Infection, Genetics and Evolution 27 (2014) 202–211

Contents lists available at ScienceDirect

Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid

Wolbachia infection status and genetic structure in natural populations of Polytremis nascens (Lepidoptera: Hesperiidae) Weibin Jiang a, Jianqing Zhu b,⇑, Minghan Chen a, Qichang Yang a, Xuan Du a, Shiyan Chen a, Lina Zhang a, Yiming Yu a, Weidong Yu a,⇑ a b

Shanghai Normal University, College of Life and Environmental Sciences, Shanghai, People’s Republic of China Shanghai Zoological Park, Shanghai, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 26 May 2014 Received in revised form 18 July 2014 Accepted 21 July 2014 Available online 29 July 2014 Keywords: Polytremis nascens Wolbachia Infection rate Mitochondrial genetic structure Selective sweep Biogeography

a b s t r a c t The maternally inherited obligate bacteria Wolbachia is known for infecting the reproductive tissues of a wide range of arthropods. In this study, we surveyed Wolbachia infections in Polytremis nascens (Lepidoptera: Hesperiidae) from 14 locations in China by amplifying the 16S rRNA gene with a nested PCR method and revealed the effect of Wolbachia on host mitochondrial DNA. The results show that 31% (21/67) are Wolbachia positive among all specimens and mainly prevails in southern populations in China. No significant difference in the prevalence is found between the sexes. Notably, the nucleotide diversity of Wolbachia infected butterflies is smaller compared to that of uninfected butterflies. The mitochondrial DNA of infected group appear to be not evolving neutrally (Tajima’s D value = 2.3303 and Fu’s F values = 3.7068). The analysis of molecular variance shows significant differentiation of mitochondrial haplotypes between infected and uninfected specimens (FST = 0.6064). The mismatch analysis speculated the different expansion pattern in Wolbachia infected specimens and all P. nascens specimens. These results suggest that the populations of P. nascens may have recently been subjected to a Wolbachiainduced sweep. Additionally, phylogenetic analysis differentiated the mitochondrial haplotypes of P. nascens into three major clades. The clades are in perfect agreement with the pattern of Wolbachia infection. One of the clades grouped with the butterflies infected with Wolbachia. The remaining two clades grouped with uninfected butterflies from the central-west of China populations and Eastern and Southern China populations respectively, which are isolated mainly by the Yangtze River. The analysis of haplotype networks, geographic distribution and population size change shows that Haplotype 1 in central-west of China is the ancestral haplotype and the populations of P. nascens are expanded. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Wolbachia, maternally inherited endosymbiotic bacteria, may be the most widespread endosymbiont in terrestrial ecosystems, infecting perhaps two-thirds of present-day insect species, as well as about 40% of terrestrial arthropod species (Hilgenboecker et al., 2008; Zug and Hammerstein, 2012). The transmission of Wolbachia is primarily vertical (i.e., by a parent to offspring via reproduction) and secondarily horizontal (i.e., by any other route, such as direct or indirect contact with a vector or the environment) (Raychoudhury et al., 2009). The bacteria manipulate the reproduction of their host to ensure their vertical transmission by cytoplasmic incompatibility, feminization, male killing and parthenogenesis (Stouthamer et al., 1999). ⇑ Corresponding authors. E-mail addresses: [email protected] (J. Zhu), [email protected] (W. Yu). http://dx.doi.org/10.1016/j.meegid.2014.07.026 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.

Wolbachia still can potentially influence mitochondrial variation of their hosts. The linkage disequilibrium is expected to occur between them since they are co-transmitted maternally. The rapidly spread of Wolbachia in the host populations can result in the hitch-hiking effect of mitochondrial DNA (mtDNA). One particular mitochondrial haplotype can sweep through a population and reduce mtDNA polymorphism in the infected population (Ballard, 2000; Charlat et al., 2009; Jiggins, 2003; Narita et al., 2006; Raychoudhury et al., 2010). Wolbachia may also drive introgression of mtDNA following hybridization events between sibling species: the introduction and spread of Wolbachia in a novel species results in spread of the mtDNA from the neighbouring species (Ballard, 2000; Gompert et al., 2008; Jackel et al., 2013; Jiggins, 2003; Narita et al., 2006). The Wolbachia surface protein (wsp), cell division protein gene (ftsZ), 16S rRNA, and other genes have been characterized and used for phylogenetic studies of this endosymbiont. The wsp gene is

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

usually regarded as the fastest evolving Wolbachia gene described (Zhou et al., 1998) and has been routinely used for supergroup designation (Haine and Cook, 2005; Kyei-Poku et al., 2005; Sintupachee et al., 2006). However, some studies showed that wsp sequences are highly recombinant and subject to strong directional selection (Baldo et al., 2005; Baldo and Werren, 2007). Thus, the patterns observed in the single locus studies may not accurately reflect the true evolutionary and demographic histories of Wolbachia isolates (Baldo et al., 2008). A multilocus sequence typing (MLST) scheme for Wolbachia was developed to overcome the recombination issue and offer an expanded data set for comparative analyses (Baldo et al., 2006). The first genome of Drosophila melanogaster wMel was released since 2004 (Wu et al., 2004) and twenty other Wolbachia genomes have been completed and deposited in GenBank (http://www.ncbi. nlm.nih.gov/genome/). The comparison of the genetic profile of strains related to various reproductive phenotypes provided important hints on the symbiont biology and evolution. Wolbachia exist in 13 recognized ‘‘supergroups’’ (A to F and H to M) and supergroup G is controversial (Augustinos et al., 2011; Baldo and Werren, 2007). Supergroups A and B were described first and are most commonly found among arthropod species, e.g., Chilo sp. of Crambidae, Triodia sylvina of Hepialidae and Culex pipiens of Culicidae (Duron et al., 2008; Lo et al., 2007; Werren et al., 1995). Other supergroups have also been described, including on Wolbachia infecting Nematodes (C and D supergroups) (Bandi et al., 1998), supergroup E in Collembola (Czarnetzki and Tebbe, 2004; Vandekerckhove et al., 1999), F in arthropods and Nematodes (Casiraghi et al., 2005), H in termites (Bordenstein and Rosengaus, 2005), I in Siphonaptera (Gorham et al., 2003), J in Spirurida (Casiraghi et al., 2005), K in Acarina (Ros et al., 2009), L in nematode (Haegeman et al., 2009), M and N in aphids (Augustinos et al., 2011). Wolbachia infections have been reported in various Lepidoptera families such as Papilionidae, Lycaenidae, Pieridae, Nymphalidae, Hesperiidae, Pyralidae, Noctuidae, and Lasiocampidae (Bipinchandra et al., 2012; Dyson et al., 2002; Hiroki et al., 2004; Jiggins et al., 2000; Russell et al., 2009; Tagami and Miura, 2004). A few butterfly species harbouring the bacteria have been thoroughly studied (Ankola et al., 2011; Charlat et al., 2007; Duplouy et al., 2010; Hornett et al., 2006; Narita et al., 2007; Nice et al., 2009). Polytremis Mabille, 1904 is a genus within the family Hesperiidae. All species are restricted to the southeastern Palaearctic and northern Oriental realm and are particularly concentrated in China (Chou, 1994; Evans, 1949; Huang and Xue, 2004; Zhu et al., 2012). We have reported the molecular phylogeny of the genus in a prior study (Jiang et al., 2013). Meanwhile, we have preliminarily screened for the presence of Wolbachia in Polytremis and found at least three species (Polytremis nascens Leech, 1893, Polytremis theca Evans, 1937 and Polytremis pellucid Murray, 1875) are infected with Wolbachia (Jiang et al., unpublished data). P. nascensis widely distributed in mountainous regions (1000– 2400 m in elevation) from western to eastern China and considered a serious pest on agricultural and horticultural crops (Zhu et al., 2012; Leech, 1892–1894; Jiang et al., 2014). Because of the phenotypes induced by Wolbachia infections, it has been suggested that the manipulation of endosymbiotic bacteria can be used as a genetic drive tool or as a controlling agent of insect vectors for the biocontrol of pest arthropods of medical, veterinary, and agricultural importance (Bian et al., 2013; Tagami et al., 2006; Xi et al., 2005; Zabalou et al., 2004). Thus, the information about its prevalence and diversity in natural populations of P. nascens is extremely relevant for further study, nevertheless it is scarce. Additionally, we also look forward to understanding the effect of Wolbachia on mtDNA evolution of P. nascens. In this study, we tested 67 specimens by molecular techniques and attempted to:

203

(i) investigate the prevalence and diversity of Wolbachia in P. nascens, (ii) assess whether the Wolbachia infection associated with mtDNA variation, and (iii) analyse the genetic structure and phylogeography of the P. nascens populations. To answer these questions, we detected and identified Wolbachia infection statuses in 67 specimens of P. nascens from 14 local regions in China. We obtained three parts of mitochondrial genes (COI, COII and ND5) from P. nascens, examined the diversity of mitochondrial haplotypes, and analysed mtDNA variation to explore the potential association with Wolbachia infection. In addition, we discussed the biogeographical implications of P. nascens according to the distribution of our specimens and experimental data. The Wolbachia analysis will enrich our knowledge of population ecology and biosystematics of P. nascens and may help for future biocontrol. 2. Materials and methods 2.1. Samples collection and DNA extraction We collected a total of 67 specimens of P. nascens from 14 local regions in China from 2006 to 2013 (Fig. 1, Table 1). All specimens were caught in the field, identified morphologically, preserved by dehydration in small envelopes and dried with silica desiccant for further processing. Two representatives of closed related species, Polytremis zina and Polytremis discreta, were chosen as outgroups for the phylogenetic analyses. The DNA was isolated from abdomen or leg tissue using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) essentially following the manufacturer’s instructions but with some modification. Briefly, after adding proteinase K and buffer AL (QIAGENÒ), the mixed homogeneous solution was incubated at 70 °C for 2 h. Subsequently, 200 lL of 100% ethanol was added and the mixture transferred to a QIAamp spin column. The mixture in the spin column was subjected to 3 cycles of centrifugation at full speed (14,000g) for 1 min and the filtrate was returned to the spin column to increase the amount of DNA obtained. 2.2. Detection and identification of Wolbachia There is an important risk to miss Wolbachia in infected specimens if using only the legs in PCR assays, because Wolbachia density is always higher in reproductive tissues than in other tissues (Chen et al., 2005; Fischer et al., 2011). In all 67 specimens, three (BSZ 1, LL 2 and LL 5, see Table 1) have lost their legs and 39 (BSZ 2, BSZ 3, etc.) have been taken the entire abdomen for observation of the female and male genitalia. Thus, we compared Wolbachia infection status in legs with abdomen in remaining 25 specimens. To screen for the presence of Wolbachia, a nested PCR method was designed according to Sakamoto et al. (2006). A region at 437 bp in length was amplified using general Wolbachia primers (WSpecF and WSpecR) for 16S rRNA (Werren and Windsor, 2000; Table 2). PCRs was performed in a 20 lL final volume under the following conditions: 1 lL template DNA, 0.4 lM concentrations of all forward and reverse primers, 200 lM each dNTP, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3) and 2.5 U Taq polymerase (Takara, Otsu, Shiga, Japan). The PCRs were run on a DNA thermal cycler (Bio-Rad, Hercules, CA, USA) with the following cycling conditions: 95 °C denaturation for 5 min followed by 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min and a final extension of 72 °C for 5 min. The second PCR was performed with two pairs of internal primers (INTF1 and INTR1; INTF2 and INTR2) respectively (Sakamoto et al., 2006; Table 2). Two microlitre of the first PCR product was transferred to a second PCR that consisted of the same reaction mixture but containing 0.2 lM of the internal primers. Positive (DNA extracted from known infected

204

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

Fig. 1. Distribution of mtDNA haplotypes among populations of P. nascens collected in China. The individuals infected with Wolbachia are marked with the slashes. The maximum parsimony networks for all haplotypes were constructed with software NETWORK4.5. The sizes of the circles are directly proportional to the number of individuals analysed and the black points indicate hypothetical ancestors. For full site names and other details see Table 1.

colony P. nascens specimens) and negative (a reaction containing all PCR ingredients except template DNA) controls were run in all reactions to control for contamination during the extraction and PCR processes. Fragments were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized by UV light. The amplification products were extracted using the Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) for sequencing and the sequences obtained were deposited in GenBank. The GenBank database was searched for homologous sequences using the Basic Local Alignment Search Tool (BLAST). Retrieved sequences were aligned with manual correction using Bioedit v.7.0 (Hall, 1999). Phylogenetic trees were constructed using maximum likelihood (ML) by the PAUP⁄ (Swofford, 2003). Modeltest 3.7 (Posada and Crandall, 2001) was used to select the most appropriate model of sequence evolution for the ML analysis. 2.3. Host mtDNA The mitochondrial genes cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII) and NADH dehydrogenase subunit 5 (ND5) were amplified using the primers described in Table 2 (Rand et al., 2000; Caterino and Sperling, 1999; Yagi et al., 1999). The PCR for COI, COII and ND5 were conducted in 20 lL final volume reactions containing 1 lL template DNA, 0.4 lM each of the amplification primers, 200 lM each dNTP, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3) and 2 U Taq polymerase (Takara, Otsu, Shiga, Japan). The thermal profile used was as follows. COI: 95 °C denaturation for 5 min followed by 35 cycles of 94 °C for 45 s, 42 °C for 1 min, 90 °C for 90 sand a final elongation step at 72 °C for 10 min. COII: 95 °C denaturation for 5 min followed by 33 cycles of 94 °C for 40 s, 45 °C for 40 s, 72 °C for 45 s and a final elongation step at 72 °C for 10 min. ND5: 95 °C denaturation for 2 min followed by 35 cycles of 94 °C for 1 min, annealing at 47 °C for1.5 min and extension at 72 °C for 1.5 min, with a final extension of 72 °C for 10 min.

