Molecular Phylogenetics and Evolution 85 (2015) 22–31

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Molecular phylogeny of Panorpidae (Insecta: Mecoptera) based on mitochondrial and nuclear genes Gui-Lin Hu, Gang Yan, Hao Xu, Bao-Zhen Hua ⇑ State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Institute of Entomology, Northwest A&F University, Yangling, Shaanxi 712100, China

a r t i c l e

i n f o

Article history: Received 5 July 2014 Revised 15 December 2014 Accepted 22 January 2015 Available online 13 February 2015 Keywords: Panorpidae Systematics Phylogenetics Divergence time Biogeography

a b s t r a c t Panorpidae are the largest family in Mecoptera, covering approximately 70% species of the order. However, the phylogenetic relationship within Panorpidae has not been adequately explored. Here we analyzed the phylogenetic relationships among 70 species of five genera in Panorpidae using maximum likelihood and Bayesian inference based on two mitochondrial (cox1 and cox2) and one nuclear (28S rRNA) gene fragments with Panorpodes kuandianensis and Brachypanorpa carolinensis in Panorpodidae as outgroups. The results show that the genera Neopanorpa, Sinopanorpa and Dicerapanorpa are monophyletic, while the widespread genus Panorpa is reconfirmed to be a paraphyletic group. The P. centralis group is monophyletic and may merit a generic status, while the P. davidi and P. amurensis groups are paraphyletic. The divergence time estimated from BEAST analysis indicates that the Panorpidae may originate in the period from early Paleogene (63.6 mya) to middle Eocene (41.2 mya), and most diversification within Panorpidae occurred in the Cenozoic. The phylogeny and biogeography of Panorpidae are briefly discussed. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Panorpidae is the most speciose family in the order Mecoptera and comprises approximately 400 described species (Byers and Thornhill, 1983; Kaltenbach, 1978; Penny and Byers, 1979). They are commonly called scorpionflies because their male genital bulb is enlarged and recurved upward, resembling the tail of scorpions. The species of Panorpidae are currently assigned to six genera, Panorpa Linnaeus, 1758; Leptopanorpa MacLachlan, 1875; Neopanorpa Weele, 1909; Sinopanorpa Cai & Hua, 2008; Furcatopanorpa Ma & Hua, 2011, and Dicerapanorpa Zhong & Hua, 2013. The monotypic Furcatopanorpa is distributed in central China (Ma and Hua, 2011). Sinopanorpa (3 species) and Dicerapanorpa (8 species) are also endemic to China (Cai et al., 2008; Zhong and Hua, 2013a). Leptopanorpa (13 species) is claimed to be exclusively distributed in Java, Indonesia (Chau and Byers, 1978). Neopanorpa (129 species) is found from India, Southeast Asia, Indo-China and southern China (Byers and Thornhill, 1983). The dominant speciose genus Panorpa (246 species) is distributed in the whole Holarctic region and northern Oriental region (Zhong and Hua, 2013a).

⇑ Corresponding author. Fax: +86 29 87091342. E-mail address: [email protected] (B.-Z. Hua). http://dx.doi.org/10.1016/j.ympev.2015.01.009 1055-7903/Ó 2015 Elsevier Inc. All rights reserved.

Panorpa and Neopanorpa are the most species-rich genera in Panorpidae and consist of more than 90% species of the family. Neopanorpa differs from Panorpa by vein 1A joining the hind margin of forewing before the origin of Rs, usually with one cross vein between 1A and 2A, tergum III of males with a developed notal organ, and the main plate short and simple, with the axis undeveloped and generally not extending beyond the main plate (Cai et al., 2008; Cheng, 1957; Ma and Hua, 2011; Ma et al., 2012; Zhong and Hua, 2013a). Panorpa is the widespread species-rich genus in Panorpidae (Penny and Byers, 1979), and is such a diverse taxon that its component species are categorized into different species groups for regional faunas based on morphological criteria (Byers, 1993; Carpenter, 1931, 1938; Cheng, 1957; Esben-Petersen, 1921; Issiki, 1933; Ward, 1983; Willmann, 1977). Previous phylogenetic studies of Panorpidae are mainly based on morphological data (Issiki, 1933; Ma et al., 2012; Willmann, 1977, 1989). Panorpa was considered paraphyletic with Neopanorpa and Leptopanorpa based on wing venation (Willmann, 1989) and female genital plates and other characters (Ma et al., 2012). Based on molecular data, Misof et al. (2000) and Whiting (2002) also concluded that Panorpa is paraphyletic with Neopanorpa. The limited studies on the phylogenetic information of Panorpidae are essentially restricted to a small number of European, American and Asian species (Ma et al., 2012; Misof et al., 2000; Whiting, 2002; Willmann, 1977, 1989). Recent studies

