Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v60i2.135-144Sociobiology 60(2): 135-144 (2013) Molecular Phylogeny of the Ant Subfamily Formicinae (Hymenoptera, Formicidae) from China Based on Mitochondrial Genes ZL Chen1, SY Zhou1, DD Ye1, Y Chen1, CW Lu1 Introduction Ants are one of the most successful groups of eusocial insects. They act as an important part of the animal biomass in tropical rainforests and occupy key positions in many ter- restrial environments (Wilson & Hölldobler 2005). Resolving the phylogeny of major ant lineages is vital for understand- ing the factors contributing to their success. Previous studies based on morphological (Baroni Urbani et al. 1992, Bolton 2003), fossil-based (Grimaldi et al. 1997, Dlussky 1999, Ward & Brady 2003, Bolton 2003), and molecular (Astruc et al. 2004, Saux et al. 2004, Ward & Brady 2003,Ward & Downie 2005, Ward et al. 2005, Brady et al. 2006, Moreau et al. 2006, Ouellette et al. 2006) data provided useful framework for un- derstanding the relationships among ant subfamilies. How- ever, relationships among genera within the subfamilies are not well understood. In addition, the genus-level phylogeny and classification of ant subfamilies remain controversial in many respects. Formicinae is one of the most abundant ant subfamilies Abstract To resolve long-standing discrepancies in the relationships among genera within the ant subfamily Formicinae, a phylogenetic study of Chinese Formicine ants based on three mitochondria genes (Cyt b, COI, COII) was conducted. Phylogenetic trees ob- tained in the current study are consistent with several previously reported trees based on morphology, and specifically confirm and reinforce the classifications made by Bol- ton (1994). The tribes Lasiini, Formicini, Plagiolepidini and Camponotini are strongly supported, while Oecophyllini has moderate support despite being consistent across all analyses. We have also established that the genus Camponotus and Polyrhachis are indeed not monophyletic. Additionally, we found strong evidence for Polyrhachis pa- racamponota, as described by Wu and Wang in 1991, to be corrected as Camponotus based on molecular, morphological and behavioral data. Sociobiology An international journal on social insects 1 - College of Life Sciences, Guangxi Normal University, Guilin, China RESEARCH ARTICLE - ANTS Article History Edited by: Gilberto M. M. Santos, UEFS - Brazil Received 26 December 2012 Initial acceptance 19 February 2013 Final acceptance 08 April 2013 Keywords Ant phylogeny; Formicidae; Cyt b, COI, COII Corresponding author: Shan-Yi Zhou College of Life Sciences Guangxi Normal University Guilin, 541004, China. E-Mail: syzhou5612@yahoo.com.cn in the Holarctic (Wilson 1955). According to Bolton (2012), Formicinae includes 49 extant genera and over 3700 species and subspecies in the world. Although the subfamily includes a large number of abundant and ecologically important spe- cies that are often subjected to ecological and sociobiological studies, little is known about their phylogeny. Although there are several classifications based on a variety of morphologi- cal characteristics, such as sexual traits and larval morphol- ogy (Wheeler 1922, Emery 1925, Wheeler & Wheeler 1985, Agosti 1991, Bolton 1994, 2003), the tribes or genus-groups represent artificial assemblages and are used inconsistently by different myrmecologists or even by the same myrmecologist at different times. In particular, some aspects of worker mor- phology show a strong tendency towards convergence, making it challenging to infer phylogenetic relationships from mor- phological characteristics alone (Ward 2007). Indeed, Bolton has acknowledged that some tribes in his tribal arrangements would likely need to be re-evaluated (Bolton 2003). No molecular phylogenetic study has been performed on the subfamily Formicinae in China to date. This study ZL Chen, SY Zhou, DD Ye, Y Chen, CW Lu - Molecular Phylogeny of Formicinae from China136 aimed to establish molecular relationships among Formicinae