Phylogenetic position of the enigmatic termite family Stylotermitidae (Insecta : Blattodea)

Abstract

Termites are eusocial insects currently classified into nine families, of which only Stylotermitidae has never been subjected to any molecular phylogenetic analysis. Stylotermitids present remarkable morphology and have the unique habit of feeding on living trees. We sequenced mitogenomes of five stylotermitid samples from China and Taiwan to reconstruct the phylogenetic position of Stylotermitidae. Our analyses placed Stylotermitidae as the sister group of all remaining Neoisoptera. The systematic position of Stylotermitidae calls for additional studies of their biology, including their developmental pathways and pheromone communication, which have the potential to change our understanding of termite evolution.

Full text

Phylogenetic position of the enigmatic termite family
Stylotermitidae (Insecta : Blattodea)
Li-Wei Wu
A
, Thomas Bourguignon
B,C
, Jan Šobotník
C
, Ping Wen
D
, Wei-Ren Liang
E
and Hou-Feng Li
E,F
A
The Experimental Forest, College of Bio-Resources and Agriculture, National Taiwan University, Nantou, Taiwan.
B
Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan.
C
Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic.
D
Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden,
Chinese Academy of Science, Kunming, Yunnan, China.
E
Department of Entomology, National Chung Hsing University, Taichung, Taiwan.
F
Corresponding author. Email: houfeng@nchu.edu.tw
Abstract. Termites are eusocial insects currently classified into nine families, of which only Stylotermitidae has never
been subjected to any molecular phylogenetic analysis. Stylotermitids present remarkable morphology and have the unique
habit of feeding on living trees. We sequenced mitogenomes of five stylotermitid samples from China and Taiwan to
reconstruct the phylogenetic position of Stylotermitidae. Our analyses placed Stylotermitidae as the sister group of all
remaining Neoisoptera. The systematic position of Stylotermitidae calls for additional studies of their biology, including
their developmental pathways and pheromone communication, which have the potential to change our understanding of
termite evolution.
Additional keywords: Asian relict, mitochondrial genome, molecular systematics, Oriental region.
Received 14 December 2017, accepted 6 April 2018, published online 9 October 2018
Introduction
Termites are eusocial cockroaches, and molecular phylogenies
have confirmed Cleveland’s original hypothesis (Cleveland 1934)
that they are the sister group of the woodroach Cryptocercus
Scudder, 1862 (Inward et al.2007;Loet al.2000). Within
termites, the relationships among the main lineages are well
resolved. Mastotermes Froggatt, 1897, an Australian relict
genus, is the sister group of all other termites. The families
Stolotermitidae Holmgren, 1910, Archotermopsidae Engel,
Grimaldi & Krishna, 2009, and Hodotermitidae Desneux, 1904,
form a monophyletic group, sister to a group composed of the
Kalotermitidae Froggatt, 1897 and the Neoisoptera Engel,
Grimaldi & Krishna, 2009 (Bourguignon et al.2015;Krishna
et al.2013). The phylogenetic positions of the main lineages
within Neoisoptera are reasonablywell resolved, except for that of
the enigmatic StylotermitidaeHolmgren & Holmgren, 1917. One
possible scenario, suggested by morphology-based phylogeny,
is that Stylotermitidae is sister to all the other Neoisoptera Engel
et al.(2009). However, sequences of Stylotermitidae have never
been included in termite molecular phylogenies, making its
phylogenetic position contentious.
Stylotermitidae comprise two fossil genera and a single living
genus, Stylotermes Holmgren & Holmgren, 1917 (45 species;
the general morphology of soldier and worker is represented in
Fig. 1). The taxonomic position of Stylotermes has long been
disputed (Chatterjee and Thakur 1964; Emerson 1971; Engel and
Krishna 2004; Holmgren and Holmgren 1917; Roonwal 1975),
and, currently, Stylotermes is placed in a family on its own
(Engel et al.2009). Stylotermitidae have been reported from
five countries: Bangladesh, China, India, Malaysia and Taiwan
(Krishna et al.2013; Liang et al.2017). Their biology is still
poorly known, except for their tendency to live in large living
trees, on which they feed (Chatterjee and Thakur 1964; Mathur
and Chhotani 1959).
The morphological and ecological characters of Stylotermitidae
are truly remarkable (Fig. 2), and call for clarification of their
phylogenetic position. In this study, we take advantage of
our recent collection of Stylotermes samples in Taiwan and
mainland China to clarify their phylogenetic position.
Materials and methods
Five samples of Stylotermes were collected in Taiwan and
China, in accordance to local regulations. Required permits
were obtained before fieldwork. No endangered or protected
species were negatively influenced by our fieldwork activities.
Journal compilation CSIRO 2018 www.publish.csiro.au/journals/is
CSIRO PUBLISHING
Invertebrate Systematics, 2018, 32, 1111–1117
https://doi.org/10.1071/IS17093