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on the morphology of the eggs (Ma et al., 2009), female reproductive system (Hou and Hua, 2008), salivary glands (Ma et al., 2011) and female genital plate (Ma et al., 2012) also support the paraphyly of Panorpa. However, the phylogenetic relationships in Panorpidae have not been satisfactorily resolved to date. The superfamily Panorpoidea (including Panorpidae) underwent an Eocene radiation, but only Panorpidae and Panorpodidae survived to present (Archibald et al., 2013). The extant Panorpidae are widely distributed throughout Asia, North America and Europe, with most species concentrated in Eastern and Southeastern Asia (Penny and Byers, 1979). Panorpidae of the Nearctic Region are, in general, more like those of the eastern Palaearctic than of the European region (Byers, 1969, 1988). The fossil record shows that Panorpidae entered North America in Early Cenozoic (Byers, 1988). But the oldest fossil record of Panorpidae was found from the Middle Jurassic in China (Ding et al., 2014). In general, the biogeography, origin, and divergence time of Panorpidae have not been satisfactorily studied to date. In this study, we analyzed phylogenetic relationships of 70 species in five genera of Panorpidae with two mitochondrial (cox1 and cox2) and one nuclear (28S rRNA) gene fragments. Two species of Panorpodidae, Panorpodes kuandianensis Zhong & Hua, 2013 and Brachypanorpa carolinensis Banks, 1905, were used as outgroups since Panorpodidae is widely accepted as the sister group of Panorpidae (Mickoleit, 1978; Pollmann et al., 2008; Willmann, 1987, 1989). The main aims of the present study were to (1) investigate the phylogenetic relationships of Panorpidae; (2) reevaluate the hypothesis of species groups division in Panorpa, in an attempt to solve the paraphyly problem of Panorpa; and (3) explore the origin, divergence time, and dispersal of Panorpidae. 2. Materials and methods 2.1. Taxon sampling We acquired a total of 70 species of Panorpidae, including 43 species of Panorpa, 20 of Neopanorpa, three of Dicerapanorpa, three of Sinopanorpa, and one of Furcatopanorpa. Nine species (P. emarginata, P. obtusa, P. byersi, P. acutipennis, P. dubia, P. nanwutaina, P. wangwushana, P. fluvicaudaria and P. arakavae) belong to the P. centralis group (Carpenter, 1938; Cheng, 1957); two species (P. liui and P. jilinensis) belong to the P. amurensis group (Issiki, 1933); four species (P. acuta, P. maculosa, P. nebulosa and P. latipennis) belong to the P. nebulosa group (Carpenter, 1931); five species (P. helena, P. speciosa, P. debilis, P. claripennis, P. carolinensis) belong to the P. refescens group (Carpenter, 1931); four species (P. communis, P. vulgaris, P. cognata and P. germanica) belong to the P. communis group (Willmann, 1977); and the remaining 18 species (excluding P. rufostigma) of Panorpa belong to the P. davidi group. We also obtained target sequences of 17 species of Panorpa, one of Neopanorpa and one of Brachypanorpa as well as the mitochondrial sequences of Neopanorpa pulchra from GenBank. Panorpodes kuandianensis and Brachypanorpa carolinensis of Panorpodidae were used as outgroups in the phylogenetic analysis. The species investigated in this study and their related information are listed in Table 1. The voucher specimens are preserved in 95–100% alcohol or dry specimens at the Entomological Museum, Northwest A&F University, China (NWAU). 2.2. DNA extraction and sequencing Total genomic DNA was extracted from the musculature of three legs on one side using traditional phenol/chloroform method or Genomic DNA Mini Preparation Kit with Spin Column

23

(Beyotime). The extracted DNA was eluted in sterile distilled H2O or TE buffer, and stored frozen. Partial sequences of one nuclear gene 28S rRNA and two mitochondrial genes, cytochrome c oxidase subunit I and II (cox1 and cox2), were amplified using primers from Whiting (2002) and Pollmann et al. (2008). An overview of primers is given in Table 2. Folmer et al.’s (1994) universal cox1 primers were used to amplify P. maculosa. Two different protocols were performed in a 25 lL PCR reaction: (1) 2.5–3.0 lL 10  PCR buffer (Mg2+ free), 2.0–2.5 lL 2.5 mM dNTPs, 2.0–2.5 lL 25 mM Mg2+, 1.5 lL DNA template, 1 lL 10 lM each primer, 0.3 lL Taq polymerase (2.5 U/lL) and the remaining was sterile distilled H2O; (2) 12.5 lL CWBIO 2  Taq MasterMix, 8.5 lL sterile distilled H2O, 1 lL 10 lM each primer, and 2 lL DNA template. PCR program for cox1 began with an initial denaturation for 5 min at 94–95 °C followed by 35–40 cycles of denaturation at 94 °C for 30 s, annealing at 50.5 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. The reaction conditions for cox2 and 28S rRNA fragments followed the above except that the annealing temperatures were modified to 45.5 °C and 58.8 °C, respectively. The PCR products were checked on a 1–2% agarose gel staining with ethidium bromide. For some specimens whose yields of DNA were very low, reamplification was necessary to obtain adequate DNA amounts for sequencing. A single bright band emerged on the target fragment size (approximately 610 bp for cox1, 630 bp for cox2, and 500 bp for 28S rRNA), and then the products were sent to Shanghai Sunny Biotechnology Co., Ltd (China) for sequencing in both directions. 2.3. Phylogenetic analyses The forward and reverse sequences were checked, assembled and edited with ChromasPro 1.5 (http://www.technelysium.com. au/ChromasPro.html). Multiple sequences alignments were performed using both MAFFT 7.037 (Katoh et al., 2009) with accurate algorithm of G-INS-i for cox1 and cox2 and ClustalX 2.0.21 (Jeanmougin et al., 1998; Thompson et al., 1997) for 28S rRNA (optimized parameters gap opening penalties of 19 and gap extension penalties of 6.66). Subsequently the gappy columns at the beginning and end of the alignment were manually deleted with BioEdit 7.0.9.0 (Hall, 1999). Molecular character statistics including parsimony informative site, base frequency and A-T content were calculated with MEGA 6.05 (Tamura et al., 2013). The level of substitution saturation of each gene and each codon position of each protein-coding gene was tested using DAMBE 5.3.74 (Xia, 2013). v2 test of homogeneity of base frequencies across taxa was implemented in PAUP⁄4.0b10 (Swofford, 2002). Phylogenetic analyses were conducted for combined genes and each individual gene. However, the phylogenetic trees obtained from single gene can hardly resolve the basal relationships of Neopanorpa and Panorpa (topology not shown). Therefore we assembled the following two datasets for comparison and analyses: (1) concatenated data of cox1, cox2 and 28S rRNA of 63 taxa; (2) combined data of cox2 and 28S rRNA of 72 taxa (in order to increase taxon sampling and include more previously published sequences of species). The most suitable partitioning strategies and the respective evolution models for each partition were estimated based on the program PartitionFinder v1.1.1 (Lanfear et al., 2012) under the Bayesian Information Criterion (BIC). For the concatenated data of cox1, cox2 and 28S rRNA, the optimal partitions and models were as follows: 28S rRNA with TVM + G; first codon position of cox1 and cox2 with TrN + I + G; second codon position of cox1 with F81; third codon position of cox1 and cox2 with HKY + G; second

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Table 1 List of species investigated and their related information. Species

Locality

GenBank accession Nos. for cox1, cox2 and 28S

Collecting date/Source

Panorpa acutipennis Hua, 1998

Huaboshan, Kuandian, Liaoning, 1000 m Baiyunshan, Henan, 1700 m Taibaishan, Shaanxi, 2100 m Huangsongpu, Jilin, 1200 m Zhuque, Shaanxi, 1390 m Wolong, Sichuan, 1700 m Baiyunshan, Henan, 1700 m Zhuque, Shaanxi, 1390 m Huashan, Shaanxi, 2070 m Liping, Shaanxi, 1500 m Zhongdian, Yunnan, 3500 m Huangsongpu, Jilin, 1200 m Xishan, Kunming, Yunnan, 2500 m Wudalianchi, Heilongjiang, 650 m Huangsongpu, Jilin, 1200 m Michigan, USA Zhuque, Shaanxi, 1390 m Taibaishan, Shaanxi, 2100 m Liuba, Shaanxi, 1600 m Lishan, Shanxi, 1950 m Zhuque, Shaanxi, 1390 m Pantiange, Zhongdian, Yunnan, 2800 m Mt. Emei, Sichuan, 2450 m Cru deu van, Neuchântel, Swizerland, 2000 m Iowa: Henry Co. Geode State Park, USA, 204 m Soisson, France Taibaishan, Shaanxi, 2100 m