members relative to previously established frameworks and to take a deeper look into species level relationships within more ambiguous assemblages. This was done by obtaining sequences of the mitochondrial genes cytochrome b (Cyt b), cytochrome oxidase subunit 1 (COI) and cytochrome oxidase subunit 2 (COII) and comparing them using Bayesian Infer- ence (BI) (Nylander 2004), Maximum Parsimony (MP) and Neighbour Joining (NJ) (Swofford 2002). Materials and Methods Taxon sampling In this study, a total of 47 species representing 14 gen- era from five tribes were selected to test the groups suggested by the tribal structure and dendrograms of Wheeler (1922), Emery (1925), Wheeler and Wheeler (1985), Agosti (1991), and Bolton (1994, 2003). Cerapachys sulcinodis from the subfamily Cerapachyinae and Radoszkowskius oculata from the family Mutillidae were added as outgroups. Apart from R. oculata, all other vouchers of Formicinae and C. sulcinodis, consisting of nestmate specimens from the same collection event have been deposited in the collection of Guangxi Nor- mal University. Detailed information of the species studied is listed in Appendix 1. DNA extraction, PCR, and sequencing alignment Total genomic DNA was extracted from ground whole workers, of which the gasters were removed to minimize con- tamination from gut bacteria, using standard CTAB methods (slightly modified from Navarro et al. 1999). DNA sequence data from three protein-coding mitochondrial genes, namely Cyt b, COI, and COII, were obtained using conventional PCR methods (Villesen et al. 2004, Ward & Downie 2005). The se- quences and positions on the mitochondrial DNA of the prim- ers used for PCR and sequencing are shown in Table 1. The primers J2791 and H3665 were used to amplify fragments of mitochondrial DNA that correspond to the 3’ end of COI, ITS, and tRNA-leucine and the 5’ end of COII. Fragments were sequenced in both directions, and the result- ing chronograms were assembled and edited using DNAStar (Bioinformatics Pioneer DNAStar, Inc., WI). Sequence for each gene fragment was aligned using CLUSTALX v.1.83 (Thompson et al. 1997). Sites from the intergenic spacer (ITS) and tRNA-leucine were not used in the analyses. All new DNA sequences generated in this study were submitted to the NCBI GenBank database. Sequence data of the outgroup R. oculata was obtained via GenBank direct submission by Wei, S.J. and Chen, X.X. All GenBank accession numbers related to this study are listed in Appendix 1. Phylogenetic analyses Reconstruction of phylogenetic relationships among taxa was conducted using NJ, MP, and BI methods. NJ analy- sis was performed using PAUP* Version 4.0b10 (PPC) (Swof- ford 2002). Estimates of nodal support on distance trees were obtained using bootstrap analyses (1000 replications). MP analysis was also unweighted and performed using PAUP* Version 4.0b10 (PPC) (Swofford 2002). It involved the use of a heuristic search with random sequence addition (10 repli- cates each) and the TBR branch-swapping algorithm. Bayes- ian phylogenetics was used to estimate tree topology using MRBAYES v.3.1.2 (Ronquist & Huelsenbeck 2003). Data were partitioned by gene to yield a total of three data par- titions, and the best-fitting model for each partition was se- lected using MRMODELTEST v. 2.2 (Nylander 2004) under Akaike information criteria (Posada & Buckley 2004). Results DNA sequence composition Table 2 shows the nucleotide content and substitution of three fragment sequences. The final data matrix contained 1830 characters (1049 variable sites, 897 parsimony-informa- tive sites, 152 singleton sites) from the following gene frag- ments: Cyt b-447 characters (270 variable sites, 232 parsi- mony-informative sites, 38 singleton sites), COI-825 aligned characters (433 variable sites, 379 parsimony-informative sites, 54 singleton sites), and COII-558 characters (341 vari- able sites, 289 parsimony-informative sites, 52 singleton Designation Sequence (5’–3’) Position Reference CB-11400 TATGTACTACCHTGAGGDCAAATATC 