http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–10 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2013.830289

ORIGINAL ARTICLE

Global haplotype analysis of the whitefly Bemisia tabaci cryptic species Asia I in Asia Jian Hu1, Yong-Dui Chen1, Zhi-Lin Jiang2, Francesco Nardi3, Tai-Yuan Yang4, Jie Jin5, and Zhong-Kai Zhang1

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1

Key Lab of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture, Yunnan Provincial Key Lab of Agricultural Biotechnology, Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming, China, 2Key Laboratory for Agricultural Biodiversity and Pest Management of China Education Ministry, Yunnan Agricultural University, Kunming, China, 3Department of Life Sciences, University of Siena, Siena, Italy, 4Plant Protection and Quarantine Station of XiShuangBanNa State, Jinghong, China, and 5Research Institute of Tropical Eco-agricultural Sciences, Yunnan Academy of Agricultural Sciences, Yuanmou, China Abstract

Keywords

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidiae), is a cryptic species complex comprising a minimum of 24 cryptic species. Some members of this complex are important agricultural pests, causing considerable damage to vegetable as well as ornamental and horticultural crops. Asia I, one of the cryptic species of B. tabaci, is widely distributed in Asia. One hundred and sixty mitochondrial cytochrome oxidase I (COI) sequences from eight countries have been analyzed to investigate the geographic origin and current genetic structure of this cryptic species. Sixty different haplotypes were identified, with levels of genetic distances ranging from 0.001 to 0.021. A sign of possible genetic differentiation emerges from the differential distribution of dominant haplotypes in Indonesia and India compared to China. A possible ancient separation between Asia I in India and Indonesia and secondary contact in China has been hypothesized.

Bemisia tabaci, cryptic species Asia I, genetic structure, haplotype, mitochondrial COI

Introduction The whitefly Bemisia tabaci (Hemiptera: Aleyrodidiae), widely distributed throughout tropical and subtropical areas (Delatte et al., 2005), is a pholem-feeding insect that causes considerable damage to ornamental, horticultural and vegetable crops (De Barro et al., 2011). Damage is caused both directly, by feeding on the plant phloem, and indirectly, by transmitting more than 110 different plant viruses (Czosnek et al., 2002; Hogenhout et al., 2008; Jones, 2003). Due to no obvious morphological differences, B. tabaci was originally subdivided in number of socalled biotypes that differ in terms of host plant range, the capacity to cause plant disorders, attraction by natural enemies, insecticide resistance, and plant virus-transmission capabilities (Bedford et al., 1994; Brown et al., 1995; Jones, 2003). Recent studies suggested that the large majority of biotypes may be in fact regarded as genetically distinct cryptic species, and B. tabaci is now considered as a cryptic species complex (Boykin et al., 2007; De Barro et al., 2011; Dinsdale et al., 2010). Boykin et al. (2007), based on a Bayesian analysis of 366 mitochondrial cytochrome oxidase I (COI) DNA sequences, initially subdivided B. tabaci species complex in 12 major well-resolved genetic groups. Subsequently, Dinsdale et al. (2010) eliminated some problematic sequences and redefined the former 12 clades into 11 major clades at411% sequence divergence. Furthermore, these Correspondence: Jian Hu & Zhong-Kai Zhang, Key Lab of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture, Yunnan Provincial Key Lab of Agricultural Biotechnology, Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, 650223 Kunming, China. Tel: +86 0871 65113769. Fax: +86 0871 65160084. E-mail: [email protected] (J. Hu), [email protected] (Z.-K. Zhang)

History Received 22 May 2013 Revised 27 July 2013 Accepted 28 July 2013 Published online 24 January 2014