JN223507; JN223493; JN223486

3. vii. 2010

GU722389; GU722405; HM061596 GU722383; GU722399; HM061590 JN223515; JN223496; JN223478 GU722394; GU722411; HM061593 JN223505; JN223489; JN223483 GU722392; GU722408; HM061601 JN223508; JN223494; JN223488 GU722381; GU722397; HM061587 JN223504; JN223491; JN223481 JN223512; JN223495; JN223476 JN223510; JN223502: JN223480 JN223513; JN223499; JN223475 GU722396; GU722409; HM061599 JN223509; JN223501; JN223479 KJ816742; KJ816708; KJ816726 JN688128; JN688140; JN688118 GU722382; GU722398; HM061588 JN223506; JN223490; JN223485 GU722384; GU722400; HM061591 GU722390; GU722406; HM061592 JN223503; JN223492; JN223474

17. viii. 2008 12. vi. 2009 18. vii. 2009 15. vii. 2008 30. vii. 2008 17. viii. 2008 11. vii. 2009 1. vi. 2009 14. ix. 2009 8. viii. 2010 16. vii. 2009 17. viii. 2010 10. vii. 2008 16. vii. 2009 15. vii. 1996 8. vii. 2009 12. vi. 2009 29. vii. 2009 10. vi. 2009 15. vii. 2008 13. viii. 2010

JN223514; JN223500; JN223477 KJ816745; KJ816711; KJ816729

29. vi. 2010 viii. 2013

KJ816743; KJ816709; KJ816727

25. viii 2004

KJ816744; KJ816710; KJ816728 GU722391; GU722407; HM061600

viii. 2012 12. vii. 2009

Mt. Emei, Sichuan, 1150 m Taibaishan, Shaanxi, 2250 m Yulongxueshan, Yunnan, 3200 m Shennongjia, Hubei, 1600 m Nangongshan, Shaanxi, 1800 m Zhuque, Shaanxi, 1250 m Yushe, Guizhou, 1900 m Mt. Emei, Sichuan, 1150 m Kathmandu, Nepal, 2015 m Diaoluoshan, Hainan,1200 m Zhongdian, Yunnan, 2800 m Leigongshan, Guizhou, 1250 m Yarlung Tsangpo Grand Canyon, Tibet, 2030 m Huoditang, Shaanxi, 1930 m Tianshui, Gansu, 1600 m Fengyangshan, Zhejiang, 1500 m Hupingshan, Hunan, 1100 m Yushe, Guizhou, 1900 m Dongpuo, Yunnan, 2800 m Lushan, Jiangxi, 1300 m Diaoluoshan, Hainan, 1200 m Leigongshan, Guizhou, 1550 m Qingliangfeng, Zhejiang, 1000 m Hupingshan, Hunan, 1100 m Xishan, Kunming, Yunnan, 2500 m Huaboshan, Kuandian, Liaoning, 1000 m USA

JQ011461; KJ816713; JQ011462 GU722385; GU722401; HM061594 KJ816730; KJ816696; KJ816714 JN688131; JN688142: JN688119 JN688127; JN688141; JN688120 GU722388; GU722404; HM061595 - - - - - - - - -; KJ830627; KJ830628 GU722395; GU722412; HM061598 KJ816741; KJ816707; KJ816725 JN688133; JN688135; JN688121 JN688130; JN688137; JN688124 KJ816731; KJ816697; KJ816715 KJ816733; KJ816699; KJ816717

8. vii. 2011 12. vi. 2009 6. vi. 2009 4. viii. 2010 18. viii. 2010 15. vii. 2008 3. vii. 2012 26. vii. 2008 24. vii. 2013 14. iv. 2008 4. viii. 2010 21. vii. 2012 12. vi. 2009

GU722387; GU722403; HM061597 KJ816732; KJ816698; KJ816716 KJ816736; KJ816702; KJ816720 KJ816740; KJ816706; KJ816724 KJ816734; KJ816700; KJ816718 JN688134; JN688138; JN688125 KJ816739; KJ816705; KJ816723 NC013180; NC013180; JN688122 KJ816738; KJ816704; KJ816722 KJ816735; KJ816701; KJ816719 KJ816737; KJ816703; KJ816721 JN688129; JN688136; JN688123 JN223516; JN223498; JN223487

2. vi. 2009 7. viii. 2011 2. viii. 2008 26. vii. 2013 4. vii. 2012 4. viii. 2010 7. vi. 2009 11. iv. 2008 20. vii. 2012 1. viii. 2011 26. vii. 2013 24. vii. 2010 10. vi. 2010

AF180070; AF424033; AF423967

Japan Japan USA USA Europe

- - - - - - - - -; AF424025; AF423959 - - - - - - - - -; AF424026; AF423960 - - - - - - - - -; AF424022; AF423955 - - - - - - - - -; AF424028; AF423962 AF180072; KJ816712; AF423954

P. communis Linnaeus, 1758*

Europe

AF180071; AF424024; AF423957

P. debilis Westwood, 1846* P. fluvicaudaria Miyake, 1910*

USA Japan

- - - - - - - - -; AF424023; AF423956 AF180073; AF424020; AF423953

Misof et al. (2000) (2002) Whiting (2002) Whiting (2002) Whiting (2002) Whiting (2002) Misof et al. (2000) (2002) Misof et al. (2000) (2002) Whiting (2002) Misof et al. (2000) (2002)