9381-9406 Modified from Folmer et al. 1994 CB- 11884 ATTACACCNCCTAATTTATTAGGRAT 9840-9865 Modified from Folmer et al. 1994 LCO1490 GGTCAACAAATCATAAAGATATTGG 117-141 Modified from Folmer et al. 1994 HCO2198 TAAACTTCAGGGTGACCAAAAAATCA 700-726 Modified from Folmer et al. 1994 J2791 ATACCHCGDCGATAYTCAGA 1300-1319 Modified from Chiotis et al. 2000 CO-R TCRTGRAAGAAGATTATTA 1650-1668 This study CO-F CTTTTATTAAAAATHAACAC 1586-1605 This study H3665 CCACARATTTCWGAACATTG 2177-2196 Modified from Chiotis et al. 2000 Table 1. Sequences of primmer used in this study. Position refers to coordinates in the Solenopsis invicta mitochondrion com- plete genome, GenBank accession numbers: HQ215540. Primer combinations are as follows, with the forward primer listed Wrst for each pair: CB-11400–CB-11884, LCO1490–HCO2198, J2791–H3665, J2791–COI-R, CO-F–H3665. Sociobiology 60(2): 135-144 (2013) 137 sites). The base composition of these three fragments varied among the studied species. On average, the base composi- tion was: T 40.8%, C 17.8%, A 31.9%, and G 9.5%, with a strong AT bias (72.7%) as is commonly found in other insect mitochondrial genomes (Vogler & Pearson 1996). The A+T contents of the third, second and first codon position from the three fragments were 84.2%, 66.2%, and 67.4%, respectively. The transitions of nucleotide substitution were more common than transversion with a transition. Numerically, the transver- sion between A and T was the highest among the four types of nucleotide transversions, whereas the transition between C and T was the highest of the two types of nucleotide transi- tions. Amino acid composition and substitution saturation The complete 1830 nucleotide sequence encoded 610 amino acids of 20 different types. Leucine (Leu) was the most frequent (13.53%) followed by isoleucine (Ile) (13.30%). Cysteine (Cys) was the least frequent, with a constant con- tent of 0.29%. All three protein-coding genes were tested for saturation. These were achieved by plotting the numbers of observed substitutions versus the uncorrected p-distance es- timates. The scattergrams (Fig. 1) show that TV increased along the uncorrected p-distance and TS reached saturation between certain pairs of taxa. Phylogenetic trees Phylogenetic analyses (Figs. 2 to 4) showed that the outgroups C. sulcinodis and R. oculata were well-resolved from the Formicinae taxa at the base of the trees with high confidence values (0.94 Bayesian posterior probability (PP), 100% NJ bootstrap, 99% MP bootstrap). As shown in Figure 5E (this Figure was synthesized from Figs. 2 to 4 ), all con- sensus trees strongly indicated that the 14 genera of Formici- nae could be divided into five lineages, which we labeled as clades I-V, and consisted of genera from the tribes Lasiini, Formicini, Oecophyllini, Plagiolepidini and Camponotini, respectively. Our findings are consistent with morphological classifications of Bolton (1994) (Figs. 5E and 5F). Clade I included four genera: Lasius, Nylanderia, Pre- nolepis, Pseudolasius (1.0 PP, 84% NJ bootstrap, 54% MP bootstrap). Pseudolasius appeared to be a sister group of 0 50 100 150 200 250 300 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 P-distance T S o r T V v a l u e s ts tv genes Cs V Pi S Nuleotide content (%) Nuleotide substitution T C A G A+T ii si sv R COI (825) 392 433 379 54 40.8 18.3 30.1 10.7 70.9 664 78 83 0.94 COII (558) 217 341 289 52 40.7 16.6 35.2 7.4 75.9 440 52 66 0.79 Cyt b (447) 177 270 232 38 40.7 18.4 31.2 9.7 71.9 349 45 52 0.88 Total (1830) 781 1049 879 152 40.8 17.8 31.9 9.5 72.7 1454 175 200 0.87 (Lasius + (Nylanderia + Prenolepis)) in all three trees. These analyses showed that Nylanderia is a sister genus of Prenole- pis with very strong support (1.0 PP, 90% NJ bootstrap, 89% MP bootstrap). A supported clade of ((Formica + Polyergus) + (Proformica + Cataglyphis)) (1.0 PP, 73% NJ bootstrap, 73% MP bootstrap) forms Clade II. Our analyses showed For- mica as a sister genus of Polyergus (1.0 PP, 97% NJ bootstrap, 97% MP bootstrap), and Proformica as a sister