11 groups were further subdivided into 24 lower level groups at 43.5% divergence, suggesting that B. tabaci may be in fact composed of at least 24 distinct cryptic species. Henceforth, the threshold of 3.5% mitochondrial COI sequence divergence has been repeatedly used for B. tabaci species level separations and classification. Later studies that included a larger geographic sampling of B. tabaci, using the same approach proposed by Dinsdale et al. (2010), increased the supposed number of B. tabaci cryptic species from 24 to 28 and to 31 (Hu et al., 2011; Wang et al., 2012). Furthermore, extensive crossing experiments among putative B. tabaci species found good support for the taxonomic criterion of 3.5% sequence divergence to identify cryptic species in this taxon (De Barro et al., 2011; Elbaz et al., 2010; Liu et al., 2012; Sun et al., 2011; Wang et al., 2011; Xu et al., 2010). The mitochondrial COI gene has become a very popular tool for molecular taxonomy and identification since Hebert et al. (2003) proposed to use it as marker of choice in the context of the DNA barcoding system. A 648 bp segment of the mitochondrial COI has been repeatedly used as an universal marker for the identification of animal species (Hebert et al., 2004a; Ratnasingham & Hebert, 2007) in different taxa with high rates of success (Armstrong & Ball, 2005; Leite, 2012; Ratnasingham & Hebert, 2007) and has proved to be particularly informative for the detection of cryptic insect species (Hausmann et al., 2011; Hebert et al., 2004b; Pfenninger et al., 2007; Smith et al., 2006), phylogenetic studies (Freeland, 2005; Galtier et al., 2009) and intraspecific studies (Hu et al., 2008). Furthermore, universal primers are available to amplify this gene segment in a large range of taxa (Folmer et al., 1994; Simon et al., 1994; Zhang & Hewitt, 1997). De Barro & Ahmed (2011) first used mitochondrial COI haplotypes to study genetic networks of Bemisia tabaci

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cryptic species. On a global scale, they dscribed 78 haplotypes of cryptic species Middle East-Asia Minor 1 (hereon MEAM1, formerly known as biotype B) and 85 haplotypes of cryptic species Mediterranean (hereon MED, formerly known as biotype Q), inferring Israel as a possible source of MEAM1 and either Sub-Saharan Africa or the Mediterranean as possible sources for MED. MEAM1 and MED are the two most notorious species of the B. tabaci complex due to their global spread during the last 20 years and the high damages they cause to agricultural production (Brown et al., 1995; Dalton, 2006; Liu et al., 2007; Naranjo et al., 2010). Other B. tabaci cryptic species were, on the other hand, generally considered as indigenous to specific areas and generally incapable of causing damage above economic threshold. As such, studies on these latter cryptic species were far less intense than on the MEAM1 and MED invasive species. A preliminary survey of COI records available in GenBank (geographic origin and host species) from B. tabaci cryptic species Asia I nevertheless revealed that it is extensively distributed throughout Asia and is capable to attack a fairly large range of plant taxa. In fact, Asia I appears to be the predominant B. tabaci species in Asia, besides MEAM1 and MED. Yet, although preliminary genetic data of Asia I were reported in De Barro & Ahmed (2011), the genetic structure of this species has never been studied in detail. We collected Asia I samples from the Yunnan Province of China, a world-renowned biodiversity hotspot (Myers et al., 2000), and complemented the dataset with all Asia I COI accessions available in GenBank. Aims in this work are: (a) to describe the genetic structure of B. tabaci cryptic species Asia I on a global scale; and (b) to identify the geographic origin of the species and the key phylogeographic events that have determined its current genetic structure.

Materials and methods Sample collection Specimens of B. tabaci (males and females) were collected from cultivated vegetables, horticultural plants and natural weeds during 2010 and 2012 from 20 locations. Details of collection are summarized in Table 1. Adult whiteflies were stored in 95%

ethanol previous to genetic analysis, and voucher speciemens are preserved in the collection of the Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences. PCR amplification and sequencing Total DNA was extracted from individual adult specimens using the DNeasyÕ Blood & Tissue Kit (QIAGEN, Hilden, Germany). A 758-bp fragment of mitochondrial gene COI was amplified using universal primers C1-J-2195 (50 -TTG ATT TTT TGG TCA TCC AGA AGT-30 ) and TL2-N-3014 (50 -TCC AAT GCA CTA ATC TGC CAT ATT A-30 ) (Simon et al., 1994) with an amplification cycle consisting of an initial denaturation of 94  C for 5 min, followed by 35 cycles of 94  C for 45 s, 50  C for 1 min and 72  C for 1 min 30 s, and a final extension of 72  C for 10 min. PCR products were gel purified using the AxyprepTM DNA Gel Extraction Kit (AXYGEN, Union City, CA) and directly sequenced using an ABI 3730 XL DNA analyzer. Following sequence correction and assembly, newly determined sequences were deposited in GenBank with accession numbers KC12318284, 88-94, KC113528, 31-32, 35-36, 38-39, 41-42, 54-57, 60, 6264, 67-68, 72-73, 75-76, HQ916817-19, 21. Dataset assembly and analysis The newly determined haplotype sequences were complemented with 123 Asia I COI sequences retrieved from GenBank (Table 2) to produce a final dataset of 160 Asia I gene sequences. Sequences of B. tabaci cryptic species MEAM1, MED and Bemisia afer (accession no. HQ916816, KC113549, AJ784260) were introduced as out-groups in some analyses. Comparisons of genetic variability were performed after subdividing the global dataset into subsamples according to the country of origin, which are here tentatively treated as populations. Sequence alignment was performed using ClustalX 1.81 (Thompson et al., 1997). Genetic distances among geographical population and out-groups were calculated based on a pairwise matrix of sequence divergence calculated using the Kimura two-parameter method as implemented in MEGA 5.05 (Tamura et al., 2011). Haplotype definition and haplotype diversity estimates were performed using DnaSP 5.10 (Librado & Rozas, 2009). A minimum spanning network of haplotypes was constructed using statistical

