). The success of termites is in large part due to
their ability to digest lignocellulose, the most abundant or-
ganic molecule on earth, and dissolved organic molecules in
soil (Norkrans 1963; Dixon et al. 1994). They play an essential
role in decomposition and nutrient recycling, consuming up
to 90% of wood, dead plant matter, and soil organic matter in
Termites move tonnes of soil per hectare per year and are
considered ecosystem engineers (Jouquet et al. 2016). Their
abundance has also allowed termites and ants to become the
only insect groups with specialist mammalian predators
(Redford 1987).
The “higher” termites (family Termitidae) constitute the
vast majority of termite species (80%) and are the most
ecologically dominant taxa. Although termites are commonly
thought to feed primarily on wood, termitids consume a
variety of organic matter and more than half of the species
feed on soil. The evolutionary history of this important and
dominant group, which spans across several biogeographic

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Mol. Biol. Evol. 34(3):589–597 doi:10.1093/molbev/msw253 Advance Access publication December 25, 2016 589
Bourguignon et al. . doi:10.1093/molbev/msw253
regions, is likely to have been complex. The termite fossil
record dates back to 140 Ma (Engel et al. 2007), but a recent
molecular analysis of 66 termite species placed the origin of
Termitidae at only 54 Ma (Bourguignon et al. 2015). For this
reason, the origin of Termitidae is believed to postdate the
breakup of Pangaea and Gondwana. Because termites are
weak flyers (Hu et al. 2007), they are expected to be poor
dispersers and the origin of their global distribution is enig-
matic. While vicariance played a critical role in the early di-
versification of termites (Bourguignon et al. 2015), the extent
to which termitids dispersed across oceans remains poorly
understood.
The poor fossil record of Termitidae (Engel et al. 2009) has
left considerable uncertainty about how and when this group
dispersed to the major landmasses. This question can be ad-
dressed in detail using molecular phylogenies inferred from a
worldwide sample of termitids. Although some studies have
included extensive taxon samples (Inward et al. 2007;
Legendre et al. 2015) and others have used large amounts
of sequence data (Cameron et al. 2012; Bourguignon et al.
2015), no studies have yet combined these two features.
To resolve the historical biogeography of higher termites,
we analyzed full mitochondrial genomes from 415 species,
including 384 termitids, representing 18% of the described
species (supplementary tables S1 and S2, Supplementary
Material online). These termites were sampled from 13 coun-
tries in Africa, Asia, South America, and Australia. By including
age calibrations from 15 fossils (supplementary table S3,
Supplementary Material online), we estimated the evolution-
ary timeframe of the group. Our specific aims were to deter-
mine: (1) the geographic origin of higher termites; (2) the
pathways and timing of dispersal events; and (3) whether
dispersal events coincided with major climatic changes and
with the origin of termite-specific predators.
Results and Discussion
Origin of Higher Termites
Our estimate of the evolutionary timescale shows that mod-
ern higher termites originated in Africa 47.6 Ma (95% cred-
ibility interval: 42.2–53.7 Ma) (fig. 1). This is consistent with
African rainforests harboring the highest diversity of
Termitidae, and comprising most of the earliest-branching
lineages in the group (Eggleton 2000). Our estimate was ob-
tained after excluding third codon positions and using the
best-fitting clock model. However, we obtained consistent
results when we repeated the analysis with and without third
codon positions, using different clock models and phylogen-
etic methods, and using different methods for reconstructing
ancestral ranges (supplementary figs. S1–S6, Supplementary
Material online).
Eocene–Oligocene out of Africa
We inferred at least five dispersals of higher termites to South
America and five dispersals to Asia, towards the end of the
Eocene through to the Oligocene, 35–23 Ma (figs. 2 and 3).
Five of these dispersal events were from Africa and five were
from undetermined origins. The inception of the “out of
590
MBE
Africa” dispersals of higher termites coincides with a sharp
drop in the atmospheric concentration of carbon dioxide
(Pagani et al. 2005) and in the global temperature at the
Eocene–Oligocene boundary, 34 Ma, as inferred from deep-
sea oxygen isotopic ratios (Zachos et al. 2001), which triggered
a mass extinction. Global temperatures remained consistently
low until the end of the Oligocene (Zachos et al. 2001), trig-
gering the retraction of megathermal rainforests towards the
equator, where they survived as tropical rainforests, albeit
experiencing numerous extinctions (fig. 3) (Morley 2011).
Termitids therefore colonized Asia and South America during
major reworking of tropical ecosystems, at the time when
tropical rainforests acquired their modern flora (Morley
2011) (fig. 2).
New Dispersal Patterns during the Miocene
Our ancestral range reconstruction shows a change in disper-
sal patterns during the Miocene, 23.0–5.3 Ma, once tempera-
tures had increased (Zachos et al. 2001). Higher termites
dispersed only once to South America and 6 times to Asia.
They also dispersed once, and for the first time, to Africa from
Asia, and 5 times to Australia.
The low rate of colonization of South America compared
with Asia in the Miocene can be explained by a combination
of ecological and tectonic factors. Termitids were already
abundant and diverse in the Dominican amber 14–
20 Ma (Krishna and Grimaldi 2009), suggesting that they
rapidly diversified following their arrival in South America,
preventing later establishments of ecologically similar species.
Although a similar situation is likely to have existed in Asia,
the Gomphotherium land bridge created a terrestrial connec-
tion between Africa and Eurasia during the early Miocene
18–20 Ma (Rögl 1998, 1999). This land bridge is likely to
have allowed the fungus-growing macrotermitine termites to
cross from Africa to Asia at this time (Aanen and Eggleton
2005), given that this group did not reach either South
America or Australia, neither of which had land bridges
with Africa.
Australia was colonized by at least five lineages, once from
Asia, once from South America and thrice from undeter-
mined regions. The Occasitermes lineage was the first to ar-
rive, at latest 19.8 Ma, followed by four more lineages that
dispersed to Australia during a short time period, 11–13 Ma.
This timing coincides with a sharp increase in aridification,
14 Ma, and the expansion of the arid biome in Australia,
during the second half of the Miocene, 15–5.3 Ma
(Martin 2006). The expansion of the arid biome and its sclero-
phyll vegetation offered ecological opportunities for new
groups to diversify and take over ecosystem functions
(Crisp and Cook 2013). Today, higher termites are the dom-
inant decomposers in the arid biome (Abensperg-Traun and
Steven 1997).
Higher Termites Dispersed between Biogeographic
Regions by Rafting
The Gomphotherium land bridge between Asia and Africa
possibly explains the colonization of Africa by
Pericapritermes from Asia. It may explain the colonization
Mitochondrial Phylogenomics Resolves the Global Spread of Higher Termites . doi:10.1093/molbev/msw253 MBE