genus of Cata- glyphis with very strong support (1.0 PP, 97% NJ bootstrap, 91% MP bootstrap) in all trees. Clade III included only one species (Oecophylla smaragdina) and was placed as a sister group to Clade II. Although this species was not supported by strong bootstrap values (0.58 PP, 54% NJ bootstrap, 16% MP bootstrap), it was a consistent feature in all reconstruc- tions. Clade IV comprised of three genera: Anoplolepis, a sis- ter group to (Plagiolepis + Lepisiota). The genus Plagiolepis and Lepisiota also formed a sister group with good support in all trees. Clade V included Camponotus and Polyrhachis with very strong support (1.0 PP, 100% NJ bootstrap, 87% MP bootstrap). However the species-level phylogeny of the genera remains unresolved except for the distinct subclade of Fig.1. Scatterplots showing the number of substitutions (y-axes; TS, transitions; TV, transversions) versus uncorrected p-distance (x-ax- es) at each codon position. Table 2. The content and substation of nucleotide sequences. Cs, conserved sites; V, variable sites; Pi, parsimony-informative sites; S, Singleton sites; ii, identical pairs; si, transitional pairs; sv, transversional pairs; R, Ts/Tv. ZL Chen, SY Zhou, DD Ye, Y Chen, CW Lu - Molecular Phylogeny of Formicinae from China138 (C. mitis + (C. vanispinus + (C. jianghuaensis + C. albospar- sus))). C. singularis is a sister species of other species of the genus Camponotus (including Polyrhachis paracamponota, excluding C. yiningensis) with very strong support (98% NJ bootstrap) in the NJ tree (Fig. 3) and modest support (67% MP bootstrap) in the MP tree (Fig. 2). However, in the BI tree (Fig. 4), C. parius first clustered with C. wasmanni with strong support (1.0 PP) and then as a sister group of C. sin- Fig. 2. Maximum-parsimony (MP) consensus tree from 1000 bootstrap replicates, obtained from 48 species of the concatenated sequences of the Cytb gene (447 bp), COI gene (825 bp) and COII gene (558 bp), with Cerapachy sulcinodis and Radoszkowskius oculata as the outgroups. gularis plus the rest of the species of Camponotus (including P. paracamponota, excluding C. yiningensis). C. yiningensis was tightly associated with Polyrhachis with very strong sup- port (1.0 PP, 100% NJ bootstrap, 87% MP bootstrap), and further studies on its status are needed. The species P. para- camponota clustered with Camponotus, and was distinct from Polyrhachis. Sociobiology 60(2): 135-144 (2013) 139 Fi g. 3 . N ei gh bo r- jo in in g (N J) c on se ns us tr ee f ro m 1 00 0 bo ot st ra p re pl ic at es , o bt ai ne d fr om 4 8 sp ec ie s of th e co nc at en at ed s eq ue nc es o f th e C yt b g en e (4 47 b p) , C O I g en e (8 25 b p) a nd C O II ge ne (5 58 b p) , w ith C er ap ac hy s ul ci no di s an d R ad os zk ow sk iu s oc ul at a as th e ou tg ro up s. Fi g. 4 . B ay es ia n (B I) m aj or ity -r ul e co ns en su s tr ee , o bt ai ne d fr om 4 8 sp ec ie s of th e co nc at - en at ed s eq ue nc es o f th e C yt b g en e (4 47 b p) , C O I ge ne ( 82 5 bp ) an d C O II g en e (5 58 b p) th re e pa rt iti on s al l u nd er th e sa m e be st -fi t m od el (G T R +I +G ) s el ec tin g by A IC in M od el te st , w ith C er ap ac hy s ul ci no di s an d R ad os zk ow sk iu s oc ul at a as th e ou tg ro up s. ZL Chen, SY Zhou, DD Ye, Y Chen, CW Lu - Molecular Phylogeny of Formicinae from China140 lasius, Prenolepis, Nylanderia and Lasius were placed and formed the tribe Lasiini, but disagrees with that of Bolton (2003), in which the genera Plagiolepis and Lepisiota were added to form the tribe Plagiolepidini. In addition, these four genera formed a strongly supported group in all trees, espe- cially in the case of the sister genus relationship between Ny- landeria and Prenolepis (1.0PP, 90% NJ bootstrap, 99% MP bootstrap). These results are consistent with those of previ- ous morphological (Emery 1925, Wheeler & Wheeler 1953, Trager 1984) and molecular studies (Brady et al. 2006), How- ever, in the study of Moreau et al. (2006), the genus Plagi- olepis, Pseudolasius and