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 Ó 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 (<Q20) were removed using the software CLC The sequencing of the ve Stylotermes samples yielded a total of Genomics Workbench 8 (CLC bio, Aarhus, Denmark). 21 551 173 reads, including 86 041 mitogenome reads (Fig. S3). Mitogenome-like reads were identified with CLC by Although representing only 0.4% of the reads, the coverage comparison to a reference dataset containing 80 termite was over 27 sequences per base in each sample. All Stylotermes mitogenomes (Table S2) using the following parameters: mitogenomes were ~15.8 kb (Table S5), and the gene order was similarity was set to 60% and minimum-length fraction was like that of other termites (Bourguignon et al. 2015; Cameron set to 50%. Mitogenome-like reads were then de novo et al. 2012). assembled using CLC and megahit 1.0 (Li et al. 2015). The Phylogenetic analyses CLC assembling parameters were set to 97% similarity and 50% overlapping, whereas the parameters of megahit were set using The results of the Bayesian and ML phylogenetic analyses were the command ‘–min-count 5 –k-min 21 –k-max 127 –k-step congruent and most nodes were strongly supported (Fig. 3). 6 –merge-level 20,0.97’. The assembly generated large Termites were monophyletic, nested within Blattodea, with scaffolds, generally over 15 kb in length, that we edited to Cryptocercus retrieved as sister group. All termite families obtain a consensus sequence with the software Sequencher 4.8 were monophyletic, with the exceptions of Archotermopsidae, (GeneCode, Boston, USA). within which Hodotermitidae were nested, and Rhinotermitidae, Genes were annotated as described by Cameron (2014). within which 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