- Email: [email protected]
Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh
Accepted Manuscript Title: Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh Authors: M.S. Fatema Khatun, S.M. Hemayet Jahan, Sukchan Lee, Kyeong-Yeoll Lee PII: DOI: Reference:
S0001-706X(18)30564-3 https://doi.org/10.1016/j.actatropica.2018.07.021 ACTROP 4726
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
5-5-2018 21-7-2018 24-7-2018
Please cite this article as: Fatema Khatun MS, Hemayet Jahan SM, Lee S, Lee K-Yeoll, Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.07.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh
Running header: Bemisia tabaci genetic diversity in Bangladesh
Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook
SC R
a
IP T
MS. Fatema Khatuna,b, S.M. Hemayet Jahanc, Sukchan Leed, Kyeong-Yeoll Leea,e,f*
National University, Daegu, Republic of Korea b
Department of Entomology, Bangabandhu Sheikh Mujibur Rahman Agricultural University,
U
Dhaka, Bangladesh c
N
Department of Entomology, Patuakhali Science and Technology University, Dumki,
A
Patuakhali, Bangladesh
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Republic of Korea
e
Institute of Plant Medicine, Kyungpook National University, Daegu, Republic of Korea
f
Institute of Agricultural Science and Technology, Kyungpook National University, Daegu,
CC E
PT
Republic of Korea
ED
M
d
*
Corresponding author:
A
Dr. Kyeong-Yeoll Lee, Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea. Tel.: 82-53-950-5759; e-mail: [email protected]
M
A
N
U
SC R
IP T
Graphical Abstract
ED
Highlights
Genetic diversity of Bemisia tabaci was determined in Bangladesh.
We identified four indigenous cryptic species but not MEAM1 and MED invasive
PT
CC E
cryptic species.
Asia I was abundant, both Asia II 1 and Asia II 5 were moderate, and Asia II 10 was found only in the central region.
A
Our study provides important information on the genetic diversity and geographic distribution of B. tabaci in Bangladesh.
Abstract Bemisia tabaci (Gennadius) is a species complex consisting of at least 40 cryptic species. Although the genetic diversity of B. tabaci has been studied in various regions, little is known
about distribution in Bangladesh, which is covered by the Bengal delta, the largest delta on Earth. We conducted an extensive survey throughout the country and determined the nucleotide sequence of mitochondrial cytochrome c oxidase subunit 1 (COI) from 110 individuals. We then examined phylogenetic relationships. The results identified four cryptic species that expressed distinct interspecific variation but low intraspecific variation. Asia I was the most
IP T
abundant, both Asia II 1 and Asia II 5 were moderately abundant, and Asia II 10 was found only in the central region. COI sequences of each cryptic species were distinctive and
SC R
differentiated into many haplotypes. Our study provides important information to better understand the genetic diversity and geographic distribution of cryptic species in Bangladesh
U
and nearby countries.
A
N
Keywords: Cryptic species; Genetic diversity; Geographic distribution; Haplotype; Invasion
M
1. Introduction
The sweet potato whitefly, Bemisia tabaci (Gennadius), is one of the most serious pests
ED
of various economically important horticultural and ornamental crop plants (Perring et al., 1993;
PT
Bellows et al., 1994; Dinsdale et al., 2010). In addition, this species acts as a vector of more than 110 viruses, most of which are begomoviruses. For example, the Tomato yellow leaf curl
CC E
virus (TYLCV) is highly viruliferous and is a major threat to crop production worldwide (Jones, 2003; Fauquet et al., 2008; Hogenhout et al., 2008). B. tabaci is distributed worldwide and is highly genetically diverse, consisting of a
A
complex of many cryptic species that are morphologically indistinguishable but genetically different (Bedford et al., 1994; Horowitz et al., 2005; Dinsdale et al., 2010; De Barro et al., 2011). The genetic diversity of B. tabaci has been examined using molecular database analysis by comparing nucleotide sequences of mitochondrial cytochrome oxidase subunit I (COI) in several studies. For example, Dinsdale et al. (2010) grouped B. tabaci into 24 cryptic species,
using 3.5% genetic distance as a species boundary threshold. Similarly, Lee et al. (2013) used 4.0% genetic distance as a species boundary based on the massive database of COI sequences, and grouped B. tabaci into 31 cryptic species. Recent studies have identified several new cryptic species from various countries and have recorded at least 40 cryptic species within the species complex of B. tabaci (Boykin and De Barro, 2014; Guo et al., 2015; Qin et al., 2016;
IP T
Zhu et al., 2016; Hu et al., 2017). Cryptic species within the species complex have different genetic characteristics, such
SC R
as environmental adaptation, host range, and pesticide resistance (Brown, 2010; De Barro et
al., 2011). Among them, both Middle East-Asia Minor 1 (MEAM1, formerly B biotype) and Mediterranean (MED, formerly Q biotype) originated from the deserts of Northeastern Africa,
U
the Arabian Peninsula, and Middle East Asia. They later spread throughout North Africa and
N
Mediterranean regions and then extensively invaded many other countries on different
A
continents (Frohlich et al., 1999; De Barro et al., 2000; Moya et al., 2001; Boykin et al.,
M
2007; Elbaz et al., 2010).
Numerous studies have shown that there are many genetically distinct cryptic species
ED
of B. tabaci in Asia. To date, at least 25 cryptic species of B. tabaci have been identified in
PT
various Asian countries, including two invasive species (MEAM1 and MED), 23 indigenous species (such as Asia I, Asia I India, 12 putative cryptic species of Asia II, Asia III, Asia IV,
CC E
and Asia V), and 6 putative cryptic species from China (Dinsdale et al., 2010; Ahmad et al., 2011; Hameed et al., 2012; Firdaus et al., 2013; Shah et al., 2013; Prasanna et al., 2015; Ellango et al., 2015; Hu et al., 2015, 2017; Gotz and Winter, 2016; Kumar et al., 2016; Jiu et al., 2017).
A
In China, 20 cryptic species (Asia I, Asia II 1, Asia II 2, Asia II 3, Asia II 4, Asia II 6, Asia II 7, Asia II 9, Asia II 10, Asia III, Asia IV, Asia V, China 1, China 2, China 3, China 4, China 5, China 6, MEAM1, and MED) have been reported, including two invasive species (Dinsdale et al., 2010; Hu et al., 2011, 2014, 2015; Firdaus et al., 2013; Jiu et al., 2017). In India, 9 cryptic species (Asia I, Asia I India, Asia II 1, Asia II 5, Asia II 7, Asia II 8, Asia II 11, China 3, and
MEAM1) have been recorded (Ellango et al., 2015; Kumar et al., 2016; Prasanna et al., 2015). Gotz and Winter, (2016) reported three cryptic species (Asia I, Asia II 6, and Asia II 10) in Thailand, and three cryptic species (Asia I, Asia II 1, Asia II 6) and two invasive species (MEAM1 and MED) in Vietnam. In Pakistan, six cryptic species (Asia I, Asia II 1, Asia II 5 Asia II 7, Asia II 8, and MEAM1) have been reported, with Asia II 1 in a major cotton-growing
IP T
region (Ahmad et al., 2011; Hameed et al., 2012; Shah et al., 2013; Islam et al., 2018). In Indonesia, both MEAM1 and MED have been invaded, but Asia I and other unidentified
SC R
indigenous cryptic species have also been reported (De Barro et al., 2008; Srinivasan et al., 2013; Rahayuwati et al., 2016). The genetic diversity and geographic distribution of each
genetic group are diverse in different countries, which may be a result of different
U
environmental conditions and plant biodiversity (Brown, 2010; De Barro et al., 2011). Like
N
other countries, Bangladesh has suffered serious damage to various horticultural crop plants
A
from B. tabaci, directly or indirectly by virus transmission. However, extensive studies on the
M
country-wide genetic diversity of B. tabaci have not been conducted in Bangladesh, which is covered by the largest delta on Earth, the Bengal delta (Hu et al., 2015; Jahan et al., 2015).
ED
The purpose of this large-scale study was to identify genetic groups of B. tabaci in
PT
Bangladesh and to understand the genetic diversity, geographical distribution, and phylogenetic
CC E
relationships with other groups that are distributed in neighboring Asian countries.
2. Materials and Methods
A
2.1 Whitefly collection Bemisia tabaci adults were collected from various crop plants (bean, brinjal, cotton,
dahlia, potato, sweetpotato, and tomato) across thirty-five different regions of Bangladesh during the peak crop growing season, from 2015 to 2017. Adult whiteflies were collected with an aspirator, then preserved in 70% alcohol in separate vials based on the crop plant and
location from which they were collected. The vials were then stored at -20 °C until further analysis (Table 1). Whiteflies from at least 20 to 100 individuals were collected from each site and at least three sites were surveyed per crop field.
2.2 DNA extraction
IP T
Genomic DNA was extracted from a single adult whitefly using the pure link genomic DNA mini kit (Invitrogen, Carlsbad, CA). The sample was placed in a 1.5 mL centrifuge tube
SC R
containing 180 µL of digestion buffer and 20 µL of proteinase K (50 µg/mL) and incubated at
55 °C for 4 h. DNA samples were extracted and purified using genomic spin columns, as described in the kit. DNA concentration was determined using a NanoPhotometer™ (Implen
N
U
GmbH, Schatzbogen, Germany).