Prenolepis emerges first, followed by Lasius along with other two genera. Besides the study by LaPolla et al. (2010) in which Prenolepis was treated as be- ing paraphyletic to the group. In addition, monophyly of the genus Lasius was strongly supported (0.99 PP, 90% NJ boot- strap, 99% MP bootstrap). The results for clade II are consistent with those of previous studies (Bolton 1994, 2003) (Figs. 5E, 5F and 5B). Genera of the tribe Formicini share the following morpholog- ical features (Bolton 1994): 12-segmented antennae, antennal Fig. 5 Classifications of Formicine genera based on the schemes of: (A) Wheeler WM 1922; (B) Bolton 2003; (C) Wheeler, WM et al. 1985; (D) Ag- osti 1991; (E) This study; (F) Bolton 1994. {NB: only positions for species of interest in this phylogeny are noted; there are changes in classifications of other genera which are not being used in this study }. Discussion Results of the phylogenetic relationships of Formici- nae in this study (Figs. 2 to 4, 5E) showed both similarities and differences compared with those of previous studies (Fig. 5A-5D, 5F). Surprisingly, results of our molecular phyloge- netic trees have better fit with the morphological cladogram of Bolton (1994), with which they are congruent, than with that of Bolton (2003). Clade I is best characterized morphologically with the worker alitrunk not conspicuously constricted or other- wise specialized and the mesonotum typically convex in pro- file view. The workers of Lasius, Nylanderia and Prenolepis shared the following morphological characters (Bolton 1994): mandibles roughly triangular with four to seven teeth, anten- nae 12-segmented, the torula close to but not touching the posterior clypeal margin. A propodeal spiracle present at or near the declivity of the propodeum, and the petiolar node in profile usually inclined forward, with a short anterior face and much longer posterior face. These data support the earlier hypothesis proposed by Bolton in 1994, into which Pseudo- Sociobiology 60(2): 135-144 (2013) 141 sockets situated close to the posterior clypeal margin. Orifices of propodeal spiracle oval, elliptical, or as elongated slits and near-vertical or inclined from the vertical. All of these analyses provided strong support for the two sister-group relationships of (Formica + Polyergus) and (Proformica + Cataglyphis), which is consistent with the molecular studies of Moreau et al. (2006). In clade III, the genus Oecophylla was separated as a distinct lineage. This result is well supported by previous mor- phological studies (Wheeler 1922, Wheeler & Wheeler 1985, Bolton 1994, 2003) (Fig. 5), which showed Oecophylla as the tribe Oecophllini. In our molecular phylogeny, Oecophylla appears to be a sister of Formicini but with low bootstrap support (0.58 PP, 0.54% NJ bootstrap, 16% MP bootstrap). However, this topology is in agreement with that of Moreau et al. (2006). Wilson and Taylor (1964) also suggested that Oecophylla and clade II cannot be given much credence con- sidering the separate placement in morphologically and parsi- mony-based phylogenies, as well as its current geographical separation. However, fossil evidence indicate that Oecophylla previously occurred in Europe, suggesting that these genera may have shared a common ancestor. Clade IV is a well supported clade consisting of mem- bers from the tribe Plagiolepidini (Anoplolepis + (Plagiolepis + Lepisiota)) (0.95 PP, 82% NJ bootstrap, 53% MP bootstrap). Bolton (1994) had previously placed the three genera into the tribe Plagiolepidini based on a morphological study (Fig. 5F) and the current study is the first to arrive at the same place- ment based on molecular phylogenetics. This tribe is distin- guished by the following features: worker with 11-segmented antennae, antennal sockets fused with the posterior clypeal margin, and palp formula of 6,4. Surprisingly, Bolton (2003) proposed the genus Plagiolepis and Lepisiota to be included in the tribe Plagiolepidini (Fig. 5B). Although Bolton (2003) represents a more