P. bifasciata Chou & Wang, 1981 P. byersi Huang & Hua, 2005 P. changbaishana Hou & Hua, 2008 P. chengi Chou, 1981 P. curva Carpenter, 1938 P. decolorata Chou & Wang, 1981 P. dubia Chou & Wang, 1981 P. emarginata Cheng, 1949 P. fulvastra Chou, 1981 P. issikiana Byers, 1970 P. jilinensis Zhou, 2000 P. kunmingensis Fu & Hua, 2009 P. liui Hua, 1997 P. macrostyla Hua, 1998 P. maculosa Hagen, 1861 P. nanwutaina Chou, 1981 P. obtusa Cheng, 1949 P. qinlingensis Chou & Ran, 1981 P. wangwushana Huang & Hua, 2004 P. sexspinosa Cheng, 1949 Panorpa sp1 Panorpa sp2 Panorpa sp3 P. speciosa Carpenter, 1931 P. vulgaris Imhoff & Labram 1845 Furcatopanorpa longihypovalva (Hua & Cai, 2009) Dicerapanorpa kimminsi (Carpenter, 1948) D. magna (Chou, 1981) D. tjederi (Carpenter, 1938) Sinopanorpa digitiformis Huang & Hua, 2008 S. nangongshana Cai & Hua, 2008 S. tincta (Navás, 1931) Neopanorpa anchoroides Zhou, 2003 N. chelata Carpenter, 1938 N. chillcotti Byers, 1971 N. hualizhongi Hua & Chou, 1998 N. lacunaris Navás, 1930 N. leigongshana Zhou & Zhou, 2012 N. lifashengi Hua & Chou, 1999 N. longiprocessa Hua & Chou, 1997 N. lui Chou & Ran, 1981 N. moganshanensis Zhou & Wu, 1993 N. nigritis Carpenter, 1938 N. pallivalva Zhou, 2003 N. pendula Qian & Zhou, 2001 N. pielina Navás, 1936 N. pulchra Carpenter, 1945# N. puripennis Chou & Wang, 1988 N. tienmushana Cheng, 1957 N. tienpingshana Chou & Wang, 1988 N. uncata Zhou, 2000 Panorpodes kuandianensis Zhong, Zhang & Hua, 2011 Panorpa acuta Carpenter, 1931* P. P. P. P. P.

arakavae Miyake, 1913* bicornuta MacLachlan, 1887* carolinensis Banks, 1905* claripennis Hine, 1901* cognata Rambur, 1842*

and Whiting

and Whiting and Whiting

and Whiting

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G.-L. Hu et al. / Molecular Phylogenetics and Evolution 85 (2015) 22–31 Table 1 (continued) Species

Locality

GenBank accession Nos. for cox1, cox2 and 28S

Collecting date/Source

Europe

AF180075; AF424032; AF423965

USA

AF180076; AF424029; AF338264

P. japonica Thunberg, 1784*

Japan

EF050552; EF050564; AF423969

P. latipennis Hine, 1901* P. nebulosa Westwood, 1846*

USA USA

- - - - - - - - -; AF424031; AF423964 KF887290; AF424030; AF423963

P. rufostigma Westwood, 1846* P. striata Miyake, 1908* P. takenouchii Miyake, 1908* Neopanorpa harmandi (Navás, 1908)* Brachypanorpa carolinensis Banks 1905*

Alepochorion, Greece Japan Japan unknown USA

EF050550; EF050562; EF050546 - - - - - - - - -; AF424036; AF423970 EF050553; EF050565; EF050548 - - - - - - - - -; AF424027; AF423961 EF050557, EF050569, EF050542

Misof et al. (2000) and Whiting (2002) Misof et al. (2000) and Whiting (2002) Pollmann et al. (2008) and Whiting (2002) Whiting (2002) Schiff et al., unpublished and Whiting (2002) Pollmann et al. (2008) Whiting (2002) Pollmann et al. (2008) Whiting (2002) Pollmann et al. (2008)

P. germanica Linnaeus, 1758* P. helena Byers, 1962

*

- - - - - - - - - The sequences are neither successfully amplified by PCR nor available on NCBI. Some of them do not contain detailed information about locations and collecting date, so we only provide their continents or countries and related references. # The mitochondrial gene fragments of N. pulchra were obtained from GenBank. * Two or three gene fragments of the species were obtained from GenBank.

Table 2 List of forward (F) and reserve (R) primers used to amplify regions of cox1, cox2 and 28S rRNA. Gene

Primer

Sequence (50 ? 30 )

Source

cox1

C1-J-1718 (F) C1-N-2329 (R) LCO1490 (F) HC02198 (R) COII-2a (F) COII-9b (R) 28S rD3.2a (F) 28S rD4.2b (R)

GGA GGA TTT GGA AAT TGA TTA GTT CC ACT GTA AAT ATA TGA TGA GCT CA GGT CAA CAA ATC ATA AAG ATA TTG G TAA ACT TCA GGG TGA CCA AAA AAT CA ATA GAK CWT CYC CHT TAA TAG AAC A GTA CTT GCT TTC AGT CAT CTW ATG AGT ACG TGA AAC CGT TCA SGG GT CCT TGG TCC GTG TTT CAA GAC GG

B. Farrel F. Sperling Folmer et al. (1994)

cox2 28S

codon position of cox2 with HKY + I + G. The combined cox2 and 28S rRNA dataset was divided into four partitions, one for 28S gene, plus first, second and third codon position for cox2. Their respective best-fit models are GTR + G, TrN + I + G, HKY + I + G and K81uf + G. To construct phylogenetic trees, maximum likelihood (ML) and Bayesian inference (BI) were performed on the datasets. Maximum likelihood analyses were executed with the program raxmlGUI 1.3, a graphical front-end for RAxML (Silvestro and Michalak, 2012). All ML analyses with thorough bootstrap were run ten times starting from random seeds under the GTRGAMMA model. The bootstrap support value (BS) was evaluated by analysis with 1000 replicates. Bayesian inference was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Two replicate runs with four independent chains were run for 4 million generations starting with random trees and sampling every 100 generations. The average standard deviation of split frequency was lower than 0.01, indicating that the sampling of posterior distribution was adequate. The stationarity was determined with the program Tracer 1.5 (Rambaut and Drummond, 2009) by plotting the log-likelihood values versus generation number. After stationarity had been reached, the first 25% of the total samples were discarded and the remaining trees were used to generate a majority rule consensus tree and calculate the posterior probabilities (PP). 2.4. Topology test To compare alternative phylogenetic hypotheses, constrained ML trees were specified based on concatenated data of three genes (cox1, cox2 and 28S rRNA) under the same partition scheme and model to the unconstrained settings except for the rapid bootstrap. Then per-site likelihood values were calculated for both uncon-