A
2.3 PCR amplification
M
Mitochondrial cytochrome oxidase subunit I (COI) DNA was amplified using the primer pair MT10/C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and MT12/L2-N-3014
ED
(5′-TCCAATGCACTAATCTGCCATATTA) (Simon et al., 1994). Polymerase chain reaction
PT
(PCR) was performed in a total reaction volume of 20 µL, containing 13 µL Smart-Taq PreMix (Solgent Co., Daejeon, Korea), 1 µL of each primer (10 pmol/µL), and 5 µl of template
CC E
DNA solution (40 ng). Reaction mixtures were amplified under the following conditions: initial denaturation at 94 °C for 2 min; followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 5 min in a Veriti 96-Well Thermal Cycler
A
(Applied Biosystems, Foster City, CA). The PCR products were separated using 1% agarose gel electrophoresis, stained with Ethidium Bromide solution, and visualized under Ultraviolet (UV) light. Amplified PCR products were excised from the gel and purified using the Wizard® PCR Preps DNA Purification System (Wizard® SV Gel, Promega Co., Madison, WI) and sequenced either directly or by cloning into the T-BluntTM easy plasmid vector (Promega Co.,
Madison, WI).
2.4 DNA sequence analysis The PCR products, cloned using the T-BluntTM vector, were determined using the BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and
IP T
analyzed with a 3100 Capillary DNA Sequencer (Applied Biosystems, Foster City, CA) at the Solgent Sequencing Facility (Solgent Co., Daejeon, Korea). The GenBank database in the
SC R
National Center for Biotechnology Information (NCBI) was searched using the BLAST algorithm (Schaffer et al., 2001) and nucleotide sequences were aligned using CLUSTAL W
(Thompson et al., 1994). Identified COI sequences from the Bangladesh samples were
N
U
submitted to the GenBank database.
A
2.5 Phylogenetic analysis
M
COI sequences were edited manually to produce consensus sequences of 817 bp for each individual whitefly using the Clustal Omega program. The aligned sequences were
ED
analyzed using mega 6.0. The phylogenetic tree constructed using the maximum likelihood
PT
method implemented in MEGA 6.0 software (Tamura et al., 2013). We used 1000 bootstrap replicates to test the robustness of the phylogeny (Felsenstein, 1985). The COI sequence of
CC E
Bemisia afer (accession number AJ784260) was used as an outgroup (Hu et al., 2015). In this study, we followed the rule of pairwise sequence divergence >3.5 to characterize different
A
cryptic species of B. tabaci (Dinsdale et al., 2010).
2.6 Genetic analysis A total of 110 COI sequences from Bangladesh were aligned using Clustal X 1.83 (Thompson et al., 1997). The sequences were analyzed using a minimum spanning network relationship among B. tabaci cryptic species to generate a haplotype network using statistical
(95% limit) parsimony in TCS software v.1.21 (Clement et al., 2000). Nucleotide polymorphisms were estimated by deliberating several parameters, including the number of polymorphic sites and haplotypes, haplotype diversity (Nei and Tajima, 1983), nucleotide diversity, singleton variable sites, and parsimony-informative sites (Jukes and Cantor, 1996). All parameters for sequence polymorphism, divergence, gene flow, neutrality tests (Fu’s Fs and
IP T
Tajima’s D), and genetic differentiation were executed using DnaSP software v.5.10 (Librado and Rozas, 2009; Tajima, 1989). The pairwise genetic distance (Fst) value was generated from
SC R
1,023 permutations and by using the K2P model (Kimura, 1980). Analysis of molecular
variance (AMOVA) with 1,023 permutations was substantiated and the populations were grouped in accordance with their locations (Excoffier et al., 1992). AMOVA analysis and F-
U
statistics of genetic variation for B. tabaci populations in Bangladesh were obtained using a
N
pairwise distance method in Arlequin software version 3.1 (Excoffier et al., 2005). Isolation by
A
distance web service (IBD version 3.23) was used in the Mantel test to determine the correlation
M
between genetic distance (Fst) and geographic distances (Jensen et al., 2005). Geographic distances among the measured populations were based on a central location of collection sites
PT
3. Results
ED
in Bangladesh.
CC E
3.1 Identification of B. tabaci cryptic species in Bangladesh In total, 110 individuals collected from 35 different locations in Bangladesh were used
to amplify 817 bp of the COI gene sequence. The identified sequences were submitted to the
A
Genbank database in NCBI, with accession numbers from MF497029 to MF497079 (Table 1; Fig. 1). The COI sequence variation among all the samples ranged from 0.12% to 18.10% (Table 2). According to the maximum likelihood phylogram of previously known sequences in the Genbank database, all samples were placed into four genetic groups, namely, Asia I, Asia
II 1, Asia II 5, and Asia II 10 (Fig. 2). Among the 110 samples, Asia I (60.9%) was the most common sample, while Asia II 1 and Asia II 5 were 18.2% and 20.0%, respectively. Asia II 10 was from only one sample (0.9%) (Table 3). Geographic analysis showed that both Asia I and Asia II 1 were widely distributed throughout the country, but that Asia II 5 was distributed in the central and southern regions of Bangladesh, while Asia II 10 was distributed in only one
IP T
location, the Bangladesh Agricultural Research Institute (BARI) in Gazipur, which is in central Bangladesh (Fig. 1). We did not detect any MEAM1 and MED cryptic species that are known
SC R
to be invasive species.
The variation in COI nucleotide sequences differed between intraspecific and interspecific levels among cryptic species. Intraspecific variation was almost two times higher
U
in Asia II 1 (0.12-1.11%) and Asia II 5 (0.12-0.99%) than in Asia I (0.12-0.49%) (Table 2).
N
Interspecific variation ranged between 10.40% and 18.10% among the 4 cryptic species (Table
A
2). The highest variation (18.10%) was observed between Asia I (MF497030) collected from
M
Brinjal in the Bandarban district and Asia II 5 (MF497078) from Brinjal in the Rangamati district, which are located in the southern region of Bangladesh (Table S1).
ED
Whitefly samples were collected from 8 different crop species—bean (Phaseolus
PT
vulgaris), brinjal (Solanum melongena), Bt brinjal, cotton (Gossypium hirsutum), dahlia (Dahlia pinnata), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), and tomato
CC E
(Solanum lycopersicum) (Table 1). Both Asia I and Asia II 1 were found in all species, but Asia II 5 was observed only on the bean, brinjal, sweet potato, and tomato crops. Asia II 10 was identified on only one sample of cotton from BARI. Two cryptic species (Asia I and Asia II 1)
A
were found together in the same field of brinjal, cotton, potato, or tomato in the Bandarban and Gazipur regions (Table 1). The variation rate of COI sequences depended on region. For instance, in the Bandarban district, which is located in the southeastern region, COI variation was less than 0.37%. However, in the Gazipur district located in the middle of the country, variation was less
than 13.83%.
3.2 Genetic diversity of B. tabaci cryptic species in Bangladesh Variation in COI gene structure was analyzed for three cryptic species (Asia I, Asia II 1, Asia II 5), excluding Asia II 10 because it had only one sample (Table 3). The number of
IP T
polymorphic sites was higher in Asia I (34) than Asia II 1 (18) and Asia II 5 (15). Asia I had a higher number (33) of singleton variable sites than parsimony informative sites (1), but both
SC R
Asia II 1 and Asia II 5 had approximately equal numbers of these two sites (Table 3). The number of haplotypes against total sequences was 28/67 (Asia I), 13/22 (Asia II 5) and 12/20 (Asia II 1). Haplotype diversity was higher in Asia II 1 (0.916) than Asia II 5 (0.896) and Asia
U
I (0.664). Asia II 5 had the highest nucleotide diversity, followed by Asia II 1 and Asia I (Table
N
3). The population genetic study was conducted within the district populations. Tajima’s D
A
statistical analysis showed that in four districts, values were negative (Dinajpur, Nilphamari,
M
Patuakhali, and Coxsbazar), and Fu’s Fs showed positive values for all districts with significant differences (Table 5).
ED
Evolutionary relationships among the haplotypes of each cryptic species were depicted
PT
using both the minimum spanning network analysis and the phylogram (Fig. 4 and Fig. S1). Three cryptic species (Asia I, Asia II 1, and Asia II 5) were diversified into many haplotypes
CC E
and they were highly distant from each cryptic species by many mutational steps (Fig. 2 and Table S3). The pattern from the minimum spanning network of haplotypes was similar to the phylogenetic tree of B. tabaci sequences. Among 28 haplotypes of Asia I, the H1 haplotype
A
occupied a central position of the network and was associated with 27 low-frequency haplotypes. H24 represented two individuals. Among 12 haplotypes of Asia II 1, the H11 haplotype occupied the central position and was diversified by 11 haplotypes. Particularly, H5 was expected to differ by 7 mutational steps. However, among 13 haplotypes of Asia II 5, most haplotypes were extended into a linear form. Hierarchical AMOVA analysis revealed that the
total genetic variation (Fst) was 42.9% within the population based on the geographic distance (Table 4). The genetic variation was low among groups (10.44). The portions of genetic variation were partitioned among groups (27.79%), among populations within groups (15.11%), and within populations (47.10%). The Mantel test showed that there was no correlation
IP T
between genetic distance and geographic distance (Fig. 3 and Table S2).