For details, see Fig. S1 and Table S1 available as Supplementary
Material to this paper.
We sequenced the mitochondrial genomes of the five
samples using a shotgun sequencing approach, as described in
previous works (Chen et al.2017; Crampton-Platt et al.2016).
In brief, genomic DNA of five samples of Stylotermes was
extracted from a single termite specimen using the Gentra
Purgene Tissue kit (Gentra Systems, Minneapolis, MN).
Extracted DNA (>100 ng per sample) was fragmented using
Covaris S220 (Covaris Inc., Woburn, MA). DNA fragments
Fig. 1. Stylotermes halumicus (specimen code: Chi15-156). Note the pleural outgrowths on the meso- and
metathorax, which are present in workers. These outgrows differ from nymphal wing-pads.
Fig. 2. Comparison of the morphology of three termite families: Kalotermitidae (top row, Incisitermes inamurai), Stylotermitidae (middle row,
Stylotermes halumicus), Rhinotermitidae (bottom row, Prorhinotermes flavus). A, Wings of imagoes; B, soldier head; C, worker left mandible;
D, worker leg. Modified from Engel et al.(2009) and Krishna et al.(2013).
1112 Invertebrate Systematics L.-W. Wu et al.

between 300 and 600 bp were selected, purified and high-
throughput sequenced with the Illumina MiSeq platform.
Sequencing libraries were constructed and tagged with different
barcodes using the NuGEN Ovation Ultralow Library System
(NuGEN Technologies, San Carlos, CA).
Raw reads of each sample were trimmed, and low-quality
regions (<Q20) were removed using the software CLC
Genomics Workbench 8 (CLC bio, Aarhus, Denmark).
Mitogenome-like reads were identified with CLC by
comparison to a reference dataset containing 80 termite
mitogenomes (Table S2) using the following parameters:
similarity was set to 60% and minimum-length fraction was
set to 50%. Mitogenome-like reads were then de novo
assembled using CLC and megahit 1.0 (Li et al.2015). The
CLC assembling parameters were set to 97% similarity and 50%
overlapping, whereas the parameters of megahit were set using
the command ‘–min-count 5 –k-min 21 –k-max 127 –k-step
6–merge-level 20,0.97’. The assembly generated large
scaffolds, generally over 15 kb in length, that we edited to
obtain a consensus sequence with the software Sequencher 4.8
(GeneCode, Boston, USA).
Genes were annotated as described by Cameron (2014).
Transfer RNAs were predicted using the MITOS webserver
(Bernt et al.2013) and the genetic code of invertebrates.
Predicted tRNA sequences were quality-checked by comparison
with tRNA sequences of Mastotermes darwiniensis (NC018120),
Zootermopsis nevadensis (NC024658), Neotermes insularis
(NC018124), Schedorhinotermes breinli (NC018126) and
Coptotermes formosanus (NC015800). Protein-coding genes
were annotated by eye, using the five species mentioned
above as references. In each case, we found no internal stop
codon. We also annotated the two rRNAs by comparison with
reference sequences. All mitogenomes sequenced in this study
are available on GenBank (accession numbers KY449045–
KY449049) (Table S3). Another 96 mitogenome sequences
were downloaded from GenBank (Table S3). Therefore, a
total of 101 mitogenomes were used to infer the phylogenetic
position of Stylotermes.
We conducted Bayesian phylogenetic analyses in MrBayes
3.2 (Ronquist et al.2012) using the best-fit partition scheme
and substitution models determined by PartitionFinder
(Lanfear et al.2012) (Table S4). The MrBayes analysis was
performed twice, with four chains (three hot and one cold),
and run for 10 million generations. We discarded 2.5 10
6
MCMC steps as burn-in based on inspection of the trace file
using Tracer 1.6 (Rambaut et al.2014). The two independent
runs mixed well (Fig. S2) and reached convergence. We also
conducted Maximum Likelihood (ML) analysis using RAxML
Pthreads-based 7.0.4 (Ott et al.2007; Stamatakis 2006). We
used the best partition scheme determined by PartitionFinder
(Table S4) and a GTRGAMMA substitution model. Branch
support values were evaluated using 1000 bootstrap replicates
with 10 ML searches for each replicate.