Whiting (2002) Whiting (2002)

strained and constrained topologies. P-values were obtained for the approximately unbiased (AU) and the Shimodaira-Hasegawa (SH) tests (Shimodaira, 2002; Shimodaira and Hasegawa, 1999) with the software CONSEL (Shimodaira and Hasegawa, 2001). 2.5. Divergence time estimation To obtain Bayesian chronogram, the program BEAST 1.8.0 (Drummond and Rambaut, 2007) was used under uncorrelated lognormal relaxed clock and a speciation Yule process with the three concatenated genes, with the same partition scheme and models. Four runs were conducted with a chain length of 100 million generations, sampling every 100 generations, and randomly generating starting tree. The stationarity and convergence of chains were checked in Tracer 1.5, ensuring the effective sample sizes >200 for all parameters (Rambaut and Drummond, 2009). In order to generate a smaller tree file, we sampled trees with the first 25% generations as burn-in and resampled the remaining trees every 1000 generations using the program LogCombiner 1.8.0 (Drummond and Rambaut, 2007). TreeAnnotator 1.8.0 was used to find a maximum credibility tree with the annotation of mean node ages and the 95% highest posterior density (HPD) intervals (Drummond and Rambaut, 2007). The results were visualized in FigTree 1.3.1 (Rambaut, 2009). The fossil evidence of scorpionflies was used as calibrations to estimate chronograms based on the protocol of Parham et al. (2012). First, the fossils of Panorpa mortua and P. obsoleta from Baltic amber dated from the lower Eocene Ypresian (50 mya) to Lutetian (40 mya) (Carpenter, 1954; Krzemin´ski and Soszyn´ska-Maj, 2012) were used to calibrate the most recent possible origin of Panorpa, with a normal distribution at 45 ± 3 mya. Second, the time to most recent common ancestor (tMRCA) was inferred for

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Panorpodidae based on the fossil record of Panorpodes weitschati in Baltic amber from upper Eocene Bartonian to Priabonian with a prior normally distributed around 37.7 ± 3 mya (Perkovsky et al., 2007; Soszyn´ska-Maj and Krzemin´ski, 2013). 3. Results 3.1. Sequence variation and alignment A total of 207 sequences (63 of cox1, 72 of cox2, 72 of 28S rRNA) were successfully obtained for five genera of Panorpidae and two species of Panorpodidae. After alignment and ends cutting, a total of 1622 base pairs (564 bp for cox1, 576 bp for cox2, and 482 bp for 28S rRNA) were used to reconstruct phylogenetic trees. The saturation test with DAMBE shows little saturation for all the three genes and each codon position of the protein-coding genes. The alignment sites, parsimony informative sites, average base frequency and A-T content are listed in Table 3. Chi-square test of homogeneity of the base frequency shows that base compositional heterogeneity is not significant (P > 0.05) across all the taxa. All the three gene fragments have high A-T preferences, 67.9% for cox1, 72.6% for cox2, and 66.7% for 28S rRNA, consistent with the situation of other insects (Frati et al., 1997; Simon et al., 1994). 3.2. Phylogenetic reconstruction based on three concatenated genes For the concatenated dataset of the three genes, phylogenetic topologies (Fig. 1) obtained with ML and BI methods are nearly identical except for the collapsed relationship of the Panorpa centralis group and the slight change of the position of Neopanorpa lui, N. chelate, N. puripennis and N. tienmushana in the BI tree. Moreover, the support values in the BI tree are generally higher than those in the ML tree. Panorpidae are well supported for their monophyly and are grouped into two principal clades with high support value (BS = 100, PP = 1). Clade A comprises all the species of Neopanorpa, implying the monophyly of the genus. In this clade, N. chillcotti forms a sister taxon to the remaining species of Neopanorpa with high support values (BS = 97, PP = 1). Clade B is further subdivided into seven subclades (C–I), clearly showing that the genus Panorpa is paraphyletic with Sinopanorpa, Dicerapanorpa and Furcatopanorpa, although Sinopanorpa and Dicerapanorpa are almost definitely monophyletic. Subclade C consists of three species of Dicerapanorpa, D. magna, D, kimminsi and D. tjederi, reconfirming the monophyly of Dicerapanorpa with high support values (BS = 100, PP = 1). Subclade D consists of the Panorpa centralis group: P. emarginata, P. wangwushana, P. dubia, P. nanwutaina, P. obtusa, P. byersi, P. acutipennis and P. fluvicaudaria. This species group is confirmed to be a monophyletic clade with moderately well support (BS = 62, PP = 1), although the relationship within the clade is collapsed.

Subclade E is formed by Panorpa sp1, P. chengi, P. sexspinosa, P. fulvastra, P. qinlingensis and P. curva with strong support values (BS = 93, PP = 1) and forms the sister group to the P. centralis group. In subclade F, Sinopanorpa is uncovered as a monophyletic group with high support values (BS = 100, PP = 1) and has a sister group relationship with P. bifasciata. Subclade G is composed of the North American species, including Panorpa helena, P. speciosa, P. acuta, P. maculosa, and P. nebulosa. P. acuta is the sister taxon to P. maculosa with high support (BS = 100, PP = 1), but the phylogenetic relationship of the other three species remains low resolution. Subclade H is mainly composed of the European species (Panorpa vulgaris, P. communis, P. cognata, P. germanica and P. rufostigma) except P. changbaishana from northeastern China. P. rufostigma branches off from the base of the subclade (BS = 81, PP = 0.99). The sister–taxon relationships of P. vulgaris + P. communis (BS = 100, PP = 1) and P. cognata + P. germanica (BS = 74, PP = 0.99) are strongly supported. Subclade I is split into two distinct groups. In the first group, Panorpa sp2, P. kunmingensis and P. decolorata, all of which are mainly distributed in southwestern China, form a monophyletic clade with strong support (BS = 95, PP = 1). The second group mainly consists of northeastern Asian species (P. takenouchii, P. japonica, P. macrostyla, P. jilinensis, P. liui) except Furcatopanorpa longihypovalva from central China. The P. amurensis group (including P. jilinensis and P. liui) seems to be paraphyletic, since P. liui receives high support to be the sister taxon to F. longihypovalva (BS = 100, PP = 1). 3.3. Phylogenetic reconstruction based on two combined genes Based on the analyses of two combined genes, the topologies (Fig. 2) are basically in accordance with the above trees except for some minor differences such as the position of the Panorpa liui and Furcatopanorpa longihypovalva cluster and the nine newly added species. In addition, some species groups are mixed together and remain incompletely resolved in the BI tree (Fig. 2B). Of the nine newly added species, the Japanese species P. arakavae forms the sister taxon to P. fluvicaudaria, P. bicornuta forms the sister species to P. takenouchii, and P. striata is the sister species to P. macrostyla (P. japonica + P. jilinensis). The four newly added North American species (P. latipennis, P. carolinensis, P. debilis, and P. claripennis) are expectedly grouped together with other North American species of Panorpa. Both ML and BI trees grouped all the species of Neopanorpa into a monophyletic clade, although the specific positions of Neopanorpa anchoroides and N. harmandi are inconsistent between the ML and BI trees. 3.4. Topology test P-values in both AU and SH tests for constrained topologies are listed in Table 4. The monophylies of the genera Neopanorpa,