Table 1. Sample collection localities and details for B. tabaci cryptic species Asia I populations in Yunnan (China).

Location Jiegang, Ruili, Dehong Maliba, Longchuan, Dehong Mangli, Mangshi, Dehong Mangdan, Baoshan Mangbang, Baoshan Dapingdi, Chuxiong Yuanmou, Chuxiong Tianxin, Yun county, Lincang Yingdi, Nanjian, Dali Xinping, Ning’er, Pu’er Jiangcheng, Jiangchuan, Yuxi Panxi, Huaning, Yuxi Dong’e, Yuanjiang, Yuxi Longtan, Yuanjiang, Yuxi Mile county town, Honghe Yusa, Kaiyuan Shisandui, Hekou Manzhen, Xishuangbanna Manhong, Xishuangbanna Gasa, Xishuangbanna

Latitude Longitude 

0

24 01 24 230 24 210 24 100 25 030 24 580 25 420 24 550 25 020 23 020 24 240 24 130 23 410 23 330 24 240 23 460 22 300 21 590 22 240 21 290



0

97 51 97 580 98 340 98 480 98 500 101 520 101 530 100 080 100 310 101 020 102 300 103 060 101 490 102 010 103 260 103 150 103 570 100 250 101 230 101 470

Host plant Laggera pterodonta Laggera pterodonta Laggera pterodonta Solanum melongena Ipomoea batatas Laggera pterodonta Solanum lycopersicum Tithonia diversifolia Phaseolus vulgaris Glycine max Ipomoea batatas Glycine max Dendrocnide urentissima Ipomoea batatas Solanum lycopersicum Ipomoea batatas Solanum melongena Glycine max Capsicum annuum Laggera pterodonta

Number of individuals Collection (number of different haplotypes) date Oct. 2010 Oct. 2010 Oct. 2010 Oct. 2010 Oct. 2010 Oct. 2010 Aug. 2012 Aug. 2012 Aug.2012 Aug. 2012 May 2012 May 2012 May 2012 May 2012 May 2012 May 2012 Sep. 2011 Aug. 2012 Aug. 2012 Sep. 2010

3 1 4 1 2 1 1 2 3 1 2 2 2 1 2 2 1 2 2 2

(3) (1) (3) (1) (1) (1) (1) (2) (3) (1) (2) (1) (1) (1) (2) (2) (1) (2) (2) (2)

Accession no. KC12318283, 93 KC123184 KC12318889, 94, HQ916818 HQ916817 KC123192, HQ916819 HQ916821 KC113572 KC11357576 KC11356264 KC113560 KC11353839 KC113531, 28 KC11356768 KC113573 KC11355455 KC11354142 KC113532 KC11355657 KC11353536 KC12319091

Haplotype analysis of Bemisia tabaci Asia I

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Table 2. List of mtDNA COI gene sequences of B. tabaci cryptic species Asia I obtained from GenBank.

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Country China China China China China China China China India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India India Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Thailand Thailand Thailand Indonesia Indonesia

Location

Host plant

Accession no.