FIG. 1. Bayesian phylogenetic tree of higher termites inferred from complete mitochondrial genomes, with third codon positions excluded.
The tree is drawn to a time scale given in millions of years. Node bars represent the 95% credibility intervals of node-time estimates.
Branches are labelled with posterior probabilities. Circles show the ancestral distribution inferred for each node using a Bayesian phylo-
genetic analysis. Clades comprising species from a single biogeographic area are collapsed into triangles. Internal branches and ancestral
states are shown in colour only when the posterior probability of the ancestral reconstruction is >0.95. The map shows the four areas
considered in the analyses.

of Asia by Microtermes, Ancistrotermes pakistanicus,
Odontotermes and Macrotermes. The last two of these genera
might be restricted to land dispersal because of their fungus-
growing behaviour (Nobre et al. 2010). It also potentially ex-
plains other colonizations of Asia, by Microcerotermes and
Termes, which dispersed from Africa, possibly after Asia and
Africa became connected by land. Alternatively, these disper-
sal events might have been by rafting in wood pieces over
water, as it must have been for the other 17 dispersal events.
Given the poor flight capability of termites, it seems likely that
termites dispersed by rafting in wood, probably logs from
trees (Bourguignon et al. 2016). This would suggest that the
dispersing termites were wood feeding, and that other feeding
habits evolved after dispersal (Inward et al. 2007). However,
our results show that extant species of some early branching
lineages are soil feeding for some comparisons, such as in the
South American Apicotermitinae and Syntermitinae. There
are two possible explanations: either that the dispersing spe-
cies were wood-feeding and are now extinct, or that soil
feeders dispersed across oceans, possibly in arboreal nests
attached to floating trees. There are modern soil-feeding,
tree-nesting species in these families, e.g., Anoplotermes
banksi and Labiotermes labralis.
Higher Termites Colonized New Continents during
Environmental Changes
Our study shows that the timing of higher termite dispersals
coincides with the opening of ecological opportunities due to
climate change. Higher termites originated in Africa and dis-
persed from there in two phases. The first phase was initiated
by the retraction of megathermal rainforests to the equatorial
region at the end of the Eocene, 34 Ma (Morley 2011).
591
Bourguignon et al. . doi:10.1093/molbev/msw253 MBE