Extraction blanks were run in all reactions to control for contamination during the extraction and PCR processes. The amplification products were subjected to electrophoresis in a 2% (w/v) agarose gel in TAE buffer (0.04 M Tris–acetate, 0.001 M EDTA) with a DL1000 ladder size marker (Takara, Otsu, Shiga, Japan) to determine whether the amplification reactions were successful. In addition, after electrophoresis in agarose gels, the amplification products were extracted using the Wizard SV Gel and PCR Cleanup System (Promega, Madison, WI, USA) for sequencing. Finally, all the sequences obtained were deposited in GenBank (Table 1). The sequence datasets of the mitochondrial COI, COII and ND5 were aligned, pruned to remove redundant sequences and translated to amino acid sequences to check for nuclear mitochondrial pseudogenes (numts) with Bioedit v.7.0 (Hall, 1999). The haplotype sequence matrix was used for all subsequent phylogenetic analyses (Table 1). The partition homogeneity/incongruencelength difference test (Farris et al., 1994; ILD) implemented in PAUP⁄ (Swofford, 2003) was used to determine whether the COI, COII and ND5 datasets were consistent and could be combined for phylogenetic analysis. Phylogenetic trees were constructed by the ML methods using the PAUP⁄ (Swofford, 2003). Modeltest 3.7 (Posada and Crandall, 2001) was used to select the most appropriate model of sequence evolution for the ML analysis. In the ML analysis, a heuristic search was conducted. The starting tree for branch-swapping was from stepwise addition. Nodal support of the ML tree was estimated by 1000 bootstraps. Relationships among the mtDNA haplotypes were represented as a haplotype network obtained by the software DnaSP4.90 (Rozas et al., 2003) and Network4.5 (fluxus-engineering.com) using the median-joining method (Bandelt et al., 1999). 2.4. Test for selective sweeps To test whether selective sweeps by Wolbachia infection decreased host mtDNA variation, we compared nucleotide diver-

Table 1 List of specimen information used for this study. Locality

Polytremis nascens Baishanzu, Qingyuan County, Zhejiang Province (N 27°440 ; E 119°100 )

Fenghuangshan, Hanyin County, Shaanxi Province (N 32°900 ; E 108°500 )

Zhanghe, Langao County, Shaanxi Province (N 32°320 ; E 108°140 )

Lianglu, Tianquan County, Sichuan Province (N 29°550 ; E 102°230 )

Baoxin, Sichuan Province (N 30°220 ; E 102°470 ) Wolong, Wenchuan County, Sichuan Province (N 31°290 ; E 103°350 ) Emeishan, Emeishan City, Sichuan Province (N 29°500 ; E 103°510 ) Shengtangshan, Jinxiu County, Guangxi Province (N 23°570 ; E 110°060 )

BSZ 1 BSZ 2 BSZ 3 BSZ 4 FHS 1 FHS 2 FHS 3 FHS 4 ZH 1 ZH 2 ZH 3 FX 1 HH 1 HLG 1 HLG 2 HLG 3 LL 1 LL 2 LL 3 LL 4 LL 5 LL 6 LL 7 LL 8 LL 9 LL 10 LL 11 LL 12 LL 13 LL 14 LL 15 LL 16 LL 17 LL 18 LL 19 LL 20 LL 21 LL 22 BX 1 WL 1 EMS 1 STS 1 STS 2 STS 3 STS 4 STS 5 STS 6 STS 7 STS 8 STS 9 STS 10 STS 11

Sex

# # # # $ $ $ # # # # # # $ # # # $ # # $ $ # # # # # # # # $ $ $ $ $ $ $ $ # # # # # # # # # # # # # #

Date

21-Jul-07 21-Jul-07 21-Jul-07 21-Jul-07 3-Aug-12 3-Aug-12 3-Aug-12 3-Aug-12 30-Jul-12 30-Jul-12 30-Jul-12 27-Jul-10 9-Jul-13 25-Jul-07 27-Jul-06 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 3-Sep-10 5-Jul-09 6-Aug-11 26-Aug-12 25-Jul-11 25-Jul-11 27-Jul-11 27-Jul-11 27-Jul-11 27-Jul-11 27-Jul-11 27-Jul-11 27-Jul-11 25-Jul-11 25-Jul-11

Altitude

1550 m 1550 m 1550 m 1550 m 1600 m 1600 m 1400 m 1400 m 1600 m 1600 m 1600 m Not recorded 1200 m 2300 m 2300 m 2300 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m 1450 m Not recorded 2000 m Not recorded 1500 m 1500 m 1900 m 1900 m 1900 m 1900 m 1900 m 1900 m 1900 m 1500 m 1500 m

Haplotype

H4 H12 H4 H4 H3 H3 H1 H3 H1 H1 H2 H1 H1 H2 H1 H1 H1 H11 H1 H1 H1 H11 H1 H11 H2 H1 H1 H2 H11 H1 H1 H2 H1 H1 H11 H11 H1 H1 H1 H1 H11 H11 H11 H9 H10 H11 H6 H11 H6 H9 H10 H11

Accession number

Infection status

COI

COII

ND5

Leg*

Abdomen*

KJ574016 KJ574022 KJ574016 KJ574016 KJ574015 KJ574015 KC684397 KJ574015 KC684397 KC684397 KJ574014 KC684397 KC684397 KJ574014 KC684397 KC684397 KC684397 KC684398 KC684397 KC684397 KC684397 KC684398 KC684397 KC684398 KJ574014 KC684397 KC684397 KJ574014 KC684398 KC684397 KC684397 KJ574014 KC684397 KC684397 KC684398 KC684398 KC684397 KC684397 KC684397 KC684397 KC684398 KC684398 KC684398 KJ574020 KJ574021 KC684398 KJ574018 KC684398 KJ574018 KJ574020 KJ574021 KC684398

KJ574025 KJ574031 KJ574025 KJ574025 KJ574024 KJ574024 KJ574023 KJ574024 KJ574023 KJ574023 KJ574023 KJ574023 KJ574023 KJ574023 KJ574023 KJ574023 KJ574023 KJ574030 KJ574023 KJ574023 KJ574023 KJ574030 KJ574023 KJ574030 KJ574023 KJ574023 KJ574023 KJ574023 KJ574030 KJ574023 KJ574023 KJ574023 KJ574023 KJ574023 KJ574030 KJ574030 KJ574023 KJ574023 KJ574023 KJ574023 KJ574030 KJ574030 KJ574030 KJ574028 KJ574029 KJ574030 KJ574026 KJ574030 KJ574026 KJ574028 KJ574029 KJ574030

KJ574033 KJ574038 KJ574033 KJ574033 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574037 KJ574032 KJ574032 KJ574032 KJ574037 KJ574032 KJ574037 KJ574032 KJ574032 KJ574032 KJ574032 KJ574037 KJ574032 KJ574032 KJ574032 KJ574032 KJ574032 KJ574037 KJ574037 KJ574032 KJ574032 KJ574032 KJ574032 KJ574037 KJ574037 KJ574037 KJ574036 KJ574036 KJ574037 KJ574035 KJ574037 KJ574035 KJ574036 KJ574036 KJ574037

/ +a,b                /   / +a  +a,b     +a      +a +a     +b +a,b +a   +a,b  +a,b    +a,b

 / / / / / / / / / / /  / / / / +a,b / /  / / +a,b / / / / +a,b /     +a,b +a,b   / / / +a,b +a,b /  +a,b  +a,b    +a,b

205

(continued on next page)

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

Feng County, Shaanxi Province (N 33°540 ; E 106°310 ) Houhe, Wufeng County, HuBei Province (N 30°220 ; E 110°680 ) Hailuogou, Tianquan County, Sichuan Province (N 29°340 ; E 102°040 )

Specimen ID

/  KJ574040 KJ574039 KC684392

3. Results

a

b

Not recorded 2-Jul-08 #

3.1. Infection rates of Wolbachia using nested PCR

Body part used for Wolbachia screen including legs and abdomens; +: amplification; —: failure to detect amplification product; /: no tissue sample. Nested PCR: first set 16S-WSpecF/WSpecR, second set INTF1/INTR1. Nested PCR: first set 16S-WSpecF/WSpecR, second set INTF2/INTR2.