Reference

Yunnan Hainan Yunnan Yunnan Guangxi Guangxi Taiwan – Kerala Kerala Arand Parchuru Lucknow Madakonduru Surat Amravati Sirsa Pedakurupadu Akola Therlapur Budurkatti Kolletigunta Kanpur Varanasi Nagpur Uas-ddu Devlinkopa Ramapuram Arand Narakoduru Yammanur Indur Ctcri Nagpur Auradhi Karnataka Karnataka Karnataka Karnataka Karnataka Amravati Lucknow Indore – Karnataka Karnataka Tamil Nadu Karnataka Karnataka Tamil Nadu Padappai Coimbatore Ahmedabad New Delhi Aurangabad Bangalore Tamilnadu Commilla Commilla Chitagong Chitagong Chitagong Joydepur Jessore Borgra Pongyang Chiangmai Suphanbure East Java West Java

Glycine max Manihot esculenta Ipomoea batatas Ipomoea batatas Gossypium spp Glycine max – Solanum lycopersicum Vigna unguiculata Solanum melongena Gossypium spp Gossypium spp Solanum melongena Gossypium spp Gossypium spp Gossypium spp Gossypium spp Gossypium spp Gossypium spp Helianthus annuus Solanum melongena Gossypium spp Solanum melongena Solanum melongena Gossypium spp Helianthus annuus Solanum melongena Arachis hypogaea Gossypium spp Gossypium spp Gossypium spp Solanum melongena Solanum melongena Gossypium spp Solanum melongena Euphorbia geniculata Solanum melongena Parthenium hysterophorus Vicia faba Solanum melongena – – – Gossypium hirsutum Brassica oleracea var.capitata Gossypium spp Hibiscus esculentus Solanum melongena Solanum melongena Solanum melongena Solanum melongena Glycine max Cucurbita moschata Solanum melongena Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum melongena Benincasa hispida Solanum melongena – Hibiscus esculentus Solanum melongena Solanum melongena Solanum melongena Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum melongena Solanum lycopersicum

HM137332 HM137323 HM137327,30, 36 EU192057,58,59 EU192044 JQ320397 FJ710454 HE653721 JQ995248 JQ995249 HM590148 HM590180 HM590162,63 HM590168 HM590164,74 HM590158,60 HM590172 HM590171 HM590155,59,61 HM590169 HM590152 HM590156 HM590173 HM590151 HM590149,54 HM590166 HM590165 HM590179 HM590157,75 HM590176 HM590167 HM590170 HM590177 HM590150 HM590153 AJ748364 AJ748359,61 AJ748369,70,71 AJ748365 AJ748360 JN855568 JN855575 JN855572 JN703454,55 HQ268810 HQ331246 HQ179952 AM040592 AM040591 AM040596,97 DQ116688 DQ116646 DQ116641 DQ116663 HE653679 HE653680 HE653681 AJ748386 AJ748387 AJ748396 AJ748397 AJ748398 AJ748401 AJ748383 AJ748388 HE653676 HE653677 HE653678 HE653701 HE653687,90,91,92

Hu et al. (2011) Hu et al. (2011) Hu et al. (2011) – – – – Firdaus et al. (2012) – – – – – – – – – – – – – – – – – – – – – – – – – – – Rekha et al. (2005) Rekha et al. (2005) Rekha et al. (2005) Rekha et al. (2005) Rekha et al. (2005) Singh et al. (2012) Singh et al. (2012) Singh et al. (2012) – – – – – – – – – – – Firdaus et al. (2012) Firdaus et al. (2012) Firdaus et al. (2012) – – – – – – – – Firdaus et al. (2012) Firdaus et al. (2012) Firdaus et al. (2012) Firdaus et al. (2012) Firdaus et al. (2012) (continued )

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Table 2. Continued.

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Country Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Indonesia Pakistan Pakistan Pakistan Cambodia Japan

Location

Host plant

Accession no.

West Java West Java West Java West Java West Java Central Java West Sumatera Central Sulawesi West Borneo West Borneo West Borneo – Bogor East Java Central Java Bali West Sumatera Multan Punjab Sindh – Ogasawara

Solanum melongena Solanum lycopersicum Amaranthus sp Capsicum annuum Capsicum annuum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum melongena Solanum lycopersicum Cucumis sativus – Capsicum annuum Capsicum annuum Solanum melongena Capsicum annuum Capsicum annuum Gossypium spp Solanum melongena – – Euphorbia pulcherrima

HE653688,93,95 HE653703,04 HE653689 HE653711 HE653686 HE653700,02 HE653709 HE653705,06,07,08 HE653682,83,98 HE653684,96,97 HE653685 HE653710 GQ900653 GQ900658 GQ900655 GQ900650,52 GQ900661 FJ025788 AJ510061 AJ510078 EU760742 AB308112

Reference Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus Firdaus

et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) et al. (2012) – – – – – – – – Gueguen et al. (2010) Ueda et al. (2009)

– Unknown locations, host plants or unpublished data.

