Molecular Phylogenetics and Evolution 98 (2016) 201–209 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev A global molecular phylogeny and timescale of evolution for Cryptocercus woodroaches q ⇑ Yanli Che a,c, Dong Wang b, Yan Shi a, Xiaohong Du a, Yongquan Zhao a, Nathan Lo c, Zongqing Wang a, a College of Plant Protection, Southwest University, Beibei, Chongqing 400716, PR China b Chongqing International Travel Healthcare Center, Jiangbei, Chongqing 400020, PR China c School of Life and Environment Sciences, University of Sydney, Sydney, NSW, Australia article info abstract Article history: Cryptocercus is a genus of sub-social wood-feeding cockroaches that represents the sister group to the Received 9 June 2015 eusocial termites. We generated mitochondrial (12S + 16S rRNA, COII), nuclear (28S rRNA) and Revised 27 January 2016 Blattbacterium endosymbiont (16S + 23S rRNA) sequence data for 8 new Chinese species, and combined Accepted 7 February 2016 these with previously available data to undertake the most extensive analysis of phylogenetic relation- Available online 11 February 2016 ships within the genus to date. As expected, phylogenetic relationships among Blattabacterium strains were found to be congruent with those of their hosts. Three major clades were found to exist in Asian Keywords: populations, one representing taxa from the Hengduan mountains in Southwestern China, a second Wood burrower including taxa from Russia, Korea, Northeastern China, and Yunnan in the Hengduan Mountains, and a Molecular phylogeny Biogeography third including taxa from the Qinling Mountains and Daba Mountains in Central China. A molecular dat- Molecular clock ing analysis using 7 termite fossils to calibrate the molecular clock indicated that the divergence of American and Asian Cryptocercus occurred 55.09 Ma (41.55–72.28 Ma 95% CI), and that the radiations of American and Asian taxa occurred 28.48 Ma (20.83–37.95 Ma 95% CI) and 20.97 Ma (15.78– 27.21 Ma 95% CI) respectively. Reconstruction of ancestral geographic distributions using S-DIVA sug- gested Cryptocercus was originally distributed across both continents, as opposed to ancestral migration of Cryptocercus from one continent to the other. The last common ancestor of Asian Cryptocercus was inferred to have existed in Central China. An examination of male chromosome numbers in Asian Cryptocercus showed that diploid numbers vary from 2n =15to2n = 41, and indicates the presence of eight new species. Our study represents the most comprehensive phylogenetic and biogeographic study yet performed for this important group of cockroaches. Ó 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Klass et al., 2008). Similar to the case for most other cockroaches and the termite Mastotermes darwiniensis, Cryptocercus species har- The wingless and wood-feeding cockroach genus Cryptocercus bor the intracellular bacterial symbiont Blattabacterium cuenoti contains representatives with a uniformly stocky and compact (hereafter Blattabacterium) in their fat-bodies. Previous studies body shape. They have a thick, scoop-shaped pronotum used as a have suggested that Blattabacterium is passed on from mother to tool in defense or digging, and their legs are powerful and fes- offspring in a strictly vertical fashion, and that the phylogenies of tooned with stout, articulated spines. Members of Cryptocercus live host and symbiont are congruent with each other (Clark et al., in biparental family groups in logs and are considered to be 2001; 2003; Lo et al., 2003; Maekawa et al., 2005). monogamous and subsocial insects (Nalepa et al., 2001). They live Before 1997, only three species of Cryptocercus were reported: in temperate forests of the Nearctic, Palaearctic (Clark et al., 2003) C. punctulatus Scudder, 1862, C. relictus Bey-Bienko, 1935 and C. and Oriental Regions (Wang, 2013). Cryptocercus is the sister group primarius Bey-Bienko, 1938. Additional species were subsequently of termites (Lo et al., 2000; Inward et al., 2007), and representa- recognized by Nalepa et al. (1997) (C. clevelandi), Burnside et al. tives of the genus are considered important models for under- (1999) (C. darwini, C. garciai and C. wrighti (but see Nalepa et al., standing the evolution of termite eusociality (Nalepa et al., 2001; 2002)) and Grandcolas et al. (2001) (C. kyebangensis), mainly on the basis of sequence divergence in mitochondrial DNA and q chromosome numbers. Some researchers distinguished additional This paper was edited by the Associate Editor Alfried Vogler. species on the basis of female genitalia (C. matilei, Grandcolas, ⇑ Corresponding author. 