CSIRO PUBLISHING Invertebrate Systematics, 2018, 32, 1111–1117 https://doi.org/10.1071/IS17093

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

AThe ExperimentalForest, College of Bio-Resources and Agriculture,National Taiwan University, Nantou, Taiwan. BOkinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan. CFaculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic. DKey Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Science, Kunming, Yunnan, China. EDepartment of Entomology, National Chung Hsing University, Taichung, Taiwan. FCorresponding author. Email: [email protected]

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 the general morphology of soldier and worker is represented in Termites are eusocial cockroaches, and molecular phylogenies Fig. 1). The taxonomic position of Stylotermes has long been have confirmed Cleveland’s original hypothesis (Cleveland 1934) disputed (Chatterjee and Thakur 1964; Emerson 1971; Engel and that they are the sister group of the woodroach Cryptocercus Krishna 2004; Holmgren and Holmgren 1917; Roonwal 1975), Scudder, 1862 (Inward et al. 2007;Loet al. 2000). Within and, currently, Stylotermes is placed in a family on its own termites, the relationships among the main lineages are well (Engel et al. 2009). Stylotermitidae have been reported from fi resolved. Mastotermes Froggatt, 1897, an Australian relict ve countries: Bangladesh, China, India, Malaysia and Taiwan genus, is the sister group of all other termites. The families (Krishna et al. 2013; Liang et al. 2017). Their biology is still Stolotermitidae Holmgren, 1910, Archotermopsidae Engel, poorly known, except for their tendency to live in large living Grimaldi & Krishna, 2009, and Hodotermitidae Desneux, 1904, trees, on which they feed (Chatterjee and Thakur 1964; Mathur form a monophyletic group, sister to a group composed of the and Chhotani 1959). Kalotermitidae Froggatt, 1897 and the Neoisoptera Engel, The morphological and ecological characters of Stylotermitidae fi Grimaldi & Krishna, 2009 (Bourguignon et al. 2015;Krishna are truly remarkable (Fig. 2), and call for clari cation of their et al. 2013). The phylogenetic positions of the main lineages phylogenetic position. In this study, we take advantage of within Neoisoptera are reasonably well resolved, except for that of our recent collection of Stylotermes samples in Taiwan and the enigmatic Stylotermitidae Holmgren & Holmgren, 1917. One mainland China to clarify their phylogenetic position. 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 Materials and methods been included in termite molecular phylogenies, making its Five samples of Stylotermes were collected in Taiwan and phylogenetic position contentious. China, in accordance to local regulations. Required permits Stylotermitidae comprise two fossil genera and a single living were obtained before fieldwork. No endangered or protected genus, Stylotermes Holmgren & Holmgren, 1917 (45 species; species were negatively influenced by our fieldwork activities.

Journal compilation CSIRO 2018 www.publish.csiro.au/journals/is 1112 Invertebrate Systematics L.-W. Wu et al.

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).