In addition, we searched the control region of each Stylotermes
mitogenome for the hairpin loop structure described by Cameron
et al.(2012). This structure is one of the rare genomic changes
(RGC), located in the A + T rich (=putative control), non-coding
region, and probably the origin of replication for the mitogenome
(Cameron et al.2012). Non-Neoisoptera Euisoptera termites
represent a RGC1-type feature, while Neoisoptera represent
a RGC1a-type feature (Cameron et al.2012).
Results
Sequence information
The sequencing of the five Stylotermes samples yielded a total of
21 551 173 reads, including 86 041 mitogenome reads (Fig. S3).
Although representing only 0.4% of the reads, the coverage
was over 27 sequences per base in each sample. All Stylotermes
mitogenomes were ~15.8 kb (Table S5), and the gene order was
like that of other termites (Bourguignon et al.2015; Cameron
et al.2012).
Phylogenetic analyses
The results of the Bayesian and ML phylogenetic analyses were
congruent and most nodes were strongly supported (Fig. 3).
Termites were monophyletic, nested within Blattodea, with
Cryptocercus retrieved as sister group. All termite families
were monophyletic, with the exceptions of Archotermopsidae,
within which Hodotermitidae were nested, and Rhinotermitidae,
within which Serritermitidae and Termitidae were nested. The
five Stylotermitidae we sequenced were grouped together with
high support value, and formed the sister clade of all remaining
Neoisoptera.
The RGC feature of dictyopteran insects is shown in Fig. 4.
The feature of Stylotermes is distinct from that of other termites,
but a Neighbour-Joining tree (Fig. S5) shows that the RGC
structure of Stylotermes is more similar to that of other
Neoisoptera.
Discussion
The monophyly of termites is not in question, but our
understanding of the interfamilial relationships has changed
considerably over time (Bourguignon et al.2015; Donovan
et al.2000; Krishna et al.2013). Stylotermes systematic
position has also been reinterpreted on several occasions
before Engel et al. (Engel et al.2009) raised the family
Stylotermitidae, suggesting it is the sister group of all other
Neoisoptera. Our results, based on full mitochondrial genomes,
validate the morphology-based phylogenetic inferences of
Engel et al. (Engel et al.2009).
The broad distribution of fossil species of Stylotermitidae
(Krishna et al.2013) contrasts with the current distribution of
Stylotermes, limited to subtropical parts and mountain ranges
of south and south-east Asia. This suggests that the present
distribution of Stylotermes is a relict of a past cosmopolitan
distribution, like that of Mastotermes, Archotermopsidae and
Stolotermitidae (Krishna et al.2013). One possible explanation
for the limited distribution of modern Stylotermes is the rise to
dominance of Termitidae during the last 30 million years
that possibly removed other termite taxa from the tropics
(Bourguignon et al.2017; Engel et al.2009).
Currently, 45 species of Stylotermitidae have been
described, but the validity of many of them is questionable,
and the type material inaccessible. A taxonomic revision of
the group is needed, but the paucity of Stylotermes material
limits the eventual initiatives. In this study, four of the five
sequenced Stylotermes samples revealed close relatedness
Phylogenetic position of Stylotermitidae Invertebrate Systematics 1113

(Kimura 2-parameter distance <2%), even though they were
sampled across a large geographic area including Taiwan,
and south-west China (Guangxi and Sichuan provinces).
Their morphological characteristics correspond to those of
S. halumicus (Liang et al.2017). S. halumicus is well separated
from the other species collected in Yunnan province, possibly
Fig. 3. Phylogenetic tree of termites reconstructed using full mitochondrial genomes (for details see Fig. S4).
Node labels: BP, Bayesian posterior probabilities; BS, ML bootstrap values.
Fig. 4. Alignment of hairpin structures in the control region of dictyopteran insects (rare genomic changes 1; RGC 1).
1114 Invertebrate Systematics L.-W. Wu et al.