Not recorded 18-Aug-09 #

Polytremis zina (Outgroup) Tianmushan, Lin’an County, Zhejiang Province (N 30°190 ; E 119°250 ) Polytremis discreta (Outgroup) Baoxing County, Sichuan Province, (N 30°220 ; E 102°480 )

Kuankuoshui, Suiyang County, Guizhou Province (N 28°140 ; E 107°110 )

Anjiangpin, Lingui County, Guangxi Province (N 25°140 ; E 110°120 )

Maoershan, Xin’an County, Guangxi Province (N 25°490 ; E 110°000 )

sity among uninfected individuals and infected individuals using the software DnaSP4.90 (Rozas et al., 2003). We calculated Tajima’s D and Fu’s F statistic and ran 10 000 coalescent simulations for each statistic to create 95% confidence intervals to test whether the mtDNA of P. nascens has evolved under neutrality, which was used to determine whether there is an excess of rare haplotypes, as expected after a selective sweep or population bottleneck (Tajima, 1989; Fu and Li, 1993). The difference of mitochondrial sequences between the infected and uninfected individuals was tested using AMOVA (Analysis of molecular variance) as implemented in the program Arlequin v3.0 (Excoffier et al., 2005). Pairwise mismatch distribution analyses were performed for P. nascens infected with Wolbachia and all P. nascens respectively to find the evidence of past demographic expansions using DnaSP4.90 (Rozas et al., 2003).

*

/ KJ574042 KC684395

KJ574041



 +a,b +a,b  / / / / / / / / / / /  +b +a  +a,b    +a,b  +a,b +a  +a,b +a,b KJ574036 KJ574037 KJ574037 KJ574036 KJ574037 KJ574034 KJ574035 KJ574035 KJ574037 KJ574034 KJ574037 KJ574037 KJ574034 KJ574037 KJ574037 # $ $ # # # # # # # # # # # $ STS 12 STS 13 STS 14 STS 15 MES 1 MES 2 MES 3 MES 4 MES 5 MES 6 AJP 1 AJP 2 KKS 1 KKS 2 KKS 3

25-Jul-11 26-Jul-11 26-Jul-11 27-Jul-11 23-Jul-12 23-Jul-12 22-Jul-12 24-Jul-12 24-Jul-12 24-Jul-12 16-Jul-11 16-Jul-11 15-Aug-10 16-Aug-10 15-Aug-10

1500 m 1500 m 1500 m 1900 m 1900 m 1900 m 2000 m 1500 m 1500 m 1500 m Not recorded Not recorded 1550 m 1550 m 1550 m

H9 H11 H11 H10 H11 H7 H6 H6 H11 H8 H11 H11 H5 H11 H11

KJ574020 KC684398 KC684398 KJ574021 KC684398 KJ574019 KJ574018 KJ574018 KC684398 KJ574019 KC684398 KC684398 KJ574017 KC684398 KC684398

KJ574028 KJ574030 KJ574030 KJ574029 KJ574030 KJ574026 KJ574026 KJ574026 KJ574030 KJ574027 KJ574030 KJ574030 KJ574026 KJ574030 KJ574030

Leg* ND5 COII COI

Accession number Haplotype Altitude Date Sex Specimen ID Locality

Table 1 (continued)

Abdomen*

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

Infection status

206

Of the butterflies examined by diagnostic PCR, 31% (21/67) are Wolbachia positive (Table 1). In the 25 specimens possessing abdomen and legs, the infection status in legs is in accord with that in abdomen (Table 1). There is also some difference in the result of the tests. In leg samples, five are positive in both nested PCR, five are positive in second set INTF1/INTR1 PCR and one is positive in second set INTF2/INTR2 PCR. These 11 specimens are positive in both nested PCR using abdomen samples (Table 1). In three specimens only possessing abdomen, one is positive in both nested PCR. In 39 specimens only possessing legs, six are positive in both nested PCR, two are positive in second set INTF1/INTR1 PCR and one is positive in second set INTF2/INTR2 PCR (Table 1). The infection rates in female and male are 39% (7/18) and 29% (14/49). No significant difference in the prevalence is found between the sexes (v2 = 0.65, P > 0.05). Fourteen specimens from seven regional populations in the central-west of China (FX, FHS, ZH, HH, WL, BX and HLG) are free from infection. Although it difficult to conclude that these populations are not infected based on one to four specimens, we can speculate that relatively low Wolbachia prevalence occurring in the central-west populations. Some populations in the south of China (STS, AJP and KKS) are relatively highly infected with Wolbachia (Table 1, Fig 1). Specific PCR targeting 16S rRNA identification shows that these sequences of Wolbachia are identical and in the A supergroup (Fig. 2). The sequences generated in this study have been deposited in the GenBank database (KJ574043). 3.2. Host mtDNA analyses All specimens and outgroups sequenced for the concatenated sequences (COI, COII and ND5), 1821 bp in size, are polymorphic in 122 nucleotide sites including 85 parsimoniously informative sites. The mean base composition of the fragment shows a strong bias of A+T (T 42.8%, C 11.9%, A 34.0% and G 11.3%), as found commonly in insect mitochondrial genomes (Simon et al., 1994). In all, twelve mitochondrial haplotypes (H1–H12) of P. nascens and two outgroups were found and deposited in GenBank with accession numbers KJ574014–KJ574042 (Table 1). According to the ILD test, partitions of the data into COI, COII, and ND5 are homogeneous (sum of gene tree lengths = 255; P = 0.76) and thus we performed the ML analysis based on the combined dataset. Optimal nucleotide substitution models (GTR + G + I) were chosen following the Akaike Information Criterion (AIC) in Modeltest 3.7. Fig. 3 shows the ML tree based on the data set of the concatenated sequences and supports the mono-

207

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211 Table 2 Primer sequences and amplicon lengths of PCR products of target genes. Gene/region COI COII ND5 16S rRNA (partial sequence 1) 16S rRNA (partial sequence 2) 16S rRNA (partial sequence 3)

Primers 0

HCO2198: 5 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 LCO1490: 50 -GGT CAA CAA ATC ATA AAG ATA TTG G-30 PIERRE: 50 -AGA GCC TCT CCT TTA ATA GAA CA-30 EVA: 50 -GAG ACC ATT ACT TGC TTT CAG TCA TCT-30 V1: 50 -CCTGTTTCTCTGCTTTAGTTTAGTTCA-30 A1: 50 -AATATDAGGTATAAATCATAC-30 WSpecF: 50 -CAT ACC TAT TCG AAG GGA TAG-30 WSpecR: 50 -AGC TTC GAG TGA AAC CAA TTC-30 INTF1: 50 -ACC CTC ATC CTT AGT TGC CAT-30 INTR1: 50 -TGT AGC ACG TGT GTA GCC CAC T-30 INTF2: 50 -AGT CAT CAT GGC CTT TAT GGA-30 INTR2: 50 -TCA TGT ACT CGA GTT GCA GAG T-30

phyly of P. nascens. On the phylogeny, the haplotypes are split into three clades supported by high bootstrap values. The clades are in perfect agreement with the pattern of Wolbachia infection. Clade I with two haplotypes (H11 and H12) consists of the butterflies infected with Wolbachia from different populations. The other two clades with ten haplotypes (H1–H10) exclusively consist of the butterflies free from Wolbachia infection, which are consistent with the distribution of geographical population: a central-west of China clade (H1–H3) and an Eastern and Southern China clade (H4–H10). All mtDNA haplotypes of P. nascens were used for network construction with the software Network4.5 using the median-joining method along with sequences of two outgroups (presented in Table 1). The results of network are shown in Fig. 1.