(95% limit) parsimony criteria as implemented in TCS 1.21 (Clement et al., 2000). In an attempt to define the ancestral haplotype in the network, this latter analysis was repeated with the addition of out-group sequences (see above). A phylogenetic tree of haplotypes (including out-groups) was also constructed by NJ clustering by bootstrap of 1000 replications in MEGA.

Results The 37 COI gene sequences generated in this study were merged with 123 COI gene sequences from GenBank to obtain a final alignment of 160 sequences and 738 bp in length. The alignment was characterized by 87 polymorphic sites (11.79 % of the total length), subdivided in 76 singletons and 11 parsimony informative sites, with no indel. Among polymorphic sites, 67 transitions (30 A/G, 37 C/T) and 23 transversions (7 A/C, 8 A/T, 6 T/G, 2 C/G) were identified, with an overall transition/transversion ratio of 2.9. A total of 60 sequence variants (haplotypes) were indentified in the global Asia I dataset (Table 3). Five haplotypes were shared by a minimum of four individuals, while 55 were unique. Genetic distances between haplotypes ranged from 0.001 to 0.021. Based on the network analysis, haplotypes differ by 1–15 mutational steps (Figure 1). The network is composed of a limited number of haplotypes that occupy a central position in the network, display a fairly large geographic distribution and a comparatively higher frequency in the dataset (H1 and H2, connected through H47; H3). Each of these (with the possible exception of H47) is connected to a crown of low frequency derived haplotypes, generally 1–3 mutational steps apart (Figure 1 and Table 3). Considering the geographic distribution of the five haplotypes (H1, H2, H3, H4 and H47) shared by multiple individuals, haplotype H1 is found in China (19 individuals), India (18), Bangladesh (4) and Indonesia (1). Haplotype H2 occurs in China (14 individuals), Indonesia (19), Thailand (2), Pakistan (1), Cambodia (1) and Japan (1). Haplotype H3 is found in China (7) and India (6). Haplotype H47 occurs in Indonesia (6), China (1) and Thailand (1) (Figures 1 and 2). Haplotype H4 is observed only in Indonesia and here shared by 4 individuals (Table 3 and Figure 1).

Attempts at defining the ancestral haplotype in the Asia I network were not conclusive, as out-group sequences were not joined to the Minimum Spanning Network due to a level of divergence above the threshold. The phylogenetic tree (Figure 3), weakly resolved due to the limited variability, nevertheless suggests a basal separation of haplotypes H1, H3 and derivates versus H47, H2 and derivates, with H47 in a basal position in the latter group. The number of different haplotypes of B. tabaci cryptic species Asia I in India is the highest (38), followed by China (13), Indonesia (8), Bangladesh (5) and Pakistan (3). The high haplotype diversity is observed in India (Hd: 0.898), followed by China (0.775) and Indonesia (0.658). Mean genetic distance between B. tabaci Asia I populations ranged from 0 to 0.005. Interspecific K2P distances ranged from 0.162 between B. tabaci Asia I and B. tabaci MEAM1, 0.169 between B. tabaci Asia I and B. tabaci MED and to 0.274 between B. tabaci Asia I and B. afer (Table 4).

Discussion Seventeen B. tabaci cryptic species have been reported to date from Asian countries (Dinsdale et al., 2010; Hu et al., 2011), including the two well-known and globally distributed invasive species MEAM1 and MED (Firdaus et al., 2012; Hu et al., 2011; Rekha et al., 2005; Shoorcheh et al., 2008) and other 15 so-called indigenous B. tabaci cryptic species (Asia II 1, Asia II 2, Asia II 3, Asia II 4, Asia II 5, Asia II 6, Asia II 7, Asia II 8, Asia II 9, Asia II 10, Asia III, China 1, China 2, China2 and Asia I). The large majority of such cryptic species are in fact indigenous species with extremely limited local distributions. On the other hand, cryptic species Asia I displays a fairly large distribution that includes China, India, Bangladesh, Pakistan, Cambodia, Thailand and Indonesia (Tables 1 and 2). This cryptic species also occurs in Turkey (Genbank acession no. AY827616), Singapore (Genbank accession no. AY686095) and Malaysia (Genbank accession no. AY686093), although such accessions have not been included in the dataset due to their limited length. Overall, it appears that B. tabaci cryptic species Asia I is one of the predominant B. tabaci species in Asia.