because of the presence of the Hengduan Mountain Range, which
has been a biological barrier for many organisms (Xie et al.2004).
The large distribution of S. halumicus also suggests the presence
of many synonyms among Chinese species. Indeed, many
species have been described based on specimens from a single
colony, collected from distinct locations and presenting only
small morphological variations among them.
The feeding biology of Stylotermes is truly remarkable
among termites. All species are associated with living trees,
mostly broadleaf, growing at low altitudes across several
mountain ranges (Table S1). All our samples originated from
trees having wounds on trunk or branches. The nesting
environment of Stylotermes is always wet (Fig. 5) (Chatterjee
and Thakur 1963; Tsai et al.1978). The gallery system is made
(A)(B)
(C)
Fig. 5. Nest structure of Stylotermes. A, Trunk of Trema orientalis (L.) wounded and infested by Stylotermes
halumicus (the rectangle indicates the area magnified in B). B, Opening to gallery system of Stylotermes halumicus
(the rectangle indicates the area magnified in C). C, Wet mud-like material deposited in the tree trunk, and two
Stylotermes soldiers defending the opening.
Phylogenetic position of Stylotermitidae Invertebrate Systematics 1115

up of inhabited tunnels of variable sizes at the margin of
living wood tissues, and abandoned galleries filled up with
mud-like material (Chatterjee and Thakur 1963; Chhotani
1983). Colonies of Stylotermes are small (Tsai et al.1978),
and likely composed of no more than a few hundred individuals.
An orphan group of S. halumicus with fewer than 50 workers and
soldiers was held in the laboratory (NCHU, Taiwan), and started
producing replacement reproductives within six months. This set
of characteristics suggests that Stylotermes has a linear
developmental pathway similar to that of the Serritermitidae
and the rhinotermitid genera Prorhinotermes Silvestri, 1909,
Psammotermes Desneux, 1902 and Termitogeton Desneux,
1904 (Barbosa and Constantino 2017; Bourguignon et al.
2009; Bourguignon et al.2012; Parmentier and Roisin 2003;
Roisin 1988).
Stylotermes have particular ecological requirements, not
observed in other termites. They live in and feed on live trees,
a strategy unique and unequalled among termites. Their
ecological requirements are probably closer to that of
Kalotermitidae, nesting in living trees (although Kalotermitidae
mostly live in dead branches), while their morphology is rather
close to that of Rhinotermitidae (Fig. 2). Stylotermes share
several synapomorphies with other Neoisoptera, including the
frontal gland that opens at the frons by a simple pore (Engel et al.
2009)(Fig.2B), imagoes showing simplified wing venation
(Fig. 2A), left mandible of workers and imagoes with three
marginal teeth (Fig. 2C), reduced tibial spurs, and missing
arolium (Fig. 2D). The caste system, as well as many other
ecological aspects of stylotermitid biology, remains unknown
and deserves additional investigation.
Data accessibility
The GenBank accession numbers of the 101 mitogenome
sequences are available as Supplementary Material to this
paper (Table S3).
Conflicts of interest
The authors declare no conflicts of interest.
Authors’contributions
L-WW conducted molecular laboratory work and data analysis.
L-WW and H-FL conceived the study and drafted the
manuscript. PW, TB, JS, W-RL, and H-FL collected samples.
TB, JS, and H-FL coordinated the project, provided critical ideas,
and revised the manuscript.
Acknowledgements
The project was supported by the Ministry of Science and Technology,
Taiwan (MOST 105-2628-B-005-003-MY3; 105-2311-B-002-021), by the
Czech Science Foundation (project No. 15-07015Y), and by the Internal
Grant Agency of Faculty of Forestry and Wood Sciences, CULS (IGA 13/17).
We thank Chun-I Chiu (NCHU) for rearing a colony in the laboratory. We
also thank David Sillam-Dussès, Yves Roisin, Valeria D. Palma-Onetto,
Chun-Wen Lu, Xiao-Lan Wen, and Ming Zhang for their help during
fieldwork.
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