4. Discussion
SC R
An extensive survey of B. tabaci in Bangladesh showed that the pairwise COI
nucleotide sequence variation reached 18.1% and the species complex was diversified into four cryptic species, namely, Asia I, Asia II 1, Asia II 5, and Asia II 10. Each cryptic species was
U
genetically distinct, possessing more than 10% sequence divergence. Asia I was the most
N
abundant. Both Asia II 1 and Asia II 5 were found in moderate levels, but Asia II 10 was found
A
in only one location. However, MEAM1 and MED cryptic species were not detected in this
M
study. The absence of these two invasive species in Bangladesh, even though they have invaded neighboring countries, is interesting. For example, MEAM1 has been found in India and
ED
Pakistan, while MED was not detected in these two countries (Ellango et al., 2015; Islam et al.,
PT
2018). It is important to monitor the abundance of these two invasive species in Asian countries for the appropriate management of whiteflies.
CC E
Asia I is one of the predominant cryptic species in Asia. Hu et al., (2015) extensively studied its presence in 7 countries, including Cambodia, China, India, Indonesia, Pakistan, Thailand, and Bangladesh. Its distribution was also reported in Turkey, Singapore, and
A
Malaysia in the GenBank database. Gotz and Winter, (2016) reported that Asia I also exists in Vietnam. Our study showed that Asia I was the most abundant cryptic species and was distributed throughout Bangladesh. Hu et al. (2011, 2015) had previously reported the presence of 5 haplotypes of Asia I from Bangladesh. Our haplotype analysis showed that Asia I was highly differentiated into 28 haplotypes. One major haplotype (H1) was associated with 27
low-frequency haplotypes. This result is consistent with Hu et al. (2015) which showed that the major haplotypes networked with many minor haplotypes. Asia II is a large but genetically diverse group, including at least 12 cryptic species (Hu et al., 2011, 2017). Among them, only three species (Asia II 1, Asia II 5, and Asia II 10) were identified in Bangladesh. Our study showed that the geographic distribution of each Asia
IP T
II cryptic species in Bangladesh differed from one another. Asia II 1 was distributed across the entire country, while Asia II 5 was present in the middle and south regions, but not in the
SC R
northern region. It is unclear why Asia II 5 was absent in the northern region of Bangladesh
given that whiteflies from various crops were intensively collected. The presence of Asia II 1 has been reported in many countries in Asia, such as India, China, Pakistan, Cambodia,
U
Thailand, Vietnam, and Indonesia (Simon et al., 2003; Haider et al., 2003; Ahmed et al., 2011;
N
Hu et al., 2011, 2015). However, Asia II 5 was reported in India (Tamil Nadu, Kerala, and West
A
Bengal) and Pakistan but not in other countries in Asia (Ellango et al., 2015; Islam et al., 2018).
M
This result indicates that Asia II 1 is distributed widely in Asia, while Asia II 5 is restricted to a certain region of Asia, including India, Pakistan, and Bangladesh. Further studies are required
ED
to monitor the geographic distribution of Asia II 5 in other Asian countries.
PT
Asia II 10 is only found in one location at BARI in Gazipur, which is near the capital of Bangladesh. This cryptic species has been identified in only two countries, the Guangdong
CC E
Province of China and one location in Thailand (Hu et al., 2011; Gotz and Winter, 2016). This finding indicates that the distribution of Asia II 10 is restricted to certain regions in Asia. The COI sequence of Asia II 10 was 100% identical to samples from Thailand, but 2.24% variable
A
compared to samples from China. We assume that Asia II 10 in Bangladesh is more genetically close to the Thailand population than the China population. The genetic structure of the COI gene in Asia II 1 and Asia II 5 in Bangladesh was highly differentiated. AMOVA analysis revealed that the highest contribution to the total variance was from differences within populations. Both Asia II 1 and Asia II 5 were highly
differentiated and possessed many polymorphic sites and haplotypes, as found in previous studies (Hu et al., 2015, 2017). However, the haplotype networks of each cryptic species indicated that their patterns were different from each other. Asia II 1 was networked with one major haplotype and differentiated into 11 haplotypes. This pattern is similar to that of Asia I. However, Asia II 5 was differentiated into a linear form of 13 haplotypes. This result suggests
IP T
that Asia II 5 was diversified in a different way from other cryptic species within the Asia II and Asia I groups (Hu et al., 2015, 2017). Genetic relationships were analyzed from different
populations from 12 districts in Bangladesh. Tajima’s D values were negative in populations
SC R
from only four districts and Fu’s Fs values were positive in populations from all districts (Table 5). According to Kumar et al. (2016), the populations from four districts showed a low
U
frequency of polymorphism, while singleton variation was deficient in populations from all
N
districts in Bangladesh.
A
The host plant range of B. tabaci is very wide, even in invasive cryptic species such as
M
MEAM1 and MED (Brown, 2010). However, little is known about the host plant range of Asian cryptic species. Our study indicates that both Asia I and Asia II 1 are present in all 8 different
ED
crop species we studied but Asia II 5 was identified on four crops (bean, brinjal, sweet potato,
PT
and tomato). De Barro and Boykin (2013) provided that Asia II 5 was found on the yard long bean in Bangladesh and on tobacco in India. It also found on cassava and sunflower, as well as
CC E
bean and brinjal in India (Ellango et al., 2015). Asia II 10 was collected from cotton in one location. Asia II 10 was also collected from squash and cabbage in China, and tomato in Thailand (Hu et al., 2011; Gotz and Winter, 2016). Thus, our results indicate that the four
A
indigenous cryptic species in this study have a wide range of host plants. This evidence suggests that indigenous Asian cryptic species are also potentially as dangerous as MEAM1 and MED, if they invade other regions where susceptible crop plants are cultivated. In conclusion, our study shows the presence of four indigenous cryptic species of B. tabaci in Bangladesh. Among them, Asia I is the most prevalent and both Asia I and Asia II 1
are widely distributed across the country. Each cryptic species was distinctive but highly differentiated into many haplotypes. Our study provides important information for a comprehensive understanding of genetic diversity and the geographic distribution of B. tabaci in Bangladesh and related Asian countries. Further, the results of this study could facilitate the
IP T
monitoring of invasion and the displacement of cryptic species of B. tabaci in the future.
Disclosure statement
SC R
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
U
Funding
N
This work was supported by the Research Program for Exportation Support of Agricultural
A
Products, Animal and Plant Quarantine Agency, in the Republic of Korea under Grant (#Z-
Acknowledgments
ED
M
1543086-2017-21-01).
PT
We thank Dr. Jae-Kyoung Shim at Kyungpook National University in the Republic of Korea for her help with sequencing and molecular analysis. We also thank Pijush Jhan at Kyungpook
A
CC E
National University in the Republic of Korea for his help with the whitefly collection.
References Ahmed, M.Z., Barro, P.J., Greeff, J.M., Ren, S.X., Naveed, M., Qiu, B.L., 2011. Genetic identity of the Bemisia tabaci species complex and association with high cotton leaf curl disease (CLCuD)
incidence
in
Pakistan.
Pest.
Manag.
Sci.
67,
307–317.
IP T
https://doi.org/10.1002/ps.2067. Bedford, I.D., Briddon, R.W., Brown, J.K., Rosell, R.C., Markham, P.G. 1994. Geminivirus
from
different
geographic
regions. Ann.
SC R
transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes App.
https://doi.org/10.1111/j.1744-7348.1994.tb04972.x.
Biol.
125,
311–325.
U
Bellows, T.S., Perring, T.M., Gill, R.J., Headrick, D.H., 1994. Description of a Species of
N
Bemisia (Homoptera: Aleyrodidae). Ann. Entomol. Soc. Am. 87, 195–206.
A
https://doi.org/10.1093/aesa/87.2.195.
M
Boykin, L.M, Shatters, R.G, Rosell, R.C., McKenzie, C.L., Bagnall, R.A., De Barro, P., Frohlich, D.R., 2007. Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae)
ED
revealed using Bayesian analysis of mitochondrial CO1 DNA sequences. Mol.
PT
Phylogenet. Evol. 44, 1306–1319. https://doi.org/10.1016/j.ympev.2007.04.020. Boykin, L.M., De Barro, P.J., 2014. A practical guide to identifying members of the Bemisia
CC E
tabaci species complex: and other morphologically identical species. Front. Ecol. Evol. 2, 1–5. https://doi.org/10.3389/fevo.2014.00045.
A
Brown, J.K., 2010. Phylogenetic biology of the Bemisia tabaci sibling species group. In Bemisia: Bionomics and Management of a Global Pest. pp. 31-67. Springer Netherlands. https://doi: 10.1007/978-90-481-2460-2_2.
Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer program to estimate gene genealogies. Mol. 294x.2000.01020.x.
Ecol.
9,
1657–1659.
https://doi.org/10.1046/j.1365-
De Barro, P.J., Boykin, L.M., 2013. Global Bemisia dataset release version 31 Dec 2012. V1. CSIRO Data Collection. 10.4225/08/50EB54B6F1042. De Barro, P.J., Driver, F., Trueman, J.W., Curran, J., 2000. Phylogenetic relationships of world populations of Bemisia tabaci (Gennadius) using ribosomal ITS1. Mol. Phylogenet. Evol. 16, 29–36. https://doi.org/10.1006/mpev.1999.0768.
IP T
De Barro, P.J., Hidayat, S.H., Frohlich, D., Subandiyah, S., Ueda, S., 2008. A virus and its vector, pepper yellow leaf curl virus and Bemisia tabaci, two new invaders of Indonesia.