Table 3 Summary of the sequence information. Taxa

cox1 cox2 28S 28S* 28S + cox2 28S* + cox1 + cox2

63 72 72 63 72 63

Length (bp)

564 576 482 482 1060 1622

Pi

188 218 234 228 451 626

Pi means parsimony informative sites. * means the data were only used in the concatenated analysis of three genes.

Average base frequency (%)

A-T content (%)

A

C

G

T

28.6 33.3 27.4 27.4 30.8 30.0

17.0 13.7 14.7 14.6 14.1 15.1

15.2 13.7 18.6 18.7 15.8 15.6

39.3 39.3 39.3 39.3 39.3 39.3

67.9 72.6 66.7 66.7 70.1 69.3

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B.carolinensis

100/1

Pd.kuandianensis N.lui 91/1

Panorpodidae

N.chelata N.moganshanensis N.pielina 62/? 94/1 N.longiprocessa N.pendula 100/1 100/1 N.pulchra N.hualizhongi 55/0.69 */- */N.uncata 66/0.97 N.lacunaris Clade A */N.puripennis 97/1 */? N.tienmushana N.lifashengi */N.tienpingshana 100/1 N.leigongshana 97/1 N.nigritis 100/1 N.pallivalva N.chillcotti D.tjederi 100/1 D.magna C 85/1 D.kimminsi P.emarginata 62/1 P.wangwushana D 51/0.94 P.dubia */P.nanwutaina P.obtusa 80/1 59/1 */0.81 P.byersi */P.acutipennis 56/1 100/1 P.fluvicaudaria P.sp1 93/1 P.fulvastra E P.chengi 76/0.98 63/0.92 P.qinlingensis 81/1 */0.96 P.curva 95/1 P.sexspinosa P.sp3 F 60/0.99 P.issikiana 63/0.99 98/1 P.bifasciata */100/1 S.nangongshana S.digitiformis */0.87 83/0.84 S.tincta P.acuta 100/1 P.maculosa 57/0.93 P.speciosa G */P.nebulosa 50/0.77 P.helena */0.97 P.rufostigma Clade B P.cognata 81/0.99 74/0.99 P.germanica H 99/0.99 100/1 P.communis */0.89 P.vulgaris P.changbaishana P.decolorata 95/1 P.kunmingensis 100/1 P.sp2 */0.97 P.takenouchii I 54/0.6 P.japonica 73/1 85/1 P.jilinensis P.macrostyla */0.97 100/1 P.liui F.longihypovalva 93/1

0.4

Fig. 1. Phylogenetic tree obtained from maximum likelihood (ML) analysis based on concatenated data of genes cox1, cox2 and 28S rRNA. ML bootstrap values and Bayesian posterior probabilities are indicated at internal nodes. Bootstrap values under 50 are replaced by ‘‘⁄’’; ‘‘–’’ indicates the clades or species are mixed together in Bayesian inference (BI); ‘‘?’’ means the positions of Neopanorpa lui, N. chelate, N. puripennis and N. tienmushana in BI tree is slightly different from those in ML tree. The right pictures are lateral views of male abdomens, from the top to bottom being N. longiprocessa, N. puripennis, Dicerapanorpa magna, Panorpa dubia, P. sexpinosa, Sinopanorpa tincta, P. nebulosa, P. helena, P. changbaishana, P. kunmingensis, P. liui and Furcatopanorpa longihypovalva, respectively.

Sinopanorpa and Dicerapanorpa as well as the Panorpa centralis and P. nebulosa groups are well supported. The genus Panorpa resolved as paraphyly in the topology of Figs. 1 and 2 is rejected as monophyly by AU and SH tests with significant P-values. The monophyly of the P. amurensis group is also rejected by the tests.

3.5. Divergence time estimation The divergence time chronogram of Panorpidae is presented in Fig. 3, with the branch length as mean age. The estimated divergence time between Panorpidae and Panorpodidae is

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8

100

N.anchoroides N.harmandi N.lui 77 N.chelata 3 23 N.moganshanensis 65 N.pielina 82 N.longiprocessa 6 N.lifashengi N.tienmushana 16 57 N.pendula 56 100 77N.lacunaris N.uncata 31 78 N.hualizhongi 100 N.pulchra 95 N.puripennis 100 N.leigongshana 96 N.tienpingshana N.nigritis 92 N.pallivalva N.chillcotti D.tjederi 100 D.magna 95 D.kimminsi P.sp1 87 P.fulvastra P.sexspinosa 80 78 P.qinlingensis 94 P.curva 49 42 P.chengi 90 P.dubia 71 22 P.nanwutaina 35 P.byersi 60 P.obtusa 28 P.emarginata P.wangwushana 16 P.acutipennis 74 99 P.arakavae P.fluvicaudaria 100 P.acuta 31 P.maculosa 33 100 P.debilis P.speciosa 75 90 P.nebulosa 62 P.latipennis 84 100 P.claripennis P.carolinensis 98 20 P.helena P.sp3 P.bifasciata 31 58 P.issikiana 99 100 S.nangongshana S.digitiformis 54 S.tincta P.changbaishana 39 48 98 P.communis 100 P.vulgaris P.cognata 91 65 P.germanica P.rufostigma 100 P.kunmingensis 86 P.sp2 P.decolorata 29 98 P.takenouchii P.bicornuta 51 P.striata 61 P.jilinensis 34 34 P.japonica 94 P.macrostyla 100 P.liui F.longihypovalva