Haplotype analysis of Bemisia tabaci Asia I

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Table 3. Distribution and frequency of different mitochondrial haplotypes of B. tabaci cryptic species Asia I.

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Location (Country) Haplotype name China

H1

Indonesia Bangladesh India

H1 H1 H1

China

H2

Indonesia

H2

Pakistan Thailand Cambodia Japan China India Indonesia China China India India India India Bangladesh China India India India India India India India India India India India China Bangladesh India India India India India India China India India India Bangladesh India Pakistan Pakistan Bangladesh India India China China China China China Indonesia Thailand India India Indonesia India India

H2 H2 H2 H2 H3 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 H33 H34 H35 H36 H37 H38 H39 H40 H41 H42 H43 H44 H45 H46 H47 H47 H47 H48 H49 H50 H51 H52

Accession no.

Number of sequences

KC123190, KC113556, KC113536, KC113560, KC113541, KC123184 KC113572, KC123188, KC113538, KC113575, KC113555, KC123182 JQ320397, HQ916821, HM137336, HM137327, EU192058, EU192057 HE653698 AJ748386, AJ748387, AJ748396, AJ748397 HE653681, HE653680, HM590175, HM590176, AM040591,HM590167 HM590170, AM040597, HM590177, HE653679, JQ995249, JQ995248 JN855572, JN855568, HM590179, HM590165, HM590166, AJ748361 AJ748364 KC113532, KC113539, KC113531, KC113542, KC113554, KC113563 KC113567, KC113568, KC113573, KC113576, KC113528, KC123183 HQ916819, EU192059 HE65371, 1HE653709, HE653708, HE653701, HE653700, HE653697 HE653692, HE653690, HE653689, HE653688, HE653686, HE653685, HE653684, HE653682, GQ900650, GQ900652, GQ900653, GQ900658, GQ900661 FJ025788 HE653678, HE653677 EU760742 AB308112 KC123191, KC123192, KC123193, KC123194, KC113562, KC123189 HQ916817 JN855575, HM590149, HM590154, HM590151, HM590155, HM590158 HE653710, HE653707, HE653705, HE653696 KC113557 HM137323 HM590173 AJ748359 HM590156 HM590152 AJ748383 KC113564 HM590169 AJ748369 AJ748371 HM590159 HM590171 HQ331246 HM590163 HM590172 JN703455 HM590160 HM590164 HM137332 AJ748388 JN703454 AJ748370 HQ179952 HM590168 AM040592 HM590174 HM137330 AJ748365 HQ268810 HM590162 AJ748398 AJ748360 AJ510061 AJ510078 AJ748401 HM590180 HM590161 HQ916818 KC113535 FJ710454 EU192044 HE653721 HE653703, HE653702, HE653695, HE653693, HE653691, HE653687 HE653676 HM590148 DQ116668 GQ900655 AM040596 HM590150

18 1 4 19 14 19 1 2 1 1 7 6 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 (continued )

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Table 3. Continued.

Location (Country) Haplotype name

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India India India Indonesia Indonesia Indonesia India India

H53 H54 H55 H56 H57 H58 H59 H60

Accession no.

Number of sequences

HM590153 HM590157 DQ116646 HE653683 HE653704 HE653706 DQ116641 DQ116663

1 1 1 1 1 1 1 1

Figure 1. Minimum spanning network depicting evolutionary relationships among B. tabaci cryptic species Asia I haplotypes. Sixty mitochondrial cytochrome oxidase I haplotypes from 160 individuals of Asia I. Area of circles is proportional to haplotype frequency in the dataset. Black circles represent unobserved intermediate haplotypes. Geographical region of each haplotype is color coded.