0

Amplicon length (bp)

Reference

487

Rand et al. (2000)

618

Caterino and Sperling (1999)

716

Yagi et al. (1999)

437

Werren and Windsor (2000)

171

Sakamoto et al. (2006)

177

Sakamoto et al. (2006)

3.3. Test for selective sweeps Nucleotide diversity in the mitochondrial region was compared between infected and uninfected butterflies. The result shows nucleotide diversity of Wolbachia infected butterflies is smaller than that of uninfected butterflies (Table 3). Neutrality tests reveal that the mtDNA of P. nascens appear to be not evolving neutrally as both D and F-values in Wolbachia infected group are negative significantly (Table 3). These results suggest that the populations of P. nascens have recently been subjected to a Wolbachia-induced sweep. Additionally, the analysis of molecular variance shows significant differentiation of mitochondrial haplotypes between infected and uninfected individuals (FST = 0.6064, P < 0.05). The mismatch analysis yielded a resemblance pattern to expected

Fig. 2. Maximum-likelihood (TVM + G + I model) phylogenetic tree of Wolbachia based on 16S rRNA gene sequences. Names at the terminal nodes are those of the host species except for the outgroup species. A–J are the supergroups and named according to Ros et al. The tree has been obtained by Maximum-likelihood phylogeny. A 50% majority-rule consensus bootstrap tree is shown, with bootstrap values over 50% at the nodes. Species in bold is newly found positive in the screening for Wolbachia performed in this study. Accession numbers are given for the sequences present in the databases.

208

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

(Sakamoto et al., 2006; Hughes et al., 2011). Some specimens, especially those from the population of QY and HLG, were stored without temperature control or ethanol for over seven years. However, even with poorly preserved material, we observed an infection rate of over 31% (21/67) and obtained all the mtDNA sequences of the hosts. The observed positives should be considered as a conservative estimate of the actual frequency of Wolbachia infection in P. nascens. The infection rates between the female and male butterflies show no significant difference (v2 = 0.65, P > 0.05). The further rearing experiment would be made to reveal whether there was a sex-ratio distortion in P. nascens induced by the strain of Wolbachia. A positive relationship has also been found between Wolbachia infections and latitudinal distribution. The populations in centralwest of China have low or no Wolbachia infection rates indicating that incidence is apparently lower in regions experiencing longer dry seasons and higher average daily temperatures, whereas higher Wolbachia prevalence occurring in more southerly moist and temperate populations (Table 1, Fig. 1). This has been observed in the beetle Chelymorpha alternans and in ants of the genus Solenopsis (Keller et al., 2004; Martins et al., 2012).

4.2. Effect of Wolbachia on host mtDNA

Fig. 3. The maximum-likelihood (GTR + G + I model) tree for mitochondrial haplotypes of P. nascens. A 50% majority-rule consensus bootstrap tree is shown, with bootstrap values over 50% at the nodes.

curves for Wolbachia infected group (Fig. 4a) compared to the unimodal distribution of pairwise differences for the group of all P. nascens specimens (Fig. 4b). According to Rogers and Harpending (1992), the observed curves with many peaks or resemblance to expected curves mean equilibrium population and unimodal curves representing population expansion.

4. Discussion 4.1. Infection rates of Wolbachia in P. nascens In this study, we surveyed Wolbachia infections in P. nascens and detected 21 specimens (31%) infected with Wolbachia in seven regional populations. However, in the other seven regional populations, all specimens are free from infection with Wolbachia (Table 1). The negative result should be considered with caution. The failure to detect Wolbachia DNA in many samples may have been due to true lack of infection, low-titer infections or, most likely, poor template quality in insufficiently preserved specimens

Many explanations have been proposed that absence of diversity in mtDNA and deviation from neutral evolution in a species can be associated with either a selective sweep on mtDNA or a genome wide bottleneck effect (Tajima, 1989; Shoemaker et al., 2004). In our study, the butterflies infected with Wolbachia show lower mtDNA polymorphism than that of uninfected butterflies (Table 3). The perfect concordance of mtDNA polymorphism and Wolbachia infection status suggests that the mitochondrial genetic structure of the host insects may be strongly affected by the Wolbachia infection. Decreased mtDNA polymorphism as a consequence of Wolbachia infection has also been reported in several other insects (Jackel et al., 2013; Raychoudhury et al., 2010; Sun et al., 2011). We also found that the mtDNA genes of the butterflies infected with Wolbachia deviated significantly from neutral evolution according to both D (2.3303, P < 0.05) and F-values (3.7068, P < 0.05) while this was not so for uninfected ones (Table 3). These results suggest that the populations of P. nascens have recently been subjected to a Wolbachia-induced sweep, making the mtDNA undergo purifying selection. Additionally, we analysed the population size change of P. nascens infected with Wolbachia and all P. nascens, respectively by the software DnaSP4.90 (Rozas et al., 2003) and got no evidence for population expansion in Wolbachia infected group (Rogers and Harpending, 1992; Fig. 4A). However, we got unimodal curves representing population expansion in P. nascens (Rogers and Harpending, 1992; Fig. 4B). A selective sweep, even under population expansion, would erase variability in the population, potentially eliminating any evidence of past demographic processes. Moreover, recent studies also revealed that there are some other endosymbionts known to manipulate host reproduction like Wolbachia, e.g. Arsenophonus, Cardinium and Rickettsia (Duron et al., 2008; Gueguen et al., 2010). A wide range of insect species can host more than one endosymbiont (Goto et al., 2006; Haine, 2008;

Table 3 Mt haplotype and nucleotide diversity estimates from infected and uninfected samples.

All Sequences Infection Free from infection

N

Number of haplotypes

Haplotype diversity

Number of variable sites (S)

p

SD (p)

D

F

67 21 46

12 2 10

0.797 0.095 0.750

119 12 77

0.0197 0.0006 0.0122

0.0012 0.0005 0.0015

1.4795 2.3303* 0.8835

2.0907* 3.7068* 1.9320

N – number of samples, p – nucleotide diversity, SD – standard deviation, D – Tajima’s D statistic, F – Fu’s F statistic. * Significant difference.

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

209

Fig. 4. The analysis of population size change with mtDNA of P. nascens by the software DnaSP4.90. X axis: pairwise differences. Y axis: frequency. The circles show the observed distribution of pairwise difference. The solid lines represent the expected equilibrium distributions. (A) No evidence for population expansion in the Wolbachia infected group of P. nascens. According to Rogers and Harpending (1992), the expected curves are free of waves in equilibrium populations. The observed curves with many peaks or resemblance to expected curves mean equilibrium population. (B) According to Rogers and Harpending (1992), unimodal curves representing population expansion in P. nascens.