0.5

N.anchoroides N.chelata N.lui 0.55 N.moganshanensis 1 N.longiprocessa 1 N.pielina N.harmandi N.tienmushana 0.98 N.pendula 1 1 1 N.lacunaris N.uncata 0.61 0.86 1 N.hualizhongi N.pulchra N.puripennis 1 1 N.leigongshana 1 0.99 N.tienpingshana N.nigritis N.pallivalva N.lifashengi N.chillcotti 0.98 D.kimminsi 1 D.magna D.tjederi P.sp3 0.97 P.bifasciata 1 P.issikiana 0.87 S.digitiformis 1 S.tincta S.nangongshana 0.88 P.chengi 0.85 P.curva 1 0.98 1 P.qinlingensis P.sexspinosa 1 P.fulvastra P.sp1 0.89 P.byersi 0.98 P.dubia P.nanwutaina 0.75 1 P.emarginata P.obtusa 0.64 P.wangwushana P.arakavae 1 1 P.fluvicaudaria 0.85 P.acutipennis 1 P.acuta P.maculosa 1 P.debilis P.speciosa 0.6 0.92 0.98 P.nebulosa P.latipennis 1 P.claripennis 0.95 P.carolinensis 1 P.helena P.changbaishana 0.66 0.84 P.communis 1 1 P.vulgaris 0.98 P.cognata 1 P.germanica P.rufostigma P.kunmingensis 1 1 P.sp2 P.decolorata 0.67 1 P.bicornuta P.takenouchii P.striata 0.97 0.9 P.jilinensis 0.63 0.59 P.japonica 1 P.macrostyla 1 P.liui F.longihypovalva 1

1

0.3

A

B

Fig. 2. Phylogenetic trees obtained from maximum likelihood (A) and Bayesian inference (B) based on combined data of cox2 and 28S rRNA (outgroups not shown). Bootstrap values and posterior probabilities are given at internal nodes. Table 4 Approximately unbiased and Shimodaria-Hasegawa tests for alternative hypotheses using the three concatenated genes. The significant values (P < 0.05 for AU and SH) are in boldface, suggesting that the monophyly is rejected. Topology constraints

ln Likelihood

AU

SH

Unconstrained tree Monophyly of Neopanorpa Monophyly of Panorpa Monophyly of Dicerapanorpa Monophyly of Sinopanorpa Monophyly of the P. centralis group Monophyly of the P. amurensis group Monophyly of the P. nebulosa group

20861.385230 20858.774404 21214.033683 20858.771959 20858.773147 20860.970717 21022.824391 20854.167577

0.592 0.001 0.611 0.591 0.503 2e072 0.660

0.597 0 0.596 0.596 0.518 0 0.670

approximately at 173.9 mya with a wide 95% HPD interval from 282.8 to 70.0 mya. The time to the most recent common ancestor of Neopanorpa is roughly at 35.7 mya, slightly later than the origin of Panorpa. The divergence of Dicerapanorpa from Panorpa is approximately dated to 31.9 mya. Sinopanorpa split from Panorpa at some 11.9 mya. The P. centralis group originated at approximately 21.0 mya. The North American species of Panorpa originated at around 22.9 mya. The most recent origin of European Panorpa species is estimated at 33.7 mya.

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37.4

Pd.kuandianensis B.carolinensis N.chillcotti N.tienmushana 19.9 N.pendula N.hualizhongi 13.6 2.1 N.pulchra 23.4 12.3 N.uncata 8.6 N.lacunaris 35.7 N.puripennis 24.7 N.lifashengi N.pallivalva 20.8 7.3 N.nigritis 26.6 15.9 N.leigongshana 6.9 N.tienpingshana 17.8 N.moganshanensis 27.8 N.pielina 12.8 N.longiprocessa N.lui 18.1 N.chelata D.tjederi 14.7 D.magna 8.5 D.kimminsi P.wangwushana 5.1 10.0 P.nanwutaina P.emarginata 6.8 3.3 P.byersi 21.0 P.obtusa 52.5 31.9 5.6 P.dubia 4.3 P.acutipennis P.fluvicaudaria 23.8 P.sp1 16.5 P.fulvastra P.sexspinosa 9.2 14.3 P.curva 6.0 P.qinlingensis 12.9 28.4 P.chengi P.bifasciata 15.0 P.issikiana 37.8 0.6 S.digitiformis 23 11.9 S.tincta 1.0 S.nangongshana P.sp3 26.0 P.nebulosa 16.3 P.acuta 9.2 P.maculosa 22.9 P.helena 16.7 P.speciosa P.rufostigma 44.5 33.7 P.cognata 14.6 P.germanica 21.0 3.6 P.communis P.vulgaris 17.5 P.changbaishana P.decolorata 25.5 14.5 P.kunmingensis P.sp2 36.1 P.takenouchii 14.6 26.9 P.japonica P.jilinensis 11.7 P.macrostyla 21.9 P.liui 6.1 F.longihypovalva

173.9

China Japan Europe North America

180.0

160.0

140.0

120.0

100.0

80.0

60.0

40.0

20.0

0.0

Time in million years before present Fig. 3. Chronogram of Panorpidae divergence time estimated from the BEAST analysis with a Bayesian relaxed lognormal clock. The numbers at nodes indicate the mean ages and blue bars represent 95% highest posterior density intervals for the node ages. Continental distributions are indicated on the right side of the taxon names.

4. Discussion 4.1. Phylogeny of Panorpidae The present study made a relatively comprehensive phylogenetic analysis of Panorpidae inferred from mitochondrial and nuclear gene sequences sampled from East Asia, North America, and Europe. Based on the phylogenetic tree (Fig. 1), the species of Panorpidae fall into two distinct clades (A and B) with high support values, suggesting that the two clades may represent two

separate subfamilies of Panorpidae, although Leptopanorpa, a closely related genus to Neopanorpa (Lieftinck, 1936; Ma et al., 2012; Willmann, 1989), is not included in the analysis. Based on the present phylogenetic analysis, Neopanorpa is definitely a monophyletic group (Figs. 1 and 2), inconsistent with the previous conclusion that Neopanorpa makes the genus Panorpa paraphyletic (Ma et al., 2012; Misof et al., 2000; Willmann, 1989). The paraphyly argument of Panorpa with Neopanorpa may partly result from the fact that Misof et al. (2000) and Whiting (2002) only include one species of Neopanorpa in their analyses.