SC R
Biol. Invasions 10, 411-433. https://doi.org/10.1007/s10530-007-9141-x
De Barro, P.J., Liu, S., Boykin, L.M., Dinsdale, A.B., 2011. Bemisia tabaci: A statement of species status. Ann. Rev. Entomol. 56, 1–19. https://doi.org/10.1146/annurev-ento-
U
112408-085504.
tabaci
(Hemiptera:
Sternorrhyncha:
Aleyrodoidea:
Aleyrodidae)
A
of Bemisia
N
Dinsdale, A., Cook, L., Riginos, C., Buckley, Y.M., Barro, P.D., 2010. Refined global analysis
M
Mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries. Ann, Entomol, Soc, Am. 103, 196–208. https://doi.org/10.1603/AN09061.
ED
Elbaz, M., Lahav, N., Morin, S., 2010. Evidence for pre-Zygotic reproductive barrier between
PT
the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae). Bull. Entomol. Res. 100, 581–590. https://doi.org/10.1017/ S0007485309990630.
CC E
Ellango, R., Singh, S.T., Rana, V.S., Priya, N.G., Raina, H., Chaubey, R., Naveen, N.C., Mahmood, R., Ramamurthy, V.V., Asokan, R., 2015. Distribution of Bemisia tabaci
A
genetic
groups
in
India.
Environ.
Entomol.
44,
1258–1264.
https://doi.org/10.1093/ee/nvv062.
Excoffier, L., Laval, G., Schneider, S., 2005. ARLEQUIN version 3.0: An integrated software package for population genetics data analysis. Evol. Bioinfo. online. 1, 47–50. https://doi.org/10.1177/117693430500100003. Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from
metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 131, 479–491. http://www.genetics.org/content/131/2/479. Fauquet, C.M., Briddon, R.W., Brown, J.K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X., 2008. Geminivirus strain demarcation and nomenclature. Arch. Virol. 153, 783–821. https://doi.org/10.1007/s00705-008-0037-6.
bootstrap. Evolution. 39, 783–791. https://doi.org/10.2307/2408678.
IP T
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the
SC R
Firdaus, S., Vosman, B., Hidayati, N., Supena, E.D., Visser, R.G., Heusden, A.W., 2013. The
Bemisia tabaci species complex: Additions from different parts of the world. Insect Sci. 20, 723–733. https://doi.org/10.1111/1744-7917.12001.
U
Frohlich, D.R., Torres-Jerez, I.I., Bedford, I.D., Markham, P.G., Brown, J.K., 1999. A
N
phylogeographical analysis of the Bemisia tabaci species complex based on
A
mitochondrial DNA markers. Mol. Ecol. 8, 1683–1691. https://doi.org/10.1046/j.1365-
M
294x.1999.00754.x.
Götz, M., Winter, S., 2016. Diversity of Bemisia tabaci in Thailand and Vietnam and of
species
replacement. J.
ED
indications
Asia. Pac.
Entomol.
19,
537–543.
PT
https://doi.org/10.1016/j.aspen.2016.04.017. Guo, T., Guo, Q., Cui, X.Y., Liu, Y.Q., Hu, J., Liu, S.S., 2015. Comparison of transmission of
CC E
papaya leaf curl China virus among four cryptic species of the whitefly Bemisia tabaci complex. Sci. Rep. 5, 15432. https://doi.org/10.1038/srep15432.
A
Haider, S.M., Bedford, I.D., Evans, A.A.F., Markham, P.G., 2003. Geminivirus transmission by different biotypes of the whitefly Bemisia tabaci (Gennadius). Pakistan. J. Zool. 35, 343–351. https://doi.org/00309923.
Hameed, S., Hameed, S., Sadia, M., Malik, S.A., 2012. Genetic diversity analysis of Bemisia tabaci populations in Pakistan using RAPD markers. Electron. J. Biotechn. 15. https://doi.org/10.2225/vol15-issue6-fulltext-5.
Hogenhout, S.A., Ammar, E., Whitfield, A.E., Redinbaugh, M.G., 2008. Insect vector interactions with persistently transmitted viruses. Annu. Rev. Phytopathol. 46, 327–359. https://doi.org/10.1146/annurev.phyto.022508.092135. Horowitz, A.R., Kontsedalov, S., Khasdan, V., Ishaaya, I., 2005. Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance. Arch. Insect
IP T
Biochem. Physiol. 58, 216–225. https://doi.org/10.1002/arch.20044. Hu, J., Barro, P.D., Zhao, H., Wang, J., Nardi, F., Liu, S.S., 2011. An extensive field survey
alien
whiteflies
in
SC R
combined with a phylogenetic analysis reveals rapid and widespread invasion of two China. PLoS
One.
https://doi.org/10.1371/journal.pone.0016061.
6,
016061.
U
Hu, J., Jiang, Z.L., Nardi, F., Liu, Y.Y., Luo, X.R., Li, H.X., Zhang, Z.K., 2014. Members of
species
in
the
Yunnan
China.
J
Insect
Sci
14:1-8.
M
https://doi.org/10.1093/jisesa/ieu143.
province
A
alien
N
Bemisia tabaci (Hemiptera: Alerodidae) cryptic species and the status of two invasive
Hu, J., Chen, Y.D., Jiang, Z.L., Nardi, F., Yang, T.Y., Jin, J., Zhang, Z.K., 2015. Global
ED
haplotype analysis of the whitefly Bemisia tabaci cryptic species Asia I in Asia.
PT
Mitochondrial DNA. 26, 232–241. https://doi.org/10.3109/19401736.2013.830289. Hu, J., Zhang, X., Jiang, Z., Zhang, F., Liu, Y., Li, Z., Zhang, Z., 2017. New putative cryptic
CC E
species detection and genetic network analysis of Bemisia tabaci (Hemiptera: Aleyrodidae) in China based on mitochondrial COI sequences. Mitochondrial DNA Part A. 29, 474-484. https://doi.org/10.1080/24701394.2017.1307974.
A
Islam, W., Lin, W., Qasim, M., Islam, S.U., Ali, H., Adnan, M., Arif, M., Du, Z., Wu, Z., 2018. A nation-wide genetic survey revealed a complex population structure of Bemisia tabaci
in
Pakistan.
Acta
trop.
183,
119-125.
https://doi.org/10.1016/j.actatropica.2018.04.015. Jahan, S.M.H., Lee, K.Y., Howlader, M.I.A., 2015. The third genotypic cluster of Bemisia
tabaci (Gennadius) (Hemiptera: Aleyrodidae) found in Bangladesh. Bangladesh. J. Agril. Res. 40, 1–16. https://doi.org/ 10.3329/bjar.v40i1.23751. Jensen, J.L., Bohonak, A.J., Kelley, S.T., 2005. Isolation by distance, web service. BMC Genet. 6, 13. https://doi.org/10.1186/1471-2156-6-13. Jiu, M., Hu, J., Wang, L.J., Dong, J.F., Song, Y.Q., Sun, H.Z., 2017. Cryptic species
IP T
identification and composition of Bemisia tabaci (Hemiptera: Aleyrodidae) complex in Henan Province, China. J. Insect Sci. 17, 1–7. https://doi.org/10.1093/jisesa/iex048.
SC R
Jones, D.R., 2003. Plant viruses transmitted by whiteflies. Eur. J. Plant Pathol. 109, 195–219. https://doi.org/10.1023/a:1022846630513.
Jukes, T.H., Cantor, C.R., 1996. Evolution of protein molecules. In: Munro HN, editor,
U
Mammalian Protein Metabolism. New York: Academic Press. Pp. 21-132.
N
https://doi.org/10.12691/jaem-3-3-4.
A
Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions
M
through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. https://doi.org/10.1007/BF01731581.
ED
Kumar, N.R., Chang, J.C., Narayanan, M.B., Ramasamy, S., 2016. Phylogeographical structure
PT
in mitochondrial DNA of whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) in southern India and Southeast Asia. Mitochondrial DNA Part A. 28, 621–631.
CC E
https://doi.org/10.3109/24701394.2016.1160075. Lee, W., Park, J., Lee, G., Lee, S., Akimoto, S., 2013. Taxonomic status of the Bemisia tabaci
A
complex (Hemiptera: Aleyrodidae) and reassessment of the number of its constituent species. PLoS One. 8, e.63817. https://doi.org/10.1371/journal.pone.0063817.
Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism
data. Bioinfo.
https://doi.org/10.1093/bioinformatics/btp187.
25,
1451–1452.
Moya, A., Guirao, P., Cifuentes, D., Beitia, F., Cenis, J.L., 2001. Genetic diversity of Iberian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) based on random amplified polymorphic
DNA-Polymerase
chain
reaction. Mol.
Ecol.
10,
891–897.
https://doi.org/10.1046/j.1365-294X.2001.01221.x. Nei, M., Tajima, F., 1983. Maximum likelihood estimation of the number of nucleotide from
restriction
sites
data.
Genetics.
105,
207–217.
IP T
substitutions
http://www.genetics.org/content/105/1/207.
species
by
genomic
and
SC R
Perring, T., Cooper, A., Rodriguez, R., Farrar, C., Bellows, T., 1993. Identification of a whitefly behavioral
studies. Science.
https://doi.org/10.1126/science.8418497.
259,
74–77.
U
Prasanna, H.C., Kanakala, S., Archana, K., Jyothsna, P., Varma, R.K., Malathi, V.G., 2015.
N
Cryptic species composition and genetic diversity within Bemisia tabaci complex in
A
soybean in India revealed by mtCOI DNA sequence. J. Integr. Agr. 14, 1786–1795.