FIG. 2. Graph summarizing dispersals between biogeographic areas. The upper curve represents the minimum partial pressure of atmospheric
CO2 as in Pagani et al. (2005). The lower curve represents deep-sea oxygen isotope records as in Zachos et al. (2001), whereby lower values
indicate warmer climate. Circles represent the maximum estimated age of the dispersal, given by the mean posterior estimates of the timing of
divergence from the most closely related foreign taxon, based on a Bayesian phylogenetic analysis. Only the dispersals with posterior prob-
ability exceeding 0.95 in all analyses are shown. Circles are coloured when the provenance of dispersers was retrieved in all analyses with a
posterior probability exceeding 0.95. Blue: Africa; red: South America; green: Asia. Blue bars represent the 95% credibility intervals of the age
estimates.

The second phase coincided with the expansion of the arid
biome in Australia during the middle Miocene, 14 Ma (Martin
2006).
Based on the evidence presented earlier, we hypothesize
that termites colonized multiple continents as they were
undergoing major environmental and climatic changes.
Ecosystems undergoing such changes may be more vulner-
able to biological invasion (Elmqvist et al. 2003; Davis 2009).
This has parallels with the success of modern invasive species
in ecosystems disturbed by human activities (King and
Tschinkel 2008). We detected one recent and unusual disper-
sal event 1.2 Ma (Holocene), Ancistrotermes pakistanicus dis-
persing from the African equatorial rainforest to tropical Asia.
Given the long connection between these continents, it
seems possible that (ancient) humans assisted this recent
592
dispersal as they have with many other termites (Evans
et al. 2013).
Influence of Higher Termite Arrival on Mammal
Predators
There is a possibility that the evolution of termites influ-
enced the evolution of other species, especially their
predators. The only groups of insects with specialist mam-
malian predators are termites and ants. As many myrme-
cophagous mammals eat both of these insect taxa, there
is a question whether these mammals evolved to eat ter-
mites first, then expanded to ants, or vice versa. We found
that of the six mammals that eat only termites, five
evolved after the appearance of the termite genera
upon which they prey (fig. 4). The sole exception, the
Mitochondrial Phylogenomics Resolves the Global Spread of Higher Termites . doi:10.1093/molbev/msw253 MBE

FIG. 3. Timing of the major phases of dispersal by higher termites during the Cenozoic. The black arrows on the left maps show the directions of
dispersal events. The green areas on the right maps show the estimated distribution of tropical rainforests (Morley 2011). The distribution of
landmasses is as inferred by Zachos et al. (2001).

numbat in Australia, has no close relatives and its timing
of origination might be overestimated by comparison
with its sister lineage in the mammal tree (dos Reis
et al. 2012). Five of 10 mammals that eat termites and
ants evolved after their termite prey. As this is the pattern
observed for the termite specialists, we suggest that they
evolved to eat termites first, then added ants to their diet
later. The other five mammal species evolved before their
termite prey, possibly adapting from an ant-based diet or
general insectivory (as suggested by armadillos in South
America and mongooses in Africa; supplementary table
S4, Supplementary Material online). Note that the esti-
mated timing of the origin of pangolins, like numbats,
suffers from a lack of close relatives (dos Reis et al.
2012). Although not definitive, our data show that the
arrival of termites predates mammalian myrmecophagy,
which suggests that termites promoted the diversification
of insectivorous mammals. This idea could be tested with
a phylo-biogeographical study on other prey species, es-
pecially ants. Further study of other predators may find
clearer patterns, confirming the major impacts that
termites have had on their ecosystems as keystone spe-
cies, as ecosystem engineers, and as the major decom-
posers of the tropics.
Materials and Methods
Mitochondrial Genome Sequencing
We carried out worldwide sampling of higher termites (family
Termitidae) and sequenced complete mitochondrial gen-
omes from 346 samples. In combination with 38 samples
sequenced in previous studies, we assembled a data set com-
prising 384 species (supplementary table S1, Supplementary
Material online). To infer the position of the root in our
phylogenetic analysis, we included the mitochondrial gen-
omes of 30 species downloaded from GenBank. These
included 25 lower termites, two cockroaches, one mantid,
one stick insect, and one grasshopper (supplementary table
S2, Supplementary Material online). All specimens were
sampled in RNA-laterV and stored at 80  C until DNA
R
extraction.
We extracted whole genomic DNA from about five
individuals, from which the digestive tract was removed,
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Bourguignon et al. . doi:10.1093/molbev/msw253 MBE