comprehensive summary of ant morpholog- ical characters assembled to date than his previous treatment (Bolton 1994), it is likely that this reflects a genuine conflict between morphology and molecular data. Clade V is strongly supported in all trees (1.0 PP, 100% NJ bootstrap, 87% MP bootstrap) and consists of Campono- tus and Polyrhachis. This result is in agreement with previ- ous morphological (Wheeler 1922, Emery 1925b, Wheeler & Wheeler 1985, Bolton 1994, 2003) (Figs. 5) and molecular studies (Astruc et al. 2004, Brady et al. 2006, Moreau et al. 2006). The tribe Camponotini can be characterized by its 12- segmented antennae, with antennal sockets situated far be- hind the posterior clypeal margin, and a palp formula of 6,4. Camponotus is however a paraphyletic group, as is noted in other studies (Brady et al. 1999, Astruc et al. 2004, Brady et al. 2006). Camponotus yiningensis has been placed outside of the genus Camponotus, which has been confirmed not to be monophyletic (Brady et al. 1999, 2000; Astruc et al. 2004, Brady et al. 2006). Morphological characters also reflected close, and sometimes overlapping, relationships between Camponotus and Polyrhachis. For instance, many species of Camponotus acquired distinctive spines, and many species of Polyrhachis have camber-shaped alitrunks. The species Polyrhachis paracamponota was first described by Wang and Wu in 1991 based on a single holotype worker which possess- es pronotal spines, and was placed in the genus Polyrhachis. But having pronotal spines is very common in Camponotus and Polyrhachis, this morphological character could not be used for distinguishing between the two genera. The original descriptions exact match with the morphological character of the genus Camponotus. In our opinion, the authorships also had the same idea, so this species be named “paracampono- ta”. Besides, this species has polymorphic workers, and they have been observed to tunnel into the soil for subterranean nesting. In contrast, the workers of Polyrhachis are exclu- sively monomorphic, and can only use existing cavities in the soil or under stones for nesting, but never excavate tunnels themselves. Our phylogenetic reconstruction indicated that this species is associated with Camponotus, and is clearly separated from Polyrhachis. As such, there is strong evidence from morphological, behavioristic and molecular data that Polyrhachis paracamponota should be placed as a member of Camponotus. Conclusion In conclusion, our study of the phylogenetic relation- ship of Formicinae from China based on sequences from three protein-coding mitochondrial genes (Cyt b, COI, COII) con- firms and reinforces the findings of previous morphological studies (Bolton 1994). The tribes Lasiini (Pseudolasius, Pre- nolepis, Paratrechina, Lasius), Formicini (Formica, Cata- glyphis, Proformica, Polyergus), Plagiolepidini (Lepisiota, Plagiolepis, Anoplolepis), and Camponotini (Camponotus, Polyrhachis) are strongly supported, while Oecophyllini has moderate support despite being consistent across all analy- ses. We have also established that the genus Camponotus and Polyrhachis are indeed not monophyletic. Additionally, evidence from molecular, morphological and behavioral data indicates that Polyhachis paracamponota should be corrected as Camponotus. Acknowledgments We sincerely thank Professor Yu-Feng Xu (National Taiwan Normal University, Taiwan), Dr. Jun-Hao Tang (Na- tional University of Singapore) and Dr. John R. Fellowes (Ka- doorie Farm and Botanic Garden, Hong Kong) for review- ing the English text. We thank two anonymous reviewers for helpful comments on the manuscript. Thanks also to Chao-Tai Wei (Guangxi Normal University) for providing us with some ant materials, De-Long Zeng (Guangxi Normal University) for helpful assistance and comments on phylogeny analysis. This study was supported by the National Natural Science ZL Chen, SY Zhou, DD Ye, Y Chen, CW Lu - Molecular Phylogeny of Formicinae from China142 Foundation of China (Project Nos. 30770258 and 31071971), Foundation of the Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Ministry of Education, Guangxi Normal University. References Agosti, D. (1991). 