M
https://doi.org/10.1016/S2095-3119(14)60931-X. Rahayuwati, S., Hidayat, S.H., Hidayat, P., 2016. Genetic identity of Bemisia tabaci
ED
(Gennadius) (Hemiptera: Aleyrodidae) from endemic area of pepper yellow leaf curl
PT
Indonesia virus based on mitochondrial cytochrome oxidase I fragment (mtCOI). J. Entomol. Indonesia 13, 156-164. https://doi.org/10.5994/jei.13.3.156
CC E
Qin, L., Pan, L.L., Liu, S.S., 2016. Further insight into reproductive incompatibility between putative cryptic species of the Bemisia tabaci whitefly complex. Insect Sci. 23, 215– 224. https://doi.org/10.1111/1744-7917.12296.
A
Schaffer, A.A., Aravind, L., Madden,T.L., Shavirin, S., Spouge, J.L., Wolf, Y.I., Koonin, E.V., Altschul, S.F., 2001. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 29, 2994– 3005. https://doi.org/10.1093/nar/29.14.2994. Shah, S.H.J., Malik, A.H., Qazi, J., 2013. Identification of new genetic variant of Bemisia
tabaci
from
Pakistan.
Int.
J.
Entomol.
Res.
1,
16–24.
http://www.escijournals.net/index.php/IJER/article/view/217/206. Simon, B., Cenis, J.L., Demichelis, S., Rapisarda, C., Caciagli, P., Bosco, D., 2003. Survey of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Italy with the description of a new biotype (T) from Euphorbia characias. Bull. Entomol. Res. 93, 259-264.
IP T
https://doi.org/10.1079/BER2003233. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution, weighting,
SC R
and phylogenetic utility of mitochondrial gene sequences and a compilation of
conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651–701. https://doi.org/10.1093/aesa/87.6.651.
U
Srinivasan, R., Hsu, Y.-C., Kadirvel, P., Lin, M.-Y., 2013. Analysis of Bemisia tabaci
N
(Hemiptera: Aleyrodidae) species complex in Java, Indonesia based on mitochondrial
A
cytochrome oxidase I sequences. Philipp. Agric. Scientist 96, 290-295.
M
Tajima, F., 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585-595. https:/doi.org/PMC1203831.
ED
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular
PT
evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. https://doi.org/10.1093/molbev/mst197.
CC E
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided
A
by
quality
analysis
tools.
Nucleic
Acids
Res.
25,
4876–4882.
https://doi.org/10.1093/nar/25.24.4876.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. https://doi.org/10.1093/nar/22.22.4673.
Zhu, D.T., Xia, W.Q., Rao, Q., Liu, S.S., Ghanim, M., Wang, X.W., 2016. Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Asia Minor I. Insect Sci. 23, 531–542. https://doi.org/10.1111/1744-7917.12367.
Figures
IP T
Fig. 1. Distribution of four cryptic species of Bemisia tabaci in Bangladesh. The colored symbols represent these four cryptic species.
A
CC E
PT
ED
M
A
N
U
SC R
Asia I Asia II 1 Asia II 5 Asia II 10
IP T SC R U N A M ED PT CC E A Fig. 2. Phylogenetic tree based on CO1 sequences of Bemisia tabaci in Bangladesh. The tree was generated using COI sequences from collected samples in this study (indicated in four different colors) and other related reference sequences (black) searched from the GenBank database using the maximum likelihood method conducted in MEGA6.
IP T SC R U N A M A
CC E
PT
ED
Fig. 3. The relationship between the genetic distance (Fst) and geographic distance (km) of all samples of Bemisia tabaci in Bangladesh.
IP T SC R
Asia II 1
U
Asia I
N
Asia II 10
PT
ED
M
A
Asia II 5
A
CC E
Fig. 4. Minimum spanning network depicting evolutionary relationships of identified haplotypes of Bemisia tabaci in Bangladesh. Circle areas are proportional to haplotype frequency in the dataset. Genetic groups of each haplotype are color-coded.
I Table 1 Information about the collection sites and host plants.
N U SC R
Tables
Collection sites*
Location coordinates
Host plants
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Panchagarh Panchagarh Thakurgaon Thakurgaon Domar Domar Dinajpur Birganj Khanshama, Nilphamari Nilphamari sadar Kaharole Saidpur Rajshahi Rajshahi BARI, Gazipur BARI, Gazipur BARI, Gazipur BARI, Gazipur BARI, Gazipur BARI, Gazipur BSMRAU, Gazipur
26°20′7.35″N 26°20′7.35″N 26°2′30.62″N 26°2′30.62″N 26°9′16.78″N 26°9′16.78″N 25°37′40.48″N 25°53′36.41″N 23°7′55.83″N 25°50′53.81″N 25°47′37.41″N 25°46′58.64″N 24°21′48.92″N 24°21′48.92″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 24°1′43.72″N
BSMRAU, Gazipur BSMRAU, Gazipur
A 22 23
Accession numbers
Solanum lycopersicum Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum lycopersicum Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Gossypium hirsutum Solanum tuberosum Ipomoea batatas Ipomoea batatas Solanum tuberosum Phaseolus vulgaris
Cryptic species Asia II 1 Asia 1 Asia II 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia II 5 Asia 1 Asia II 1 Asia II 10 Asia II 1 Asia II 1 Asia II 5 Asia 1 Asia II 5
24°1′43.72″N 90°23′10.57″E
Solanum tuberosum
160103
Asia II 1
MF497060
24°1′43.72″N 90°23′10.57″E
Ipomoea batatas
160103
Asia II 1
MF497061
M
ED
PT
CC E
Panc-To-281 Panc-Br-283 Thak-Br-4 Thak-Br-5 Doma-Br-2 Doma-To-7 Dina-Br-2 Birg-Br-3 Khan-Br-4 Nilp-Br-1 Kaha-Br-1 Said-Br-1 Rajs-To-341 Rajs-To-342 BARI-To-15 BARI-Co-17 BARI-Po-18 BARI-Sp-19 BARI-Sp-20 BARI-Po-21 BSMRAUBe-11 BSMRAUPo-13 BSMRAUSp-33
Collection dates 160602 160602 160611 160611 160617 160617 170119 170124 170124 170121 170125 170120 160723 160723 160118 160111 160104 160110 160110 160118 151229
A
No Samples
88°33′6.11″E 88°33′6.11″E 88°25′41.74″E 88°25′41.74″E 88°48′25.52″E 88°48′25.52″E 88°37′59.43″E 88°40′20.49″E 91°56′56.48″E 88°56′29.09″E 88°36′4.36″E 88°53′53.76″E 88°37′26.89″E 88°37′26.89″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°23′10.57″E
MF497064 MF497042 MF497067 MF497043 MF497040 MF497041 MF497039 MF497033 MF497049 MF497050 MF497045 MF497052 MF497076 MF497051 MF497057 MF497079 MF497058 MF497059 MF497068 MF497032 MF497069
N U SC R
I PT
A
23°48′37.2″N 23°46′18.3″N 23°52′55.25″N 23°52′55.25″N 23°49′54.91″N 22°21′31.02″N 22°21′31.02″N 22°21′31.02″N 22°43′56.7″N 22°43′56.7″N 22°43′56.7″N 22°20′51.13″N 22°20′51.13″N 22°20′51.13″N 22°20′51.13″N 22°20′51.13″N 23°7′55.83″N 23°14′27.05″N 23°7′55.83″N 23°7′55.83″N 21°49′51.96″N 21°49′51.96″N 21°49′51.96″N 21°49′51.96″N 21°49′51.96″N 21°26′22.07″N 21°26′22.07″N 21°26′22.07″N
M
ED
SAU, Dhaka SAU, Dhaka Savar, Dhaka Savar, Dhaka Khustia Patuakhali Patuakhali Patuakhali Rangamati Rangamati Rangamati Chittagang Chittagang Chittagang Chittagang Chittagang Khagrachori Khagrachori Khagrachori Khagrachori Bandarban Bandarban Bandarban Bandarban Bandarban Coxsbazar Coxsbazar Coxsbazar
CC E
Dhak-Br-10 SAU-To-351 Sava-To-361 Sava-To-363 Kust-Br-2 Patu-To-1 Patu-Br-3 Patu-Br-8 Rang-To-1 Rang-Br-3 Rang-To-4 Chit-Br-1 Chit-Br-3 Chit-Po-5 Chit-Po-6 Chit-To-7 Khag-To-A Digh-To-C Khag-To-E Khag-To-F Band-Dah-1 Band-Br-4 Band-Br-5 Band-Co-8 Band-Co-10 Coxs-Sp-1 Coxs-Sp-5 Coxs-Br-9
A
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
90°24′45.07″E 90°22′37.88″E 90°16′50.83″E 90°16′50.83″E 89°4′30.9″E 90°19′54.19″E 90°19′54.19″E 90°19′54.19″E 92°17′54.65″E 92°17′54.65″E 92°17′54.65″E 91°48′44.4″E 91°48′44.4″E 91°48′44.4″E 91°48′44.4″E 91°48′44.4″E 91°56′56.48″E 92°3′50.65″E 91°56′56.48″E 91°56′56.48″E 92°22′7.08″E 92°22′7.08″E 92°22′7.08″E 92°22′7.08″E 92°22′7.08″E 92°0′27.83″E 92°0′27.83″E 92°0′27.83″E
Solanum melongena Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum melongena Solanum lycopersicum Solanum melongena Solanum melongena Solanum lycopersicum Solanum melongena Solanum lycopersicum Solanum melongena Bt Solanum melongena Solanum tuberosum Solanum tuberosum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Dahlia pinnata Solanum melongena Solanum melongena Gossypium hirsutum Gossypium hirsutum Ipomoea batatas Ipomoea batatas Solanum melongena
160825 160825 160825 160825 160525 160721 160721 160721 170204 170204 170204 170205 170205 170205 170205 170205 170117 170117 170117 170117 170203 170203 170203 170203 170203 170202 170202 170202
Asia 1 Asia 1 Asia II 1 Asia 1 Asia II 5 Asia II 5 Asia II 5 Asia II 1 Asia II 5 Asia II 5 Asia II 1 Asia 1 Asia 1 Asia II 1 Asia II 5 Asia II 5 Asia 1 Asia 1 Asia 1 Asia II 5 Asia 1 Asia II 1 Asia 1 Asia 1 Asia II 1 Asia 1 Asia 1 Asia 1
MF497036 MF497037 MF497063 MF497038 MF497073 MF497074 MF497075 MF497065 MF497077 MF497078 MF497066 MF497034 MF497035 MF497062 MF497070 MF497071 MF497046 MF497047 MF497048 MF497072 MF497029 MF497055 MF497030 MF497031 MF497056 MF497053 MF497044 MF497054
*The full names are as follow: Bangladesh Agriculture Research Institute (BARI), Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Sher-e-Bangla Agricultural University (SAU).