FIG. 4. The timing of the origins of termite-eating mammals and their termite prey: (a) mammals that specialize on termites only; (b) mammals
that specialize on ants and termites. The shaded area indicates that the origin of termite prey antedates that of the mammal predators, suggesting
that the mammals evolved to eat the termites. The number of termite origin dates (horizontal standard error bars) varies between mammals. A
mammal species has either one termite origin date if it targets just one termite genus or subfamily (e.g., aardwolves and Trinervitermes), or two
termite origin dates if it eats many (the two origin dates are for the most commonly eaten termite species). See supplementary table S4,
Supplementary Material online, for the complete list of termite prey.

using phenol-chloroform extraction procedure. The com-
plete mitochondrial genomes were amplified with
TaKaRa LA Taq in two long-PCR reactions using the pri-
mers described by Bourguignon et al. (2015). We mixed
the two PCR fragments in equimolar concentration, then
multiplexed and paired-end sequenced them with
Illumina HiSeq2000.
594
We de novo assembled separately 88-bp paired-end reads
using the organelle assembler ORG.asm (available at http://
metabarcoding.org/asm). Each resulting assembly was then
checked by read mapping with the CLC Assembly Cell suite
of programs. ORG.asm failed to assemble 30% of the mito-
chondrial genomes, which were mapped to reference gen-
omes and assembled using the CLC Assembly Cell suite of
Mitochondrial Phylogenomics Resolves the Global Spread of Higher Termites . doi:10.1093/molbev/msw253
programs. Briefly, reads were mapped onto the mitochondrial
genomes of closely related species that served as references.
The sequence of the reference was then replaced by the con-
sensus inferred from the reads. In all cases of polymorphic
bases, we selected the base with the highest representation.
Reads were mapped again onto the new consensus sequence,
and this procedure was repeated until we reached stability.
We inspected by eye all assembled mitochondrial genomes,
with the reads mapped onto them.
We omitted control regions of the mitochondrial genomes
from the final matrix, as they present repetitive patterns that
are generally poorly assembled with short reads, and thus
provide no useful information. We annotated the 22 tRNA
genes, the 13 protein-coding genes, and the two ribosomal
RNA genes by manual checking, aided by previously pub-
lished sequences that we aligned to each mitochondrial gen-
ome using the Muscle algorithm implemented in MEGA 5.2.1
(Tamura et al. 2011), taking into account the codon structure
of the protein-coding genes. The resulting sequence align-
ments were concatenated with SequenceMatrix (Vaidya
et al. 2011).
Phylogenetic Analyses and Molecular Dating
We partitioned the sequence alignment into five subsets:
one for each codon position of the concatenated protein-
coding genes, one for the 12S and 16S rRNA genes, and
one for the tRNA genes. We found no evidence of satur-
ation at the third codon positions of the protein-coding
genes using Xia’s method in DAMBE (NumOTU ¼ 32,
ISS ¼ 0.478, ISS.CAsym ¼ 0.517) (Xia et al. 2003; Xia and
Lemey 2009). However, we detected heterogeneity in
base composition between samples at the third codon
positions (chi-square ¼ 11771.72; df ¼ 672; P < 106).
Therefore, we ran each analysis with and without the
third codon positions, giving preference to the analyses
in which the third codon positions were excluded. This
strategy allowed us to determine the impact of base com-
positional heterogeneity at third codon positions on our
estimates of the tree topology and evolutionary timescale.
We analyzed the concatenated sequence alignment using
the Bayesian phylogenetic software BEAST 1.8.0 (Drummond
and Rambaut 2007). For each subset of the data, we assigned
an independent GTR model of nucleotide substitution with
gamma-distributed rate variation across sites. We imple-
mented two models of rate variation across branches: a strict
clock and an uncorrelated lognormal relaxed clock
(Drummond et al. 2006). In both of these models, we allowed
each subset of the data to have a distinct relative rate. In both
the analyses with and without third codon positions, com-
parison of the marginal likelihood indicated decisive support