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The separation of Neopanorpa from other genera of Panorpidae is supported by characters of a developed notal organ, distinctive male genitalia, undeveloped salivary glands, and adopting a nonnuptial-providing mating behaviour (Byers, 1965, 1994; Chau and Byers, 1978; Hua and Chou, 1997; Ma et al., 2011; Zhong and Hua, 2013b). Neopanorpa chillcotti reasonably forms the sister species to all the other species of Neopanorpa, and is only known to occur in Nepal with abdominal segments VI–VIII of males extremely elongated, together about twice the length of segments I–V combined (Byers, 1971). The topologies of the phylogenetic trees show that Panorpa is reconfirmed to be a paraphyletic assemblage with Sinopanorpa and Dicerapanorpa, both of which are separated from Panorpa recently (Cai et al., 2008; Zhong and Hua, 2013a). The Chinese species of Panorpa were once categorized into the P. diceras, P. centralis and P. davidi groups (Carpenter, 1938; Cheng, 1957). The P. diceras group is recognized mainly by the double anal horns on tergum VI and trifurcate parameres in males, and the genital plate with the axis not extending beyond the main plate in females (Zhong and Hua, 2013a). This species group was recently raised to generic status (Dicerapanorpa Zhong & Hua, 2013), which receives a high support from the present molecular phylogenetic analysis. The species of the P. centralis group are distributed in China, Korea and Japan, and are quite uniform in appearance, especially the presence of a single anal horn on the posterior margin of male tergum VI (Cheng, 1957). From the phylogenetic trees of the three combined genes, the eight species of the P. centralis group studied are all clustered into a clade (subclade D, Fig. 1). The AU and SH tests accept the monophyly of the P. centralis group (Table 4). In combination with other morphological characters (Ma et al., 2009, 2012), the P. centralis group may very likely merit a generic status. The species examined for the P. davidi group are diverse in morphology and are topologically scattered along the phylogenetic trees (in subclades E, F and I, Fig. 1), indicating the P. davidi group is apparently a paraphyletic grade, which may need further split. With respect to the monotypic genus Furcatopanorpa, its position on the phylogenetic tree differ greatly from the result of Ma et al. (2012) from morphological data. In the topology of our phylogram (Fig. 1), it is strange enough that P. liui forms a sister taxon relationship with Furcatopanorpa longihypovalva rather than P. jilinensis, a member of the P. amurensis group, to which P. liui belongs. This result is inconsistent with Ma et al. (2012). The genus Furcatopanorpa was erected on a suite of autopomorphy characters (Ma and Hua, 2011). According to their phylogenetic analysis on morphological data, Furcatopanorpa forms a sister group relationship with all the other genera of Panorpidae (Ma et al., 2012). Furcatopanorpa needs further study for its phylogenetic position. Based on our analysis, the North American species of Panorpa form sister group relationship with the East Asian species instead of the European species, indicating the Nearctic fauna is more closely related to the east Palaearctic fauna rather than the European fauna.

4.2. Divergence time and biogeography of Panorpidae Based on our relaxed molecular clock analysis, most diversification in Panorpidae occurred in the Cenozoic and the family Panorpidae likely originated in the period from Early Paleogene (63.6 mya) to middle Eocene (41.2 mya) (Fig. 3). The origin of Panorpidae estimated from the molecular data is younger than the oldest fossil record of Panorpidae (Ding et al., 2014), indicating the discrepancy between the known fossil record and molecular estimates.

The Asian, North American and European species of Panorpa are generally clustered in their own clades except for a few cases of intercontinental mixing. The Asian species of Panorpa exhibit the greatest diversity, suggesting that the genus may have an Asian origin, as suggested by Byers (1988). Some North American species of Panorpa exhibit structural similarities to those in the Chinese fauna, reinforcing the argument that Panorpidae originated in Asia and migrated into North American via the Bering land bridge (Byers, 1988), since Asia remained in contact with North America through the Bering land bridge during the period from at least early Paleocene to Pliocene (Downes and Kavanaugh, 1988; Sanmartin et al., 2001; Tiffney and Manchester, 2001). The faunal exchange between Europe and Asia was strengthened during the Oligocene (30 mya) after the Turgai Strait dried up (Sanmartin et al., 2001). However, the uplift of the Himalaya Mountains in the Late Oligocene isolated Europe and West Asia from East Asia (Macey et al., 1999). Our BEAST analysis (Fig. 3) indicates that the occurrence of Panorpa in Europe was earlier than North America, thus the North American species of Panorpa are more closely related to the Asian than European species. The species of Panorpa from Japan and China are mixed together, and their close relationship may result from the closely geographical location. According to Sanmartin et al. (2001), the vicariance between the Japanese and Chinese biotas resulted from the opening of the Japanese Sea, which initiated in the Miocene but did not completely isolate the Japanese island until the Late Miocene–Early Pliocene (6 mya). Thus, the faunal exchange between China and Japan was facilitated in the early evolutionary history of Panorpa. Currently, the morphological similarities of the two isolated regions are retained, and may arise through morphological stasis and/or low rates of morphological evolution (Wen, 2001). 5. Conclusions A phylogenetic analysis of Panorpidae was conducted based on three genes and partly resolved the phylogenetic relationships of Panorpidae, although it failed to contain representatives of Leptopanorpa. The monophylies of Neopanorpa, Sinopanorpa and Dicerapanorpa, and the P. centralis group are strongly supported, and the paraphyly of the genus Panorpa is reconfirmed. Nevertheless, Panorpidae are such a complicated family that the combination of DNA sequences with morphological data may be needed to satisfactorily resolve the phylogeny and make the generic revision of Panorpidae. Acknowledgments We thank Na Ma, Jing Chen, Qingxiao Chen, Beibei Zhang, Lu Jiang, Meng Wang, Guowei Zhang, Ying Miao, Mei Liu and Chao Gao for assistance in specimen collection, and Wen Zhong for taking digital photos and identifying some specimens. We also thank the two anonymous reviewers for their valuable comments and suggestions on the revision of the manuscript. This research was financially supported by the National Natural Science Foundation of China (Grant no. 31172125). References Archibald, S.B., Mathewes, R.W., Greenwood, D.R., 2013. The Eocene apex of panorpoid scorpionfly family diversity. J. Paleontol. 87, 677–695. Byers, G.W., 1965. The Mecoptera of Indo-China. Pac. Insects 7, 705–748. Byers, G.W., 1969. Ecological and geographical relationships of southern Appalachian Mecoptera (Insecta). In: Holt, P.C., Hoffman, R.L., Hart, C.W. (Eds.), The distributional history of the biota of the southern Appalachians. Part I: Invertebrates Res. Div. Monogr. Virginia Polytechnic Institute, Blacksburg, pp. 265–276. Byers, G.W., 1971. A new Neopanorpa from Nepal. J. Kans. Entomol. Soc. 44, 534– 539.

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