Martin et al., 2013). The infection status of the secondary endosymbionts in P. nascens needs to be further studied. 4.3. Biogeographical implications Although there seems to be a strong association existed between mtDNA haplotypes and Wolbachia infection status, the association between mtDNA haplotypes, Wolbachia infection and geographical distribution is weak (Fig. 3). In our preliminary experiment, we found two sympatrically distributed sister species of P. nascens (P. theca and P. pellucid) are infected with Wolbachia. We could not eliminate the possibility of the multiple introgression events, hybridizations between the species pairs (Jiggins, 2003; Jackel et al., 2013; Whitworth et al., 2007). Thus, we excluded the infected group (Clade I) in the analysis of biogeographical implications of P. nascens. It is notable that if we only take the uninfected group into account, the conclusion can be drawn that P. nascens probably has two genetically diverse and geographically localized clades in China based on the mtDNA haplotype phylogeny and networks (Figs. 1 and 3). They are the central-west of China clade (H1–H3) and the Eastern and Southern China clade (H4–H10) (Fig. 3). These two clades are isolated mainly by the Yangtze River, with the exception of a specimen in HH (Fig. 1). Our results were similar to those obtained from the striped stem borer, Chilo suppressalis (Meng et al., 2008) and the melon fly, Bactrocera cucurbitae (Zhu et al., 2005), which suggest that the Yangtze River Range has acted

as a substantial barrier to gene flow. This contrasts with studies of the beet armyworm, Spodoptera exigua (Niu et al., 2006) and the migratory locust, Locusta migratoria (Zhang and Kang, 2005) and the cotton bollworm Helicoverpa armigera (Xu et al., 2002), which showed little or no evidence that the Yangtze River Range limits gene flow. Statistical parsimony network reflects genealogical relationships of the mtDNA haplotypes, that is, single mutation steps separate adjacent haplotypes in the network and older haplotypes are placed at internal branching points whereas younger ones occur toward the tip positions (Narita et al., 2007). The haplotypes network of P. nascens displays a star-like pattern (Fig. 1). Haplotype 1 (H1), the most common and geographically widespread in central-west of China, is in the star’s centre and derivatives are connected to it by short branches. Based on coalescence theory, the star-like topologies for this cluster strongly suggest the effect of a population expansion (Slatkin and Hudson, 1991). We still confirm the result of the population size change of P. nascens with the software DnaSP4.90 and got unimodal curves representing population expansion (Fig. 4B). The most common H1 had strong support as the ancestral haplotype due to its representation in a significant proportion of individuals in all populations and its basal location in the network. A reticulation connecting multiple haplotypes from the Eastern and Southern China clade (H4–H10) was formed in the network. In conclusion, this work provides a conservative estimate that the Wolbachia infection rates in P. nascens is 31% and no significant

210

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211

difference in the prevalence is found between the sexes. The Wolbachia infection mainly prevails in populations of P. nascens in southern China, which influence the diversity of mtDNA in P. nascens by a Wolbachia-induced sweep. Additionally, we revealed two genetically diverse and geographically localized clades of P. nascens in China, which are isolated mainly by the Yangtze River. The analysis of molecular phylogeny, haplotype networks, geographic distribution and population size change shows that Haplotype 1 in central-west of China is the ancestral haplotype and the populations of P. nascens are expanded. Acknowledgements We thank for Huang Hao (Qingdao, China), Li Yufei (Xi’an Jiao Tong University, Shanxi, China), Tang Liang, Hu Jiayao, Yin Ziwei, Peng Zhong, Li Lizhen and their colleagues (Shanghai Normal University, Shanghai, China) for providing valuable comments and specimens throughout the project. This study was financially supported by grants from Innovation Program of Shanghai Municipal Education Commission - China (No. 14YZ068), Innovation Program for University Student in Shanghai, China (No. B-9117-13-007058) and Shanghai Normal University (No. SK201340). References Ankola, K., Brueckner, D., Puttataju, H.P., 2011. Wolbachia endosymbiont infection in two Indian butterflies and female-biased sex ratio in the Red Pierrot, Talicada nyseus. J. Biosci. 36, 845–850. Augustinos, A.A., Santos-Garcia, D., Dionyssopoulou, E., Moreira, M., Papapanagiotou, A., Scarvelakis, M., Doudoumis, V., Ramos, S., Aguiar, A.F., 2011. Detection and characterization of Wolbachia infections in natural populations of Aphids: is the hidden diversity fully unraveled? PLoS ONE 6, e28695. Bandelt, H.J., Foster, P., Rohl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37–48. Baldo, L., Lo, N., Werren, J.H., 2005. Mosaic nature of wsp (Wolbachia surface protein). J. Bacteriol. 187, 5406–5418. Baldo, L., Werren, J.H., 2007. Revisiting Wolbachia supergroup typing based on WSP: spurious lineages and discordance with MLST. Curr. Microbiol. 55, 81–87. Baldo, L., Ayoub, N.A., Hayashi, C.Y., Russell, J.A., Stahlhut, J.K., Werren, J.H., 2008. Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity. Mol. Ecol. 17, 557–569. Baldo, L., Dunning, H.J.C., Jolley, K.A., Bordenstein, S.R., Biber, S.A., Choudhury, R.R., Hayashil, C., Maiden, M.C.J., Tettelin, H., Werren, J.H., 2006. Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl. Environ. Microbiol. 72, 7098–7110. Ballard, J.W., 2000. When one is not enough: introgression of mitochondrial DNA in Drosophila. Mol. Biol. Evol. 17, 1126–1130. Bandi, C., Anderson, T., Genchi, C., Blaxter, M., 1998. Phylogeny of Wolbachia in filarial nematodes. Proc. R. Soc. Lond. B Biol. Sci. 265, 2407–2413. Bian, G.W., Joshi, D., Dong, Y.M., Lu, P., Zhou, G.L., Pan, X.L., Xu, Y., Dimopoulos, G., Xi, Z.Y., 2013. Wolbachia Invades Anopheles stephensi populations and induces refractoriness to plasmodium infection. Science 340, 748–751. Bipinchandra, K.S., Rahul, C.S., Dhiraj, P.D., Sandeep, A.W., Avinash, B.K., Rahul, C., Rakesh, K.C., Hemant, V.G., Milind, S.P., John, H.W., Yogesh, S.S., 2012. Determination of Wolbachia diversity in butterflies from Western Ghats, India, by a multigene approach. Appl. Environ. Microbiol. 78, 4458–4467. Bordenstein, S., Rosengaus, R.B., 2005. Discovery of a novel Wolbachia supergroup in Isoptera. Curr. Microbiol. 51, 393–398. Casiraghi, M., Bordenstein, S.R., Baldo, L., Lo, N., Beninati, T., Wernegreen, J.J., Werren, J.H., Bandi, C., 2005. Phylogeny of Wolbachia pipientis based on gltA, groEL and ftsZ gene sequences: clustering of arthropod and nematode symbionts in the F supergroup, and evidence for further diversity in the Wolbachia tree. Microbiology 151, 4015–4022. Caterino, M.S., Sperling, F.A.H., 1999. Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes. Mol. Phylogenet. Evol. 11, 122–137. Charlat, S., Duplouy, A., Hornett, E.A., 2009. The joint evolutionary histories of Wolbachia and mitochondria in Hypolimnas bolina. BMC Evol. Biol. 9, 64. Charlat, S., Hornett, E.A., Fullard, J.H., Davies, N., Roderick, G.K., Wedell, N., Hurst, G.D.D., 2007. Extraordinary flux in sex ratio. Science 317, 214. Chen, W.J., Tsai, K.H., Cheng, S.L., Huang, C.G., Wu, W.J., 2005. Using in situ hybridization to detect endosymbiont Wolbachia in dissected tissues of mosquito host. J. Med. Entomol. 42, 120–124. Chou, I., 1994. Monographia Rhopalocerorum Sinensium. Henan Scientific and Technological Press, Zhengzhou, p. 854. Czarnetzki, A.B., Tebbe, C.C., 2004. Detection and phylogenetic analysis of Wolbachia in Collembola. Environ. Microbiol. 6, 35–44.