Table 2 Summary of the pairwise comparison of COI nucleotide sequences among four cryptic species of Bemisia tabaci identified in Bangladesh. The full data is listed in Table S1 of Supplementary data. Asia II 10 is only one sequence obtained. Asia II 1
Asia II 5
0.12-0.49
Asia II 1
15.0-15.80
0.12-1.11
Asia II 5
17.10-18.10
10.40-11.30
0.12-0.99
Asia II 10
14.9-15.40
13.8-14.10
13.6-13.90
SC R
Asia 1
Asia II 10
IP T
Asia 1
-
Asia II 1 20 18 10 8 12 0.916 0.00396
A
M
ED
Number of sequences Number of polymorphic sites (S) Singleton variable sites (Svs) Parsimony informative sites (Pis) Number of haplotypes Haplotype diversity (Hd) Nucleotide diversity (Pi)
Asia I 67 34 33 1 28 0.664 0.00135
N
U
Table 3 Comparison of the genetic structure of COI sequences among four cryptic species of Bemisia tabaci identified in Bangladesh. Asia II 5 22 15 8 7 13 0.896 0.00424
Asia II 10 1 0 0 0 1 0 0
A
CC E
PT
Table 4 Genetic variation partitioning of Bemisia tabaci in Bangladesh at different hierarchical levels. Source of variation d.f. Sum of Variance Percentage of Fixation squares components variation indices (Fstatistics) Among groups 13 1482.73 10.44va 27.79 Fct = 0.278 (=regions) Among populations 40 1318.19 5.68vb 15.11 Fsc = 0.209 within groups Within populations 56 1200.91 21.44vc 47.10 Fst = 0.429 (=individuals) Total 109 4001.84 37.56 Highly significant at P< 0.01 level
29
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Table 5 The number of samples, number of haplotypes, Tajima’s D and Fu’s Fs of Bemisia tabaci from different districts of Bangladesh. District No. of samples No. of Tajima’s D Fu’s Fs haplotypes Dhaka 6 2 1.5455 13.695 Gazipur 14 10 0.6717 5.597 Thakurgaon 6 6 2.3335 1.427 Dinajpur 8 5 -1.8951 5.433 Nilphamari 10 7 -1.8733 4.035 Rajshahi 10 9 0.3912 1.824 Khustia 13 4 1.950 22.126 Patuakhali 5 4 -1.0767 3.745 Bandarban 10 6 0.1613 6.945 Chittagang 12 12 1.9258 0.277 Khagrachori 6 5 1.5470 3.318 Coxsbazar 10 5 -1.8391 1.430
30
S0001-706X(18)30564-3 https://doi.org/10.1016/j.actatropica.2018.07.021 ACTROP 4726
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
5-5-2018 21-7-2018 24-7-2018
Please cite this article as: Fatema Khatun MS, Hemayet Jahan SM, Lee S, Lee K-Yeoll, Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.07.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Genetic diversity and geographic distribution of the Bemisia tabaci species complex in Bangladesh
Running header: Bemisia tabaci genetic diversity in Bangladesh
Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook
SC R
a
IP T
MS. Fatema Khatuna,b, S.M. Hemayet Jahanc, Sukchan Leed, Kyeong-Yeoll Leea,e,f*
National University, Daegu, Republic of Korea b
Department of Entomology, Bangabandhu Sheikh Mujibur Rahman Agricultural University,
U
Dhaka, Bangladesh c
N
Department of Entomology, Patuakhali Science and Technology University, Dumki,
A
Patuakhali, Bangladesh
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Republic of Korea
e
Institute of Plant Medicine, Kyungpook National University, Daegu, Republic of Korea
f
Institute of Agricultural Science and Technology, Kyungpook National University, Daegu,
CC E
PT
Republic of Korea
ED
M
d
*
Corresponding author:
A
Dr. Kyeong-Yeoll Lee, Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea. Tel.: 82-53-950-5759; e-mail: [email protected]
M
A
N
U
SC R
IP T
Graphical Abstract
ED
Highlights
Genetic diversity of Bemisia tabaci was determined in Bangladesh.
We identified four indigenous cryptic species but not MEAM1 and MED invasive
PT
CC E
cryptic species.
Asia I was abundant, both Asia II 1 and Asia II 5 were moderate, and Asia II 10 was found only in the central region.
A
Our study provides important information on the genetic diversity and geographic distribution of B. tabaci in Bangladesh.
Abstract Bemisia tabaci (Gennadius) is a species complex consisting of at least 40 cryptic species. Although the genetic diversity of B. tabaci has been studied in various regions, little is known
about distribution in Bangladesh, which is covered by the Bengal delta, the largest delta on Earth. We conducted an extensive survey throughout the country and determined the nucleotide sequence of mitochondrial cytochrome c oxidase subunit 1 (COI) from 110 individuals. We then examined phylogenetic relationships. The results identified four cryptic species that expressed distinct interspecific variation but low intraspecific variation. Asia I was the most
IP T
abundant, both Asia II 1 and Asia II 5 were moderately abundant, and Asia II 10 was found only in the central region. COI sequences of each cryptic species were distinctive and
SC R
differentiated into many haplotypes. Our study provides important information to better understand the genetic diversity and geographic distribution of cryptic species in Bangladesh
U
and nearby countries.
A
N
Keywords: Cryptic species; Genetic diversity; Geographic distribution; Haplotype; Invasion
M
1. Introduction
The sweet potato whitefly, Bemisia tabaci (Gennadius), is one of the most serious pests
ED
of various economically important horticultural and ornamental crop plants (Perring et al., 1993;
PT
Bellows et al., 1994; Dinsdale et al., 2010). In addition, this species acts as a vector of more than 110 viruses, most of which are begomoviruses. For example, the Tomato yellow leaf curl
CC E
virus (TYLCV) is highly viruliferous and is a major threat to crop production worldwide (Jones, 2003; Fauquet et al., 2008; Hogenhout et al., 2008). B. tabaci is distributed worldwide and is highly genetically diverse, consisting of a
A
complex of many cryptic species that are morphologically indistinguishable but genetically different (Bedford et al., 1994; Horowitz et al., 2005; Dinsdale et al., 2010; De Barro et al., 2011). The genetic diversity of B. tabaci has been examined using molecular database analysis by comparing nucleotide sequences of mitochondrial cytochrome oxidase subunit I (COI) in several studies. For example, Dinsdale et al. (2010) grouped B. tabaci into 24 cryptic species,
using 3.5% genetic distance as a species boundary threshold. Similarly, Lee et al. (2013) used 4.0% genetic distance as a species boundary based on the massive database of COI sequences, and grouped B. tabaci into 31 cryptic species. Recent studies have identified several new cryptic species from various countries and have recorded at least 40 cryptic species within the species complex of B. tabaci (Boykin and De Barro, 2014; Guo et al., 2015; Qin et al., 2016;
IP T
Zhu et al., 2016; Hu et al., 2017). Cryptic species within the species complex have different genetic characteristics, such
SC R
as environmental adaptation, host range, and pesticide resistance (Brown, 2010; De Barro et
al., 2011). Among them, both Middle East-Asia Minor 1 (MEAM1, formerly B biotype) and Mediterranean (MED, formerly Q biotype) originated from the deserts of Northeastern Africa,
U
the Arabian Peninsula, and Middle East Asia. They later spread throughout North Africa and
N
Mediterranean regions and then extensively invaded many other countries on different
A
continents (Frohlich et al., 1999; De Barro et al., 2000; Moya et al., 2001; Boykin et al.,
M
2007; Elbaz et al., 2010).
Numerous studies have shown that there are many genetically distinct cryptic species
ED
of B. tabaci in Asia. To date, at least 25 cryptic species of B. tabaci have been identified in
PT
various Asian countries, including two invasive species (MEAM1 and MED), 23 indigenous species (such as Asia I, Asia I India, 12 putative cryptic species of Asia II, Asia III, Asia IV,
CC E
and Asia V), and 6 putative cryptic species from China (Dinsdale et al., 2010; Ahmad et al., 2011; Hameed et al., 2012; Firdaus et al., 2013; Shah et al., 2013; Prasanna et al., 2015; Ellango et al., 2015; Hu et al., 2015, 2017; Gotz and Winter, 2016; Kumar et al., 2016; Jiu et al., 2017).
