Phylogenetic Analysis and Rapid Identification of the Whitefly, Bemisia afer, in China

The phylogenetic relationship between the whitefly Bemisia afer (Priesner & Hosny) (Hemiptera: Aleyrodidae) from China and other populations among the world were analyzed based on the mitochondrial cytochrome oxidase I (mtCOI) gene. Phylogenetic analysis of mtCOI sequences and those of reference B. afer sequences showed that the populations of the species could be separated into 5 clades (I–V). There were at least two clades of the species from China (IV and V). These data suggested that B. afer might be a species complex. The Chinese B. afer populations were most divergent with B. afer from the United Kingdom and African countries. The distance between the Chinese B. afer (IV and V) and clades I, II, and III is more than 32%, while the distance among clades I, II, III is lower than 7.7%. A new set of primers specific to B. afer was designed to amplify a region of approximately 400 bp to discriminate B. afer from other Bemisia species in China based on mtCOI sequences.


Introduction
During recent years, the outbreak of Bemisia tabaci (Priesner & Hosny) (Hemiptera: Aleyrodidae) and the serious damage caused by this whitefly pest in many countries have required researchers to study the biological and ecological characteristics of and effective control strategies against it (Brown et al. 2000;Brown 2007; De Barro et al. 2000Barro et al. , 2003. The identification of species in the genus Bemisia is the basis of this research, but the taxonomy of whiteflies has long been problematic because of similarities in the morphology of pupae and adults. Pupae of Bemisia species exhibit phenotypic variation in response to differences in leaf surface topology and to environmental and physical factors (Maruthi et al. 2007).
In China, B. tabaci became the major pest of the fiber crops, ornamental plants and vegetables (Chu et al. 2006(Chu et al. , 2007(Chu et al. , 2008. During field research, a Bemisia species, which was difficult to distinguish from B. tabaci, was discovered on Broussonetia papyrifera (Linn.) Vent.
(Urticales: Moraceae). Based on the morphological characteristics of pupae and adults, the species was identified as B. afer. As B. tabaci has several close relatives and numerous biotypes, B. afer also is likely to have many forms and cryptic species. Earlier studies indicated that B. afer exhibits much greater morphological variation than does B. tabaci and its variants (Anderson et al. 2001;Maruthi et al. 2007). The Chinese B. afer has slightly different morphology compared to B. afer from other geographical locations, and it is expected that these differences would be reflected at the molecular level.
The mitochondrial cytochrome oxidase I (mtCOI) gene has been used extensively as a molecular marker to identify B. tabaci variants that exhibit rich biological differences (Frohlich et al. 1999;Hsieh et al. 2007) but lack distinguishing morphological features. Previous studies have shown that mtCOI sequences also are informative for identifying B. afer variants, which lack distinguishing morphological features (Maruthi et al. 2007). In this study, the mtCOI gene of B. afer was sequenced using the primer set (C1-J-2195 and L2-N-3014) that has been used extensively on B. tabaci, and the fragments were also sequenced. The phylogenetic relationships among the world populations were analyzed. The infection status of an endosymbiont Wolbachia of Chinese B. afer was studied because it often causes reproductive incompatibilities between infected and uninfected hosts, which can affect the divergence of mtDNA and can facilitate or even cause host speciation (Werren 1997;Ballard et al. 2004;Shoemaker et al. 2004). Finally, the specific primers to Chinese B. afer were designed based on the sequences of the mtCOI gene of Chinese B. afer and B. tabaci biotypes B and Q.
The objectives of the paper are: 1) to further analyze the phylogenetic relationships, based on the mitochondria COI gene, between Chinese populations of B. afer on B. papyrifera with other populations of B. afer from the United Kingdom and African countries and to discuss the relationship between the divergence of B. afer and the endosymbiont, Wolbachia; 2) to develop a rapid molecular marker based on the mtCOI gene to distinguish B. afer from B. tabaci biotypes B and Q, which are the predominate biotypes in China, especially the biotypes in the Shandong province. The aim is to contribute to the understanding of the systematic status of B. afer populations in China and the genetic differentiation of B. afer worldwide.

Collection of the samples and species identification
During 2006 and 2007, pupae and adults of Bemisia species on B. papyrifera were collected alive and placed singly into tubes containing 95% ethanol. The species were identified based on the pupae and adults.