2000; C. hirtus, C. meridianus and C. parvus, Grandcolas et al., E-mail address: [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.ympev.2016.02.005 1055-7903/Ó 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 202 Y. Che et al. / Molecular Phylogenetics and Evolution 98 (2016) 201–209 2005; C. convexus and C. arcuatus, Wang et al., 2015). Currently the to increase the accuracy of inferred evolutionary timescales (Ho genus comprises 14 species (Beccaloni, 2014; Wang et al., 2015), and Lo, 2013). eight of which are from China. The phylogenetic relationships and divergence times within 2. Materials and methods and between Palaearctic and Nearctic lineages based on molecular data have been examined on multiple occasions, with variable 2.1. Taxon sampling results. Grandcolas (1999) investigated Palaearctic and Nearctic taxa and hypothesized that the American lineage evolved from The taxa used in this study include all known Cryptocercus spe- the Asian lineage. Grandcolas et al. (2001) inferred the splitting cies. Specimens used in the analysis, and their collection localities event between Asian and American Cryptocercus group to have are shown in Table S1 and Fig. 1. Specimens of Cryptocercus from occurred 18–2 Ma based on mitochondrial 12S and 16S rRNA. A China were collected during June 2011 and October 2014. All spec- molecular clock analysis based on Blattabacterium endosymbiont imens studied were deposited in the Institute of Entomology, DNA performed by Clark et al. (2001) indicated that the divergence Southwest University (SWU), Chongqing, or Museum of Hebei times between Asian and American species were much older, at University (HBU), Baoding, China. Specimens were preserved in 115–70 Ma. The divergence between Asian and American lineages 100% ethanol (SWU) or pinned (HBU) for subsequent sequencing. was inferred by Park et al. (2004) to have occurred 61–26 Ma based Extensive field sampling has shown that populations of Cryptocer- on the COII gene. Maekawa et al. (2005) inferred the divergence cus exist in most of the high-forested regions from the North (Gaol- times between Asian and American lineages at between the late ingzi, Heilongjiang Province) to the South (Yunshanping, Cretaceous and early Tertiary (77.8–58.7 Ma). Yulongxueshan, Yunnan Province) of China. They can live either No study to date has included all known Cryptocercus spp. at a relatively low altitude (702 m, Gaolingzi, Heilongjiang Pro- worldwide to infer the phylogenetic relationships between host vince) or a high altitude (3756 m, Shikaxueshan, Yunnan Province) and Blattabacterium, the timescale of their evolution, and the evo- (Table S1, Fig. 1). During our survey from 2006 to 2014, we failed to lution of their karyotypes. In this study, we sequenced three mito- find Cryptocercus in a number of localities; these are listed in chondrial (12S rRNA, 16S rRNA and COII) genes and one nuclear Table S2. All type specimens and samples of Cryptocercus used in (28S rRNA) gene of all Cryptocercus spp. from China, and two genes this study were deposited in the Institute of Entomology, College of their associated Blattabacterium symbionts (16S rRNA and 23S of Plant Protection, Southwest University (SWU) except some indi- rRNA), including eight new species inferred on the basis of their cated as ‘Museum of Hebei University’ (HBU). karyotypes. Combining these sequences with previously published sequences, we performed phylogenetic and divergence date analy- 2.2. Karyotype analysis ses, and inferred the biogeographic history and evolution of chro- mosome number across all known species within the genus. To For karyotype analysis, a total of 13 species were collected from calibrate evolutionary rates, we used fossils from termites, the 18 locations shown in Table S1. For each of the species described closest relatives of Cryptocercus. Calibrating evolution on the basis (see Supplementary Material), 3 individuals were used. Mitotic of fossils closely related to the taxa under investigation is thought chromosomes from the testes of males (thirteen species except C. Fig. 1. Distribution and collection localities of analyzed specimens of Cryptocercus. Numbers for sampling localities are as indicated in Table S1 (blue circle meaning samples from central and southwestern China, purple circle meaning samples from Manchuria and Korean Peninsula) and Table S2 (empty circle). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Y. Che et al. / Molecular Phylogenetics and Evolution 98 (2016) 201–209 203 hirtus) and from follicle cells