A
In China, 20 cryptic species (Asia I, Asia II 1, Asia II 2, Asia II 3, Asia II 4, Asia II 6, Asia II 7, Asia II 9, Asia II 10, Asia III, Asia IV, Asia V, China 1, China 2, China 3, China 4, China 5, China 6, MEAM1, and MED) have been reported, including two invasive species (Dinsdale et al., 2010; Hu et al., 2011, 2014, 2015; Firdaus et al., 2013; Jiu et al., 2017). In India, 9 cryptic species (Asia I, Asia I India, Asia II 1, Asia II 5, Asia II 7, Asia II 8, Asia II 11, China 3, and
MEAM1) have been recorded (Ellango et al., 2015; Kumar et al., 2016; Prasanna et al., 2015). Gotz and Winter, (2016) reported three cryptic species (Asia I, Asia II 6, and Asia II 10) in Thailand, and three cryptic species (Asia I, Asia II 1, Asia II 6) and two invasive species (MEAM1 and MED) in Vietnam. In Pakistan, six cryptic species (Asia I, Asia II 1, Asia II 5 Asia II 7, Asia II 8, and MEAM1) have been reported, with Asia II 1 in a major cotton-growing
IP T
region (Ahmad et al., 2011; Hameed et al., 2012; Shah et al., 2013; Islam et al., 2018). In Indonesia, both MEAM1 and MED have been invaded, but Asia I and other unidentified
SC R
indigenous cryptic species have also been reported (De Barro et al., 2008; Srinivasan et al., 2013; Rahayuwati et al., 2016). The genetic diversity and geographic distribution of each
genetic group are diverse in different countries, which may be a result of different
U
environmental conditions and plant biodiversity (Brown, 2010; De Barro et al., 2011). Like
N
other countries, Bangladesh has suffered serious damage to various horticultural crop plants
A
from B. tabaci, directly or indirectly by virus transmission. However, extensive studies on the
M
country-wide genetic diversity of B. tabaci have not been conducted in Bangladesh, which is covered by the largest delta on Earth, the Bengal delta (Hu et al., 2015; Jahan et al., 2015).
ED
The purpose of this large-scale study was to identify genetic groups of B. tabaci in
PT
Bangladesh and to understand the genetic diversity, geographical distribution, and phylogenetic
CC E
relationships with other groups that are distributed in neighboring Asian countries.
2. Materials and Methods
A
2.1 Whitefly collection Bemisia tabaci adults were collected from various crop plants (bean, brinjal, cotton,
dahlia, potato, sweetpotato, and tomato) across thirty-five different regions of Bangladesh during the peak crop growing season, from 2015 to 2017. Adult whiteflies were collected with an aspirator, then preserved in 70% alcohol in separate vials based on the crop plant and
location from which they were collected. The vials were then stored at -20 °C until further analysis (Table 1). Whiteflies from at least 20 to 100 individuals were collected from each site and at least three sites were surveyed per crop field.
2.2 DNA extraction
IP T
Genomic DNA was extracted from a single adult whitefly using the pure link genomic DNA mini kit (Invitrogen, Carlsbad, CA). The sample was placed in a 1.5 mL centrifuge tube
SC R
containing 180 µL of digestion buffer and 20 µL of proteinase K (50 µg/mL) and incubated at
55 °C for 4 h. DNA samples were extracted and purified using genomic spin columns, as described in the kit. DNA concentration was determined using a NanoPhotometer™ (Implen
N
U
GmbH, Schatzbogen, Germany).
A
2.3 PCR amplification
M
Mitochondrial cytochrome oxidase subunit I (COI) DNA was amplified using the primer pair MT10/C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′) and MT12/L2-N-3014
ED
(5′-TCCAATGCACTAATCTGCCATATTA) (Simon et al., 1994). Polymerase chain reaction
PT
(PCR) was performed in a total reaction volume of 20 µL, containing 13 µL Smart-Taq PreMix (Solgent Co., Daejeon, Korea), 1 µL of each primer (10 pmol/µL), and 5 µl of template
CC E
DNA solution (40 ng). Reaction mixtures were amplified under the following conditions: initial denaturation at 94 °C for 2 min; followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 5 min in a Veriti 96-Well Thermal Cycler
A
(Applied Biosystems, Foster City, CA). The PCR products were separated using 1% agarose gel electrophoresis, stained with Ethidium Bromide solution, and visualized under Ultraviolet (UV) light. Amplified PCR products were excised from the gel and purified using the Wizard® PCR Preps DNA Purification System (Wizard® SV Gel, Promega Co., Madison, WI) and sequenced either directly or by cloning into the T-BluntTM easy plasmid vector (Promega Co.,
Madison, WI).
2.4 DNA sequence analysis The PCR products, cloned using the T-BluntTM vector, were determined using the BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and
IP T
analyzed with a 3100 Capillary DNA Sequencer (Applied Biosystems, Foster City, CA) at the Solgent Sequencing Facility (Solgent Co., Daejeon, Korea). The GenBank database in the
SC R
National Center for Biotechnology Information (NCBI) was searched using the BLAST algorithm (Schaffer et al., 2001) and nucleotide sequences were aligned using CLUSTAL W
(Thompson et al., 1994). Identified COI sequences from the Bangladesh samples were
N
U
submitted to the GenBank database.
A
2.5 Phylogenetic analysis
M
COI sequences were edited manually to produce consensus sequences of 817 bp for each individual whitefly using the Clustal Omega program. The aligned sequences were
ED
analyzed using mega 6.0. The phylogenetic tree constructed using the maximum likelihood
PT
method implemented in MEGA 6.0 software (Tamura et al., 2013). We used 1000 bootstrap replicates to test the robustness of the phylogeny (Felsenstein, 1985). The COI sequence of
CC E
Bemisia afer (accession number AJ784260) was used as an outgroup (Hu et al., 2015). In this study, we followed the rule of pairwise sequence divergence >3.5 to characterize different
A
cryptic species of B. tabaci (Dinsdale et al., 2010).
2.6 Genetic analysis A total of 110 COI sequences from Bangladesh were aligned using Clustal X 1.83 (Thompson et al., 1997). The sequences were analyzed using a minimum spanning network relationship among B. tabaci cryptic species to generate a haplotype network using statistical
(95% limit) parsimony in TCS software v.1.21 (Clement et al., 2000). Nucleotide polymorphisms were estimated by deliberating several parameters, including the number of polymorphic sites and haplotypes, haplotype diversity (Nei and Tajima, 1983), nucleotide diversity, singleton variable sites, and parsimony-informative sites (Jukes and Cantor, 1996). All parameters for sequence polymorphism, divergence, gene flow, neutrality tests (Fu’s Fs and
IP T
Tajima’s D), and genetic differentiation were executed using DnaSP software v.5.10 (Librado and Rozas, 2009; Tajima, 1989). The pairwise genetic distance (Fst) value was generated from
SC R
1,023 permutations and by using the K2P model (Kimura, 1980). Analysis of molecular
variance (AMOVA) with 1,023 permutations was substantiated and the populations were grouped in accordance with their locations (Excoffier et al., 1992). AMOVA analysis and F-
U
statistics of genetic variation for B. tabaci populations in Bangladesh were obtained using a
N
pairwise distance method in Arlequin software version 3.1 (Excoffier et al., 2005). Isolation by
A
distance web service (IBD version 3.23) was used in the Mantel test to determine the correlation
M
between genetic distance (Fst) and geographic distances (Jensen et al., 2005). Geographic distances among the measured populations were based on a central location of collection sites
PT
3. Results
ED
in Bangladesh.
CC E
3.1 Identification of B. tabaci cryptic species in Bangladesh In total, 110 individuals collected from 35 different locations in Bangladesh were used
to amplify 817 bp of the COI gene sequence. The identified sequences were submitted to the
A
Genbank database in NCBI, with accession numbers from MF497029 to MF497079 (Table 1; Fig. 1). The COI sequence variation among all the samples ranged from 0.12% to 18.10% (Table 2). According to the maximum likelihood phylogram of previously known sequences in the Genbank database, all samples were placed into four genetic groups, namely, Asia I, Asia
II 1, Asia II 5, and Asia II 10 (Fig. 2). Among the 110 samples, Asia I (60.9%) was the most common sample, while Asia II 1 and Asia II 5 were 18.2% and 20.0%, respectively. Asia II 10 was from only one sample (0.9%) (Table 3). Geographic analysis showed that both Asia I and Asia II 1 were widely distributed throughout the country, but that Asia II 5 was distributed in the central and southern regions of Bangladesh, while Asia II 10 was distributed in only one
IP T
location, the Bangladesh Agricultural Research Institute (BARI) in Gazipur, which is in central Bangladesh (Fig. 1). We did not detect any MEAM1 and MED cryptic species that are known
SC R
to be invasive species.
The variation in COI nucleotide sequences differed between intraspecific and interspecific levels among cryptic species. Intraspecific variation was almost two times higher
U
in Asia II 1 (0.12-1.11%) and Asia II 5 (0.12-0.99%) than in Asia I (0.12-0.49%) (Table 2).
N
Interspecific variation ranged between 10.40% and 18.10% among the 4 cryptic species (Table
A
2). The highest variation (18.10%) was observed between Asia I (MF497030) collected from
M
Brinjal in the Bandarban district and Asia II 5 (MF497078) from Brinjal in the Rangamati district, which are located in the southern region of Bangladesh (Table S1).