In our dataset, haplotypes H1-H47-H2 of B. tabaci cryptic species Asia I occupy the central position in the network and are associated to a large number of low frequency haplotypes, 1–11 mutational steps away, that likely from these originated (Figure 1). Notably, this structure is generally associated with a population expansion following separation and/or invasion of a new area (Avise, 2000; Dlugosch & Parker, 2008; Herborg et al., 2007; Hu et al., 2008; Malacrida et al., 2007; Nardi et al., 2005). These results can be compared in their general structure with the work by De Barro & Ahmed (2011), that constructed 34 networks to analyze COI gene sequence variants in each of 26 B. tabaci cryptic species. Based on a larger dataset (160 sequences compared to 36) we could support their observation of a central position of haplotype A1Ind7 (corresponding to our H1) and relatively low Kimura distances among haplotypes (0.001–0.021

in our dataset, compared to 0.002–0.015 in De Barro & Ahmed, 2011). Taking into account the differential distribution and frequency of haplotypes, the most notable sign of differentiation is observed between India and Indonesia, with China lying someway in between. Indonesia is characterized by haplotypes H2, H47 and H4, the latter being exclusive of this area, while haplotype H1 is here very rare. On the other hand, India and Bangladesh are characterized by haplotypes H1 and H3, with H47, H2 and H4 totally missing. Noteworthy both haplotype groups are observed in China at similar – and fairly high – frequencies. Although excluded from the analysis due to limited length, the three additional accessions available are compatible with this pattern, as the one from Turkey is probably haplotype H1 and two from Singapore and Malaysia appear to be derived from H2.

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Figure 2. Distributions of four dominant haplotypes of B. tabaci cryptic species Asia I in Asia. Area of circles are proportional to individual frequency in the populations (Details of individual per population correspond to Table 4). The colored pie charts represent the frequency of dominant haplotype in population. BGD in the map stands for Bangladesh, KHM stands for Cambodia.

Our attempts at the identification of the ancestral haplotype in the Asia I network were not conclusive, nevertheless the basal dichotomy in the phylogenetic tree would indicate H47 as the ancestral haplotype, a possibility that is also supported by its central position between H1 and H2. This suggests a pristine separation of the Asia I genetic stock in the two major groups characterized by haplotype groups derived from H1 and H2. Nevertheless, the presence of both haplotype groups in China is not compatible with a simple event of differentiation in allopatry, suggesting a more complex evolutionary history. Two alternative scenarios can be envisioned for the origin and spread of cryptic species Asia I in Asia. On one hand, the occurrence of all major haplotypes in China, alongside the relatively high genetic variability observed here, would suggest China as a possible source of Asia I. India and Indonesia may have been colonized in two distinct – and independent – waves, with drift accounting for near fixation of haplotypes H2-H47 in Indonesia and H1 in India and subsequent local evolution for the differentiation of these into larger haplotype groups. On the other hand, the possibility of a primary and old separation between Indonesia and India is suggested by the observation that these latter areas and neighboring regions are characterized by the two haplotype groups H47-H2 and H1-H3, respectively, that based on the rooting analysis, are suggested as the initial offshoot of ancestral haplotype H47. The occurrence of both groups in China – at a similar and relatively high frequency – may be accounted

for by a secondary contact of the two groups in a northward expansion. Although no definitive evidence could be found to support either view, some support for the latter can be found in the observation that the unique hapotypes centered on H1 and H2 that are present in China tend to be very close to the parent haplotypes (eight of nine are a single step apart, one is two step apart), while unique hapotypes similarly centered on H1 and H2 in India and Indonesia are much more differentiated from the parent haplotypes, up to 11 steps and with the possibility of additional substructures. As the crown of ancillary haplotypes likely developed in loco, this points to an older presence in India and Indonesia than in China. At the same time, the low frequency of putatively ancestral haplotype H27 in China suggests a secondary colonization of this area. In order to further explore this possibility, the distribution of the two haplotype groups in China was carefully reconsidered in the light of a possible unequal distribution arising from a secondary contact, both at a country scale (for which sampling is nevertheless limited) and at a regional scale inside Yunnan. Although the two regions facing S/E towards India (Dehong and Xishuangbanna) display a much higher frequency of haplogroup H1 than H2 (Table 1) compared to others (Yuxi) where haplogroup H2 predominates, a much longer transect and more uniform sampling would be necessary to actually test this hypothesis.

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H59_India 72 52 H60_India 55 H39_Pakistan

Figure 3. Phylogenetic reconstruction based on haplotypes of Asia I, MEAM1, MED and B. afer.