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ZL Chen, SY Zhou, DD Ye, Y Chen, CW Lu - Molecular Phylogeny of Formicinae from China144 Appendix 1 Species Collection locality Voucher specimen GenBank accession numbers Cyt b COI COI & COII Lepisiota xichangensis Jingxi, Guangxi GXJX0006 JQ681097 JQ681046 JQ680992 Plagiolepis manczshurica Helan Mt, Inner Mongolia NMHL0422 JQ681098 JQ681047 JQ680993 Plagiolepis rothneyi Xiangtou Mt, Guangdong GDXT0122 JQ681099 JQ681048 JQ680994 Anoplolepis gracilipes Beiliu, Guangxi GXBL0001 JQ681100 JQ681049 JQ680995 Anoplolepis sp. Bohai, Yunnan YNBH0003 JQ681101 JQ681050 JQ680996 Pseudolasius cibdelus Jingxi, Guangxi GXJX0031 JQ681102 JQ681051 JQ680997 Pseudolasius similus Jingxi, Guangxi NMHL0269 JQ681103 JQ681052 JQ680998 Prenolepis sphingthorax Jingxi, Guangxi GXJX0144 JQ681104 JQ681053 JQ680999 Cataglyphis aenescens Heze, Shandong Shandong_70 JQ681105 HQ619705 JQ681000 Cataglyphis sp. Yangling, Shanxi SXYL0007 JQ681106 JQ681054 JQ681001 Formica candida Xiaowutai Mt, Hebei Hebei_50 JQ681107 HQ619704 JQ681002 Formica longicepes Helan Mt, Inner Mongolia NMHL0227 JQ681108 JQ681055 JQ681003 Formica cunicularia Xiaowutai Mt, Hebei Hebei_307 JQ681109 HQ619714 JQ681004 Formica lemani Xiaowutai Mt, Hebei Hebei_251 JQ681110 HQ619712 JQ681005 Proformica mongolica Helan Mt, Inner Mongolia NMHL0045 JQ681111 JQ681056 JQ681006 Proformica jacoti Xiaowutai Mt, Hebei HBXW0039 JQ681112 JQ681057 JQ681007 Nylanderia flavipes Heze, Shandong SDHZ0104 JQ681113 JQ681058 JQ681008 Nylanderia vividula Guilin, Guangxi GXGL0111 JQ681149 JQ681093 JQ681044 Nylanderia bourbonica Jingxi, Guangxi GXJX0022 JQ681114 JQ681059 JQ681009 Lasius niger Xiaowutai Mt, Hebei HBXW0263 JQ681115 JQ681060 JQ681010 Lasius flavus Helan Mt, Inner Mongolia NMHL0320 JQ681116 JQ681061 JQ681011 Lasius fuliginosus Xiaowutai Mt, Hebei HBXW0266 JQ681117 JQ681062 JQ681012 Lasius alienus Helan Mt, Inner Mongolia NMHL0316 JQ681118 JQ681063 JQ681013 Oecophylla smaragdina Xiangtou Mt, Guangdong GDXT0104 JQ681119 JQ681064 JQ681014 Polyrhachis illaudata Jingxi, Guangxi GXJX0141 JQ681120 JQ681065 JQ681015 Polyrhachis halidayi Jingxi, Guangxi GDJX0024 JQ681121 JQ681066 JQ681016 Polyrhachis rastellata Rong’an, Guangxi GXRA0045 JQ681122 JQ681067 JQ681017 Polyrhachis dives Beiliu, Guangxi GXGL0099 JQ681123 JQ681068 JQ681018 Polyrhachis jianghuaensis Beiliu, Guangxi GXBL0006 JQ681124 JQ681069 JQ681019 Polyrhachis paracampponota Jingxi, Guangxi GXJX0009 JQ681125 JQ681070 JQ681020 Camponotus variegatus Jingxi, Guangxi GXJX0155 JQ681126 JQ681071 JQ681021 Camponotus herculeanus Helan Mt, Inner Mongolia NMHL0273 JQ681127 JQ681072 JQ681022 Camponotus albosparsus Jingxi, Guangxi GXJX0130 JQ681128 JQ681073 JQ681023 Camponotus vanispinus Jingxi, Guangxi GXJX0007 JQ681129 JQ681074 JQ681024 Camponotus wasmanni Xiangtou Mt, Guangdong GDXT0102 JQ681130 JQ681075 JQ681025 Camponotus dolendus Jingxi, Guangxi GXJX0036 JQ681131 JQ681076 JQ681026 Camponotus jianghuaensis Rong’an, Guangxi GXRA0010 JQ681132 JQ681077 JQ681027 Camponotus mitis Bohai, Yunnan YNBH0111 JQ681133 JQ681078 JQ681028 Camponotus helvus Jingxi, Guangxi GXJX0015 JQ681134 JQ681079 JQ681029 Camponotus yiningensis Jingxi, Guangxi GXJX0013 JQ681135 JQ681080 JQ681030 Camponotus albivillosus Helan Mt, Inner Mongolia NMHL2122 JQ681136 JQ681081 JQ681031 Camponotus lasiselene Jingxi, Guangxi GXJX0012 JQ681137 JQ681082 JQ681032 Camponotus parius Beiliu, Guangxi GXBL0009 JQ681138 JQ681083 JQ681033 Camponotus singularis Beiliu, Guangxi GXBL0008 JQ681139 JQ681084 JQ681034 Camponotus sp. 1 Jingxi, Guangxi GXJX0017 JQ681140 JQ681085 JQ681035 Camponotus sp. 2 Jingxi, Guangxi GXJX0123 JQ681141 JQ681086 JQ681036 Polyergus samurai Beiliu, Guangxi GXBL0212 JQ681142 JQ681087 JQ681037 Out-group Cerapachys sulcinodis Beiliu, Guangxi GXBL0095 JQ681145 JQ681090 JQ681040 Radoszkowskius oculata From GenBank NC_014485 NC_014485 NC_014485