Duplouy, A., Hurst, G.D.D., O’Neill, S.L., Charlat, S., 2010. Rapid spread of male-killing Wolbachia in the butterfly Hypolimnas bolina. J. Evol. Biol. 23, 231–235. Duron, O., Bouchon, D., Boutin, S., Bellamy, L., Zhou, L., Engelstädter, J., Hurst, G.D., 2008. The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone. BMC Biol. 6, 27. Dyson, E.A., Kamath, M.K., Hurst, G.D., 2002. Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae): evidence for horizontal transmission of a butterfly male-killer. Heredity 88, 166–171. Evans, W.H., 1949. A Catalogue of the Hesperiidae from Europe, Asia and Australia in the British Museum (Natural History). Trustees of the British Museum, London, p. 12458. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol. Bioinform. 1, 47–50. Farris, J.S., Kallersjo, M., Kluge, A.G., Bult, C., 1994. Testing significance of congruence. Cladistics 10, 315–319. Fischer, K., Beatty, W.L., Jiang, D., Weil, G.J., Fischer, P.U., 2011. Tissue and stagespecific distribution of Wolbachia in Brugia malayi. PLoS Negl. Trop. Dis. 5, e1174. Fu, Y.X., Li, W.H., 1993. Statistical tests of neutrality of mutations. Genetics 133, 693–709. Gompert, Z., Forister, M.L., Fordyce, J.A., Nice, C.C., 2008. Widespread mito-nuclear discordance with evidence for introgressive hybridization and selective sweeps in Lycaeides. Mol. Ecol. 17, 5231–5244. Gorham, C.H., Fang, Q.Q., Durden, L.A., 2003. Wolbachia endosymbionts in fleas (Siphonaptera). J. Parasitol. 89, 283–289. Goto, S., Anbutsu, H., Fukatsu, T., 2006. Asymmetrical interactions between Wolbachia and Spiroplasma endosymbionts coexisting in the same insect host. Appl. Environ. Microbiol. 72, 4805–4810. Gueguen, G., Vavre, F., Gnankine, O., Peterschmitt, M., Charif, D., Chiel, E., Gottlieb, Y., Ghanim, M., Zchori-Fein, E., Fleury, F., 2010. Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex. Mol. Ecol. 19, 4365–4378. Haegeman, A., Vanholme, B., Jacob, J., Vandekerckhove, T.T., Claeys, M., Borgonie, G., Gheysen, G., 2009. An endosymbiotic bacterium in a plant-parasitic nematode: member of a new Wolbachia supergroup. Int. J. Parasitol. 39, 1045–1054. Haine, E.R., Cook, J.M., 2005. Convergent incidences of Wolbachia infection in fig wasp communities from two continents. Proc. Biol. Sci. 272, 421–429. Haine, E.R., 2008. Symbiont-mediated protection. Proc. R. Soc. Lond. B Biol. Sci. 275, 353–361. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor an analysis program for Windows 95/98/NT. Nucleic Acids Symp. 41, 95–98. Hilgenboecker, K., Hammerstein, P., Schlattmann, P., Telschow, A., Werren, J.H., 2008. How many species are infected with Wolbachia?—a statistical analysis of current data. FEMS Microbiol. Lett. 281, 215–220. Hiroki, M., Tagami, Y., Miura, K., Kato, Y., 2004. Multiple infection with Wolbachia inducing different reproductive manipulations in the butterfly Eurema hecabe. Proc. R. Soc. Lond. B Biol. Sci. 271, 1751–1755. Hornett, E.A., Charlat, S., Duplouy, A.M.R., Davies, N., Roderick, G.K., Wedell, N., Hurst, G.D.D., 2006. Evolution of male-killer suppression in a natural population. PLoS Biol. 4, 283. Huang, H., Xue, Y.P., 2004. Notes on some Chinese butterflies (Lepidoptera, Rhopalocera). Neue Entomologische Nachrichte 57, 238–239. Hughes, G.L., Allsopp, P.G., Brumbley, M., Woolfit, M., McGraw, E.A., O’Neill, S.L., 2011. variable infection frequency and high diversity of multiple strains of Wolbachia pipientis in Perkinsiella Planthoppers. Appl. Environ. Microbiol. 77, 2165–2168. Jackel, R., Mora, D., Dobler, S., 2013. Evidence for selective sweeps by Wolbachia infections: phylogeny of Altica leaf beetles and their reproductive parasites. Mol. Ecol. 22, 4241–4255. Jiang, W.B., Zhu, J.Q., Song, C., Li, X.Y., Yang, Y., Yu, W.D., 2013. Molecular phylogeny of the butterfly genus Polytremis (Hesperiidae, Hesperiinae, Baorini) in China. PLoS ONE 8, e84098. Jiang, W.B., Zhu, J.Q., Zhan, L., Chen, M.H., Song, C., Yu, W.D., 2014. Isolation and characterization of microsatellite loci in Polytremis nascens (Lepidoptera: Hesperiidae), and their cross-amplification in related species. Appl. Entomol. Zool. 49, 177–181. Jiggins, F.M., Hurst, G.D., Dolman, C.E., Majerus, M.E., 2000. High prevalence male-killing Wolbachia in the butterfly Acraea encedana. J. Evol. Biol. 13, 495–501. Jiggins, F.M., 2003. Male-killing Wolbachia and mitochondrial DNA: selective sweeps, hybrid introgression and parasite population dynamics. Genetics 164, 5–12. Keller, G.P., Windsor, D.M., Saucedo, J.M., Werren, J.H., 2004. Reproductive effects and geographical distributions of two Wolbachia strains infecting the neotropical beetle, Chelymorpha alternans Boh. (Chrysomelidae, Cassidinae). Mol. Ecol. 13, 2405–2420. Kyei-Poku, G.K., Colwell, D.D., Coghlin, P., Benkel, B., Floate, K.D., 2005. On the ubiquity and phylogeny of Wolbachia in lice. Mol. Ecol. 14, 285–294. Leech, J.H., 1892–1894. Butterflies from China, Japan and Corea. RH Porter, London, p. 614. Lo, N., Paraskevopoulos, C., Bourtzis, K., O’Neill, S.L., Werren, J.H., Bordenstein, S.R., Bandi, C., 2007. Taxonomic status of the intracellular bacterium Wolbachia pipientis. Int. J. Syst. Evol. Microbiol. 57, 654–657. Martin, O.Y., Puniamoorthy, N., Gubler, A., Wimmer, C., Bernasconi, M.V., 2013. Infections with Wolbachia, Spiroplasma, and Rickettsia in the Dolichopodidae and other Empidoidea. Infect. Genet. Evol. 13, 317–330.