For details, see Fig. S1 and Table S1 available as Supplementary In brief, genomic DNA of five samples of Stylotermes was Material to this paper. extracted from a single termite specimen using the Gentra We sequenced the mitochondrial genomes of the five Purgene Tissue kit (Gentra Systems, Minneapolis, MN). samples using a shotgun sequencing approach, as described in Extracted DNA (>100 ng per sample) was fragmented using previous works (Chen et al. 2017; Crampton-Platt et al. 2016). Covaris S220 (Covaris Inc., Woburn, MA). DNA fragments Phylogenetic position of Stylotermitidae Invertebrate Systematics 1113 between 300 and 600 bp were selected, purified and high- represent a RGC1-type feature, while Neoisoptera represent throughput sequenced with the Illumina MiSeq platform. a RGC1a-type feature (Cameron et al. 2012). Sequencing libraries were constructed and tagged with different barcodes using the NuGEN Ovation Ultralow Library System Results (NuGEN Technologies, San Carlos, CA). Sequence information Raw reads of each sample were trimmed, and low-quality fi regions (Serritermitidae and Termitidae were nested. The Transfer RNAs were predicted using the MITOS webserver five Stylotermitidae we sequenced were grouped together with (Bernt et al. 2013) and the genetic code of invertebrates. high support value, and formed the sister clade of all remaining Predicted tRNA sequences were quality-checked by comparison Neoisoptera. with tRNA sequences of Mastotermes darwiniensis (NC018120), The RGC feature of dictyopteran insects is shown in Fig. 4. Zootermopsis nevadensis (NC024658), Neotermes insularis The feature of Stylotermes is distinct from that of other termites, (NC018124), Schedorhinotermes breinli (NC018126) and but a Neighbour-Joining tree (Fig. S5) shows that the RGC Coptotermes formosanus (NC015800). Protein-coding genes structure of Stylotermes is more similar to that of other were annotated by eye, using the five species mentioned Neoisoptera. above as references. In each case, we found no internal stop codon. We also annotated the two rRNAs by comparison with Discussion reference sequences. All mitogenomes sequenced in this study The monophyly of termites is not in question, but our are available on GenBank (accession numbers KY449045– understanding of the interfamilial relationships has changed KY449049) (Table S3). Another 96 mitogenome sequences considerably over time (Bourguignon et al. 2015; Donovan were downloaded from GenBank (Table S3). Therefore, a et al. 2000; Krishna et al. 2013). Stylotermes systematic total of 101 mitogenomes were used to infer the phylogenetic position has also been reinterpreted on several occasions position of Stylotermes. before Engel et al. (Engel et al. 2009) raised the family We conducted Bayesian phylogenetic analyses in MrBayes Stylotermitidae, suggesting it is the sister group of all other 3.2 (Ronquist et al. 2012) using the best-fit partition scheme Neoisoptera. Our results, based on full mitochondrial genomes, and substitution models determined by PartitionFinder validate the morphology-based phylogenetic inferences of (Lanfear et al. 2012) (Table S4). The MrBayes analysis was Engel et al. (Engel et al. 2009). performed twice, with four chains (three hot and one cold), The broad distribution of fossil species of Stylotermitidae and run for 10 million generations. We discarded 2.5 Â 106 (Krishna et al. 2013) contrasts with the current distribution of MCMC steps as burn-in based on inspection of the trace file Stylotermes, limited to subtropical parts and mountain ranges using Tracer 1.6 (Rambaut et al. 2014). The two independent of south and south-east Asia. This suggests that the present runs mixed well (Fig. S2) and reached convergence. We also distribution of Stylotermes is a relict of a past cosmopolitan conducted Maximum Likelihood (ML) analysis using RAxML distribution, like that of Mastotermes, Archotermopsidae and Pthreads-based 7.0.4 (Ott et al. 2007; Stamatakis 2006). We Stolotermitidae (Krishna et al. 2013). One possible explanation used the best partition scheme determined by PartitionFinder for the limited distribution of modern Stylotermes is the rise to (Table S4) and a GTRGAMMA substitution model. Branch dominance of Termitidae during the last 30 million years support values were evaluated using 1000 bootstrap replicates that possibly removed other termite taxa from the tropics with 10 ML searches for each replicate. (Bourguignon et al. 2017; Engel et al. 2009). In addition, we searched the control region of each Stylotermes Currently, 45 species of Stylotermitidae have been mitogenome for the hairpin loop structure described by Cameron described, but the validity of many of them is questionable, et al.(2012). This structure is one of the rare genomic changes and the type material inaccessible. A taxonomic revision of (RGC), located in the A + T rich (=putative control), non-coding the group is needed, but the paucity of Stylotermes material region, and probably the origin of replication for the mitogenome limits the eventual initiatives. In this study, four of the five (Cameron et al. 2012). Non-Neoisoptera Euisoptera termites sequenced Stylotermes samples revealed close relatedness 1114 Invertebrate Systematics L.-W. Wu et al.

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).