for the uncorrelated lognormal relaxed clock over the strict
clock. Therefore, we only present the trees inferred using an
uncorrelated lognormal relaxed clock. A birth–death speci-
ation process was used for the tree prior (Gernhard 2008).
The molecular clock was calibrated using 15 minimum
age constraints (supplementary table S3, Supplementary
Material online). These constraints were based on the
fossil record, and we systematically selected the youngest
MBE
possible age for each fossil as mentioned on the
Paleobiology Database (www.paleobiodb.org; last ac-
cessed on 15 July 2015). Fossil calibrations were imple-
mented as exponential priors on node times (Ho and
Phillips 2009). We allowed the calibrated nodes to be
considerably older than the oldest known fossils because
the termite fossil record is fragmentary and often under-
estimates node age (Ho and Phillips 2009). We chose the
values for the 97.5% soft maximum bounds based on a
combination of phylogenetic bracketing and absence of
fossil evidence (supplementary table S3, Supplementary
Material online).
Posterior distributions of parameters, including the
tree, were estimated using Markov chain Monte Carlo
(MCMC) sampling. We performed three replicate
MCMC runs, with the tree and parameter values sampled
every 20,000 steps over a total of 200 million generations
in each case. Acceptable sample sizes and convergence to
the stationary distribution were checked using Tracer 1.5
(Rambaut and Drummond 2007), with a proportion of
samples removed as burn-in. The remaining samples
from the three MCMC runs were combined and the
maximum-clade-credibility tree was obtained using
TreeAnnotator in BEAST.
To test the sensitivity of our estimates to the choice of
phylogenetic method, we also analyzed the concatenated
sequence alignment using maximum likelihood in RAxML
version 7.7.1 (black-box webserver; http://embnet.vital-it.ch/
raxml-bb/) (Stamatakis et al. 2008). We used a gamma distri-
bution to model rate heterogeneity across sites. Node support
was estimated using 100 bootstrap replicates. We ran analyses
with and without the third codon positions of the protein-
coding genes.
Biogeographic Analyses
We reconstructed the evolution of termite geographic ranges
as a discrete trait using a Bayesian phylogenetic approach in
BEAST 1.8.0 (Drummond and Rambaut 2007). This method
reconstructs the ancestral range as a trait on each tree during
the MCMC, taking into account phylogenetic uncertainty.
We used sample locations to assign a biogeographic area to
each tip. We distinguished four biogeographic areas: Africa,
Australia, South America, and Asia.
We also reconstructed the evolution of termite geographic
ranges using a Bayesian binary model implemented in RASP
2.1 (Yu et al. 2015). We used a fixed (JC) model of state
frequencies with equal rates across sites. The analysis was
performed with the default chain parameters (10 chains of
50,000 steps, with samples drawn every 100 steps and with a
temperature of 0.1). Using an estimated model of state fre-
quencies and gamma-distributed rates across sites did not
substantially change the results. The root distribution was
set to Null and the maximum number of areas for each
node was set to 1. These analyses were carried out for the
trees inferred in our BEAST analysis using the uncorrelated
lognormal relaxed clock, and for the trees inferred using
RAxML.
595
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Supplementary Material
Supplementary figures S1–S6 and tables S1–S4 are available at
Molecular Biology and Evolution online.
Acknowledgments
We are grateful to Tamara Hartke for providing specimens,
and to Jan Krecek for help with species identification. This
work was supported by the LHK fund of the National
University of Singapore; by the Singapore-MIT Alliance for
Research and Technology; by the Alliance National
University of Singapore—Université Sorbonne Paris Cité; by
the Czech Science Foundation (project No. 15-07015Y); by
the Internal Grant Agency of Faculty of Forestry and Wood
Sciences, CULS (IGA A11/16); and by the Grand Agency of the
Czech University of Life Sciences (project CIGA No.
20154320). T.B. was supported by the University of Sydney
through a postdoctoral fellowship.
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