H38_Pakistan H19_India H7_India H35_India H31_India H30_India H20_India H22_India H23_India H40_Bangladesh H42_India H34_India H17_India 66

H18_India

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H29_India H8_India H55_India H13_India H14_India H25_Bangladesh H33_India H15_India H26_India H27_India H32_China H1 H51_India H6_China H5_China H24_China H41_India H16_India H21_India H36_Bangladesh H37_India H12_China H3 H53_India H9_India H10_India 59

H54_India H48_India H52_India H11_Bangladesh H47 H28_India

100

H49_India H45_China H56_Indonesia H44_China H50_Indonesia H2

65

H4_Indonesia H58_Indonesia H46_China H43_China H57_Indonesia

B. tabaci MEAM1 100

B. tabaci MED B. afer

0.02

Haplotype analysis of Bemisia tabaci Asia I

DOI: 10.3109/19401736.2013.830289

9

Table 4. Mean K2P genetic distances between populations of B. tabaci cryptic species Asia I and outgroup species.

1 China 2 India 3 Bangladesh 4 Cambodia 5 Pakistan 6 Thailand 7 Indonesia 8 Japan 9 B. tabaci MEAM1 10 B. tabaci MED 11 B. afer

1

2

0 0.003 0.002 0.002 0.003 0.002 0.002 0.002 0.161 0.170 0.273

0 0.003 0.005 0.004 0.004 0.005 0.005 0.162 0.172 0.274

3

0 0.004 0.003 0.003 0.004 0.004 0.161 0.171 0.273

4

0 0.003 0.001 0.001 0.000 0.164 0.169 0.274

5

0 0.003 0.003 0.003 0.162 0.170 0.274

6

0 0.001 0.001 0.163 0.169 0.274

7

0 0.001 0.164 0.169 0.275

8

0 0.164 0.169 0.274

9

0 0.053 0.259

10

n

Pi

Hd

0 0.253

49 61 8 1 3 3 34 1 1 1 1

0.00219 0.00367 0.00169 N/A 0.00361 0.00090 0.00128 N/A N/A N/A N/A

0.775 0.898 0.786 N/A 1.000 0.067 0.658 N/A N/A N/A N/A

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The number of individuals analyzed (n), nucleotide diversity (Pi) and haplotype diversity (Hd) inside populations are indicated.

Yunnan is considered as a global biodiversity hotspot (Myers et al., 2000) due to its diversified climate and vegetation, and lies at the crossroads between the Indonesian region, the Indian subcontinent and central China. Different pest species have been described as expanding north-ward to China or in China from S/E costal regions to China mainland, including B. dorsalis (Wan et al., 2011) and the invasive B. tabaci cryptic species MED (Hu et al., 2011). At the same time, India is commonly considered as the native home of many important agricultural pests, such as the melon fly, Bactrocera cucurbitae (Hu et al., 2008; Prabhakar et al., 2012; Virgilio et al., 2010) and the sweet potato weevil, Cylas formicarius (Kawamura et al., 2009). In the end, based on its large geographic range, capability to successfully colonize new areas, genetic variability, large host plant range and the possibility to transmit pathogenic begomoviruses, B. tabaci cryptic species Asia I may be regarded as a major agricultural pest throughout Asia. As such, any effort to develop more appropriate and effective control programs to prevent the dispersal and the possible establishments of stable population of cryptic species Asia I in new areas may be warranted.

Acknowledgements The authors are grateful to the colleagues Yuan-Yuan Liu, Jin-Gang Cha, Chong-Hui Qi, Kun Yang, Hong-Xiang Li, Jian-Chun Zhao, Guang-Xi Li, Zhan Li for sample collections for the study. The authors wish to thank two anonymous reviewers for their insightful comments.

Declaration of interest Financial support for this study was provided by the Key Program of Joint Funds of National Natural Science Foundation of China and Yunnan Province (Grant U1136606), the Fund for Applied Basic Research of Yunnan Province (Grant 2011FB122) and the Fund for Reserve Talents of Young and Middle-aged Academic and Technical Leaders of Yunnan Province (Grant 2012HB038). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

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Global haplotype analysis of the whitefly Bemisia tabaci cryptic species Asia I in Asia.

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidiae), is a cryptic species complex comprising a minimum of 24 cryptic species. Some members of this ...
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