(Kimura 2-parameter distance <2%), even though they were Their morphological characteristics correspond to those of sampled across a large geographic area including Taiwan, S. halumicus (Liang et al. 2017). S. halumicus is well separated and south-west China (Guangxi and Sichuan provinces). from the other species collected in Yunnan province, possibly Phylogenetic position of Stylotermitidae Invertebrate Systematics 1115 because of the presence of the Hengduan Mountain Range, which The feeding biology of Stylotermes is truly remarkable has been a biological barrier for many organisms (Xie et al. 2004). among termites. All species are associated with living trees, The large distribution of S. halumicus also suggests the presence mostly broadleaf, growing at low altitudes across several of many synonyms among Chinese species. Indeed, many mountain ranges (Table S1). All our samples originated from species have been described based on specimens from a single trees having wounds on trunk or branches. The nesting colony, collected from distinct locations and presenting only environment of Stylotermes is always wet (Fig. 5) (Chatterjee small morphological variations among them. 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. 1116 Invertebrate Systematics L.-W. Wu et al. up of inhabited tunnels of variable sizes at the margin of Bernt, M., Donath, A., Jühling, F., Externbrink, F., Florentz, C., Fritzsch, G., living wood tissues, and abandoned galleries filled up with Pütz, J., Middendorf, M., and Stadler, P. F. (2013). MITOS: improved mud-like material (Chatterjee and Thakur 1963; Chhotani de novo metazoan mitochondrial genome annotation. Molecular – 1983). Colonies of Stylotermes are small (Tsai et al. 1978), Phylogenetics and Evolution 69, 313 319. doi:10.1016/j.ympev.2012. and likely composed of no more than a few hundred individuals. 08.023 Bourguignon, T., Šobotník, J., Hanus, R., and Roisin, Y. (2009). An orphan group of S. halumicus with fewer than 50 workers and Developmental pathways of Glossotermes oculatus (Isoptera, soldiers was held in the laboratory (NCHU, Taiwan), and started Serritermitidae): at the cross-roads of worker caste evolution in producing replacement reproductives within six months. This set termites. Evolution & Development 11, 659–668. doi:10.1111/j.1525- of characteristics suggests that Stylotermes has a linear 142X.2009.00373.x developmental pathway similar to that of the Serritermitidae Bourguignon, T., Šobotník,J., Sillam-Dussès, D., Jiroš, P., Hanus, R., Roisin, and the rhinotermitid genera Prorhinotermes Silvestri, 1909, Y., and Miura, T. (2012). Developmental pathways of Psammotermes Psammotermes Desneux, 1902 and Termitogeton Desneux, hybostoma (Isoptera: Rhinotermitidae): old pseudergates make up 1904 (Barbosa and Constantino 2017; Bourguignon et al. a new sterile caste. PLoS One 7, e44527. doi:10.1371/journal.pone. 2009; Bourguignon et al. 2012; Parmentier and Roisin 2003; 0044527 Š Roisin 1988). Bourguignon, T., Lo, N., Cameron, S. L., obotník, J., Hayashi, Y., Shigenobu, S., Watanabe, D., Roisin, Y., Miura, T., and Evans, T. A. Stylotermes have particular ecological requirements, not (2015). The evolutionary history of termites as inferred from 66 observed in other termites. They live in and feed on live trees, mitochondrial genomes. Molecular Biology and Evolution 32, a strategy unique and unequalled among termites. Their 406–421. doi:10.1093/molbev/msu308 ecological requirements are probably closer to that of Bourguignon, T., Lo, N., Šobotník, J., Ho, S. Y. W., Iqbal, N., Coissac, E., Kalotermitidae, nesting in living trees (although Kalotermitidae Lee, M., Jendryka, M., Sillam-Dussès, D., Krížková, B., Roisin, Y., and mostly live in dead branches), while their morphology is rather Evans, T. A. (2017). Mitochondrial phylogenomics resolves the global close to that of Rhinotermitidae (Fig. 2). Stylotermes share spread of higher termites, ecosystem engineers of the tropics. Molecular several synapomorphies with other Neoisoptera, including the Biology and Evolution 34, 589–597. frontal gland that opens at the frons by a simple pore (Engel et al. Cameron, S. L. (2014). How to sequence and annotate insect mitochondrial 2009)(Fig.2B), imagoes showing simplified wing venation genomes for systematic and comparative genomics research. Systematic Entomology 39, 400–411. doi:10.1111/syen.12071 (Fig. 2A), left mandible of workers and imagoes with three Cameron, S. L., Lo, N., Bourguignon, T., Svenson, G. J., and Evans, T. A. marginal teeth (Fig. 2C), reduced tibial spurs, and missing (2012). A mitochondrial genome phylogeny of termites (Blattodea: arolium (Fig. 2D). The caste system, as well as many other Termitoidae): robust support for interfamilial relationships and molecular ecological aspects of stylotermitid biology, remains unknown synapomorphies define major clades. Molecular Phylogenetics and and deserves additional investigation. Evolution 65, 163–173. doi:10.1016/j.ympev.2012.05.034 Chatterjee, P.N., and Thakur, M.L. (1963). Biology and ecology of Oriental Data accessibility termites (Isoptera). Some observations on Sarvaritermes faveolus The GenBank accession numbers of the 101 mitogenome Chatterjee and Thakur (Isoptera: Stylotermitidae). Indian Forester 89, sequences are available as Supplementary Material to this 635–637. paper (Table S3). Chatterjee, P. N., and Thakur, M. L. (1964). 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