W. Jiang et al. / Infection, Genetics and Evolution 27 (2014) 202–211 Martins, C., Souza, R.F., Bueno, O.C., 2012. Presence and distribution of the endosymbiont Wolbachia among Solenopsis spp. (Hymenoptera: Formicidae) from Brazil and its evolutionary history. J. Invertebr. Pathol. 109, 287–296. Meng, X.F., Shi, M., Chen, X.X., 2008. Population genetic structure of Chilo suppressalis (Walker) (Lepidoptera: Crambidae): strong subdivision in China inferred from microsatellite markers and mtDNA gene sequences. Mol. Ecol. 17, 2880–2897. Narita, S., Nomura, M., Kato, Y., Fukatsu, T., 2006. Genetic structure of sibling butterfly species affected by Wolbachia infection sweep: evolutionary and biogeographical implications. Mol. Ecol. 15, 1095–1108. Narita, S., Nomura, M., Kato, Y., Yata, O., Kageyama, D., 2007. Molecular phylogeography of two sibling species of Eurema butterflies. Genetica 131, 241–253. Nice, C.C., Gompert, Z., Forister, M.L., Fordyce, J.A., 2009. An unseen foe in arthropod conservation efforts: the case of Wolbachia infections in the Karner blue butterfly. Biol. Conserv. 142, 3137–3146. Niu, C.W., Zhang, Q.W., Ye, Z.H., Luo, L.Z., 2006. Analysis of genetic diversity in different geographic populations of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) with AFLP technique. Acta Entomol. Sin. 49, 867–873. Posada, D., Crandall, K.A., 2001. Selecting the best-fit model of nucleotide substitution. Syst. Biol. 50, 580–601. Rand, D.B., Heath, A., Suderman, T., Pierce, N.E., 2000. Phylogeny and life history evolution of the genus Chrysoritis within the Aphnaeini (Lepidoptera: Lycaenidae), inferred from mitochondrial cytochrome oxidase I sequences. Mol. Phylogenet. Evol. 17, 85–96. Raychoudhury, R., Baldo, L., Oliveira, D.C.S.G., Werren, J.H., 2009. Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex. Evolution 63, 165–183. Raychoudhury, R., Grillenberger, B.K., Gadau, J., 2010. Phylogeography of Nasonia vitripennis (Hymenoptera) indicates a mitochondrial-Wolbachia sweep in North America. Heredity 104, 318–326. Rogers, A.R., Harpending, H., 1992. Population growth makes waves in the distribution of pairwise genetic differences. Mol. Bio. Evol. 9, 552–569. Ros, V.I., Fleming, V., Feil, E.J., Breeuwer, J.A., 2009. How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae). Appl. Environ. Microbiol. 4, 1036–1043. Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X., Rozas, R., 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496–2497. Russell, J.A., Goldman-Huertas, B., Moreau, C.S., Baldo, L., Stahlhut, J.K., Werren, J.H., Pierce, N.E., 2009. Specialization and geographic isolation among Wolbachia symbionts from ants and lycaenid butterflies. Evolution 63, 624–640. Sakamoto, J.M., Feinstein, J., Jason, L.R., 2006. Wolbachia infections in the Cimicidae: museum specimens as an untapped resource for endosymbiont surveys. Appl. Environ. Microbiol. 72, 3161–3167. Shoemaker, D.D., Dyer, K.A., Ahrens, M., McAbee, K., Jaenike, J., 2004. Decreased diversity but increased substitution rate in host mtDNA as a consequence of Wolbachia endosymbiont infection. Genetics 168, 2049–2058. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651– 701. Sintupachee, S., Milne, J.R., Poonchaisri, S., Baimai, V., Kittayapong, P., 2006. Closely related Wolbachia strains within the pumpkin arthropod community and the potential for horizontal transmission via the plant. Microb. Ecol. 51, 294–301.

211

Slatkin, M., Hudson, R.R., 1991. Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations. Genetics 129, 555–562. Stouthamer, R., Breeuwer, J.A., Hurst, G.D., 1999. Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu. Rev. Microbiol. 53, 71–102. Sun, X.J., Xiao, J.H., Cook, J.M., Feng, G., Huang, D.W., 2011. Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity. BMC Evol. Biol. 11, 86. Swofford, D.L., 2003. PAUP⁄. Phylogenetic Analysis Using Parsimony (⁄and Other Methods). Version 4. Sinauer Associates, Sunderland. Tagami, Y., Miura, K., 2004. Distribution and prevalence of Wolbachia in Japanese populations of Lepidoptera. Insect Mol. Biol. 13, 359–364. Tagami, Y., Doi, M., Keitaro, S., Tatara, A., Saito, T., 2006. Wolbachia-induced cytoplasmic incompatibility in Liriomyza trifolii and its possible use as a tool in insect pest control. Biol. Control 38, 205–209. Tajima, F., 1989. Statistical-method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585–595. Vandekerckhove, T.T., Watteybe, S., Willems, A., Swings, J.G., Mertens, J., Gillis, M., 1999. Phylogenetic analysis of the 16S rDNA of the cytoplasmic bacterium Wolbachia from the novel host Folsomia candida (Hexapoda, Collembola) and its implications for Wolbachia taxonomy. FEMS Microbiol. Lett. 180, 279–286. Werren, J.H., Zhang, W., Guo, L.R., 1995. Evolution and phylogeny of Wolbachia: reproductive parasite of arthropod. Proc. R. Soc. Lond. B Biol. Sci. 161, 55–63. Werren, J.H., Windsor, D.M., 2000. Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proc. Biol. Sci. 267, 1277–1285. Whitworth, T.L., Dawson, R.D., Magalon, H., Baudry, E., 2007. DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae). Proc. R. Soc. Lond. B Biol. Sci. 274, 1731–1739. Wu, M., Sun, L.V., Vamathevan, J., Riegler, M., Deboy, R., Brownlie, J.C., McGraw, E.A., Martin, W., Esser, C., Ahmadinejad, N., 2004. Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2, E69. Xi, Z., Khoo, C.C., Dobson, S.L., 2005. Wolbachia establishment and invasion in an Aedes aegypti laboratory population. Science 310, 326–328. Xu, G., Wang, G.R., Wu, K.M., Guo, Y.Y., 2002. Gene flow analysis among different geographical populations of Helicoverpa armigera (Hübner) by restriction fragment length polymorphisms. Cotton Sci. 14, 352–355. Yagi, T., Sasaki, G., Takebe, H., 1999. Phylogeny of Japanese Papilionid butterflies inferred from nucleotide sequences of the mitochondrial ND5 gene. J. Mol. Evol. 48, 42–48. Zabalou, S., Riegler, M., Theodorakopoulou, M., Stauffer, C., Savakis, C., Bourtzis, K., 2004. Wolbachia-induced cytoplasmic incompatibility as a means for insect pest population control. Proc. Natl. Acad. Sci. U.S.A. 101, 15042–15045. Zhang, M.Z., Kang, L., 2005. The genetic differentiation among geographic populations of Locusta migratoria in China. Scichina 35, 220–230. Zhou, W., Rousset, F., O’Neill, S., 1998. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc. R. Soc. Lond. B Biol. Sci. 265, 509–515. Zhu, J.Q., Chen, Z.B., Li, L.Z., 2012. Polytremis jigongi: a new skipper from China (Lepidoptera: Hesperiidae). Zootaxa 3274, 63–68. Zhu, Z.H., Ye, H., Zhang, Z., 2005. Genetic relationships among four Bactrocera cucurbitae geographic populations in Yunnan Province. Chin. J. Appl. Ecol. 16, 1889–1892. Zug, R., Hammerstein, P., 2012. Still a host of hosts for Wolbachia: analysis of recent data Suggests that 40% of terrestrial arthropod species are infected. PLoS ONE 7, e38544.