DNA extraction and PCR
Genomic DNA was extracted from individual adults according to the method described previously by Frohlich et al. (1999). Polymerase chain reaction (PCR) was employed to amplify fragments of the B. afer mitochondrial COI gene (800-820 bp), using parameters and PCR primers (C1-J-2195 and L2-N-3014) as described by Frohlich et al. (1999).
PCR assays were conducted using 2 μl of each template DNA in a total reaction volume of 25 μl. PCR conditions follow Frohlich et al. (1999), with 1 unit of Taq DNA polymerase. PCR products were separated on 1.0% agarose gel. The bands were visualized by ethidium bromide staining and viewed with a UV light source.
Cytochrome oxidase I sequencing and phylogenetic analysis PCR products were purified using an EZ Spin Column DNA Gel Extraction Kit (Sangon Technology Company, www.sangon.com/ index.htm) according to the manufacturer's instructions. The DNA sequence for each PCR product was determined from the 5'end at the Sangon Technology Company. The mtCOI sequences determined were deposited in GenBank.
Phylogenetic analysis included all available mtCOI sequences from GenBank and sequences from this study, with the sequences of Bemisia tabaci (mainly including the indigenous biotypes from China), B. tuberculata, B. berbericola, Trialeurodes vaporariorum, and Trialeurodes abutilonea as the outgroup (Table 1). The mtCOI sequences were aligned using the CLUSTAL W algorithm (Thompson et al. 1994). The aligned mtCOI sequences of ~600 bp are presented. Distances based on the mtCOI sequences of ~600 bp were calculated based on the Kimura 2-parameter model using MEGA 4.1 (Tamura et al. 2007). The ME (Molecular Evolution) and MP (Maximum Parsimony) algorithms available in MEGA 4.1 were used to infer phylogenetic relationships from the sequences. One thousand Bootstrap replicates were performed for each analysis.
On the basis of the results of phylogenetic analysis, the B. afer specimens were separated into five subclades. The sequences in the subclades were selected to further calculate distances within and between group average calculations using MEGA 4.1.

Wolbachia detection of Chinese B. afer
All B. afer were also screened for Wolbachia infection by PCR, employing the primers wsp81F and wsp691R (Zhou et al. 1998), which amplify part of the Wolbachia surface protein gene (wsp). The study included 15 Chinese B. afer individuals, and the PCR was repeated three times.

Development of the specific diagnostic test
Experiments showed that the primers C1-J-2195 and L2-N-3014 did not always amplify products of the expected size, although the DNA was useful. A set of primers specific to the B. afer mtCOI gene was designed, which included the newly designed forward primer Bafer-J2 (5'-GTTAGTTTTGGGGATTAGTC-3') by aligning in CLUSTAL W (Thompson et al. 1994) and the reverse primer L2-N-3014 (5'-TCCAATGCACTAATCTGCCATATTA-3'). The other whitefly species (Table 2) were used in PCR reactions to test the specificity of primers to B. afer. The PCR reaction mix followed the method previously described by Frohlich et al. (1999). Reactions used the following PCR program: 94!"# for 2 min; followed by 30 cycles of 94!" #" for 1 min, 54!" # for 1 min, and 72!" # for 1 min; and ending  Maruthi et al.(2007) -, indicates unknown with 72!"# for 5 min. PCR products were separated on 1.0% agarose gel. The bands were visualized by ethidium bromide staining and viewed with a UV light source.

Morphology of B. afer
Adults of New World specimens of B. afer have the upper and lower eyes completely separate in both sexes, as contrasted with B. tabaci adults,which have one ommatidium connection. Some B. afer from the Macaronesian Islands have the male eyes connected by one ommatidium but separated in the female, while others from different hosts have both eyes separate as in the North American forms (RJ Gill, unpublished data). In the specimens for this study, the female had a one ommatidium connection, while the male had both eyes connected positively with one ommatidium, but also another ommatidium almost but not touching. In the adults, there were clear morphological differences. The phylogenetic tree generated with the Minimum Evolution (ME) method is shown in Figure 1. The tree generated with MP (Maximum Parsimony) method (not shown) is similar to Figure 1. The tree that was generated by heuristic research had 65% confidence level. Based on the trees, B. afer could be separated into 5 clades. The populations from Xinjiang, China (AJ784260Xinjiang) and Shandong, China (Bafer1-Bafer8) were grouped in clade IV and V, respectively. The Chinese B. afer populations (clade IV and V) were most divergent with clades I, II, and III.

Genetic differentiation of the B.afer in China and Wolbachia infection
Average distances within and between clades based on mtCOI of whiteflies are summarized in Table 3. The Chinese B. afer populations were most divergent with B.afer from the United Kingdom and African countries. The distance between the Chinese B. afer (clade IV and V) and clades I, II, and III was more than 32%, while the distance among clades I, II, and III were lower than 7.7%. No Wolbachia could be detected in Chinese  Chu et al.(2008) b Described in Hsieh et al.(2007) Figure 1. ME (Minimum Evolution) tree based on 600-bp fragment of the mtCOI sequences. Numbers at nodes indicate bootstrap scores using 1000 replicates. Abbreviations are as described in Table 1. indicates sequences obtained in this study. High quality figures are available online.
populations of B.afer (clade V). Figure 2 shows the PCR products generated from the DNA of several whitefly species using Bafer-J2 and L2-N-3014 primers. A band of approx 400 bp was obtained from DNA of B. afer from Shandong, China. No specific PCR products were obtained from DNA of B. tabaci biotype B, Q1, Q2, or Nauru.

Discussion
The phylogenetic analysis based on the mtCOI sequences suggested that the B. afer is a species complex that includes many genetically divergent clades. The result based on the molecular marker is consistent with the analysis based on the morphological characteristics. This study revealed the presence of at least 5 clades in B. afer worldwide. The