ED
Whitefly samples were collected from 8 different crop species—bean (Phaseolus
PT
vulgaris), brinjal (Solanum melongena), Bt brinjal, cotton (Gossypium hirsutum), dahlia (Dahlia pinnata), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), and tomato
CC E
(Solanum lycopersicum) (Table 1). Both Asia I and Asia II 1 were found in all species, but Asia II 5 was observed only on the bean, brinjal, sweet potato, and tomato crops. Asia II 10 was identified on only one sample of cotton from BARI. Two cryptic species (Asia I and Asia II 1)
A
were found together in the same field of brinjal, cotton, potato, or tomato in the Bandarban and Gazipur regions (Table 1). The variation rate of COI sequences depended on region. For instance, in the Bandarban district, which is located in the southeastern region, COI variation was less than 0.37%. However, in the Gazipur district located in the middle of the country, variation was less
than 13.83%.
3.2 Genetic diversity of B. tabaci cryptic species in Bangladesh Variation in COI gene structure was analyzed for three cryptic species (Asia I, Asia II 1, Asia II 5), excluding Asia II 10 because it had only one sample (Table 3). The number of
IP T
polymorphic sites was higher in Asia I (34) than Asia II 1 (18) and Asia II 5 (15). Asia I had a higher number (33) of singleton variable sites than parsimony informative sites (1), but both
SC R
Asia II 1 and Asia II 5 had approximately equal numbers of these two sites (Table 3). The number of haplotypes against total sequences was 28/67 (Asia I), 13/22 (Asia II 5) and 12/20 (Asia II 1). Haplotype diversity was higher in Asia II 1 (0.916) than Asia II 5 (0.896) and Asia
U
I (0.664). Asia II 5 had the highest nucleotide diversity, followed by Asia II 1 and Asia I (Table
N
3). The population genetic study was conducted within the district populations. Tajima’s D
A
statistical analysis showed that in four districts, values were negative (Dinajpur, Nilphamari,
M
Patuakhali, and Coxsbazar), and Fu’s Fs showed positive values for all districts with significant differences (Table 5).
ED
Evolutionary relationships among the haplotypes of each cryptic species were depicted
PT
using both the minimum spanning network analysis and the phylogram (Fig. 4 and Fig. S1). Three cryptic species (Asia I, Asia II 1, and Asia II 5) were diversified into many haplotypes
CC E
and they were highly distant from each cryptic species by many mutational steps (Fig. 2 and Table S3). The pattern from the minimum spanning network of haplotypes was similar to the phylogenetic tree of B. tabaci sequences. Among 28 haplotypes of Asia I, the H1 haplotype
A
occupied a central position of the network and was associated with 27 low-frequency haplotypes. H24 represented two individuals. Among 12 haplotypes of Asia II 1, the H11 haplotype occupied the central position and was diversified by 11 haplotypes. Particularly, H5 was expected to differ by 7 mutational steps. However, among 13 haplotypes of Asia II 5, most haplotypes were extended into a linear form. Hierarchical AMOVA analysis revealed that the
total genetic variation (Fst) was 42.9% within the population based on the geographic distance (Table 4). The genetic variation was low among groups (10.44). The portions of genetic variation were partitioned among groups (27.79%), among populations within groups (15.11%), and within populations (47.10%). The Mantel test showed that there was no correlation
IP T
between genetic distance and geographic distance (Fig. 3 and Table S2).
4. Discussion
SC R
An extensive survey of B. tabaci in Bangladesh showed that the pairwise COI
nucleotide sequence variation reached 18.1% and the species complex was diversified into four cryptic species, namely, Asia I, Asia II 1, Asia II 5, and Asia II 10. Each cryptic species was
U
genetically distinct, possessing more than 10% sequence divergence. Asia I was the most
N
abundant. Both Asia II 1 and Asia II 5 were found in moderate levels, but Asia II 10 was found
A
in only one location. However, MEAM1 and MED cryptic species were not detected in this
M
study. The absence of these two invasive species in Bangladesh, even though they have invaded neighboring countries, is interesting. For example, MEAM1 has been found in India and
ED
Pakistan, while MED was not detected in these two countries (Ellango et al., 2015; Islam et al.,
PT
2018). It is important to monitor the abundance of these two invasive species in Asian countries for the appropriate management of whiteflies.
CC E
Asia I is one of the predominant cryptic species in Asia. Hu et al., (2015) extensively studied its presence in 7 countries, including Cambodia, China, India, Indonesia, Pakistan, Thailand, and Bangladesh. Its distribution was also reported in Turkey, Singapore, and
A
Malaysia in the GenBank database. Gotz and Winter, (2016) reported that Asia I also exists in Vietnam. Our study showed that Asia I was the most abundant cryptic species and was distributed throughout Bangladesh. Hu et al. (2011, 2015) had previously reported the presence of 5 haplotypes of Asia I from Bangladesh. Our haplotype analysis showed that Asia I was highly differentiated into 28 haplotypes. One major haplotype (H1) was associated with 27
low-frequency haplotypes. This result is consistent with Hu et al. (2015) which showed that the major haplotypes networked with many minor haplotypes. Asia II is a large but genetically diverse group, including at least 12 cryptic species (Hu et al., 2011, 2017). Among them, only three species (Asia II 1, Asia II 5, and Asia II 10) were identified in Bangladesh. Our study showed that the geographic distribution of each Asia
IP T
II cryptic species in Bangladesh differed from one another. Asia II 1 was distributed across the entire country, while Asia II 5 was present in the middle and south regions, but not in the
SC R
northern region. It is unclear why Asia II 5 was absent in the northern region of Bangladesh
given that whiteflies from various crops were intensively collected. The presence of Asia II 1 has been reported in many countries in Asia, such as India, China, Pakistan, Cambodia,
U
Thailand, Vietnam, and Indonesia (Simon et al., 2003; Haider et al., 2003; Ahmed et al., 2011;
N
Hu et al., 2011, 2015). However, Asia II 5 was reported in India (Tamil Nadu, Kerala, and West
A
Bengal) and Pakistan but not in other countries in Asia (Ellango et al., 2015; Islam et al., 2018).
M
This result indicates that Asia II 1 is distributed widely in Asia, while Asia II 5 is restricted to a certain region of Asia, including India, Pakistan, and Bangladesh. Further studies are required
ED
to monitor the geographic distribution of Asia II 5 in other Asian countries.
PT
Asia II 10 is only found in one location at BARI in Gazipur, which is near the capital of Bangladesh. This cryptic species has been identified in only two countries, the Guangdong
CC E
Province of China and one location in Thailand (Hu et al., 2011; Gotz and Winter, 2016). This finding indicates that the distribution of Asia II 10 is restricted to certain regions in Asia. The COI sequence of Asia II 10 was 100% identical to samples from Thailand, but 2.24% variable
A
compared to samples from China. We assume that Asia II 10 in Bangladesh is more genetically close to the Thailand population than the China population. The genetic structure of the COI gene in Asia II 1 and Asia II 5 in Bangladesh was highly differentiated. AMOVA analysis revealed that the highest contribution to the total variance was from differences within populations. Both Asia II 1 and Asia II 5 were highly
differentiated and possessed many polymorphic sites and haplotypes, as found in previous studies (Hu et al., 2015, 2017). However, the haplotype networks of each cryptic species indicated that their patterns were different from each other. Asia II 1 was networked with one major haplotype and differentiated into 11 haplotypes. This pattern is similar to that of Asia I. However, Asia II 5 was differentiated into a linear form of 13 haplotypes. This result suggests
IP T
that Asia II 5 was diversified in a different way from other cryptic species within the Asia II and Asia I groups (Hu et al., 2015, 2017). Genetic relationships were analyzed from different
populations from 12 districts in Bangladesh. Tajima’s D values were negative in populations
SC R
from only four districts and Fu’s Fs values were positive in populations from all districts (Table 5). According to Kumar et al. (2016), the populations from four districts showed a low
U
frequency of polymorphism, while singleton variation was deficient in populations from all
N
districts in Bangladesh.
A
The host plant range of B. tabaci is very wide, even in invasive cryptic species such as
M
MEAM1 and MED (Brown, 2010). However, little is known about the host plant range of Asian cryptic species. Our study indicates that both Asia I and Asia II 1 are present in all 8 different
ED
crop species we studied but Asia II 5 was identified on four crops (bean, brinjal, sweet potato,
PT
and tomato). De Barro and Boykin (2013) provided that Asia II 5 was found on the yard long bean in Bangladesh and on tobacco in India. It also found on cassava and sunflower, as well as
CC E
bean and brinjal in India (Ellango et al., 2015). Asia II 10 was collected from cotton in one location. Asia II 10 was also collected from squash and cabbage in China, and tomato in Thailand (Hu et al., 2011; Gotz and Winter, 2016). Thus, our results indicate that the four
A
indigenous cryptic species in this study have a wide range of host plants. This evidence suggests that indigenous Asian cryptic species are also potentially as dangerous as MEAM1 and MED, if they invade other regions where susceptible crop plants are cultivated. In conclusion, our study shows the presence of four indigenous cryptic species of B. tabaci in Bangladesh. Among them, Asia I is the most prevalent and both Asia I and Asia II 1
are widely distributed across the country. Each cryptic species was distinctive but highly differentiated into many haplotypes. Our study provides important information for a comprehensive understanding of genetic diversity and the geographic distribution of B. tabaci in Bangladesh and related Asian countries. Further, the results of this study could facilitate the
IP T
monitoring of invasion and the displacement of cryptic species of B. tabaci in the future.
Disclosure statement
SC R
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
U
Funding
N
This work was supported by the Research Program for Exportation Support of Agricultural
A
Products, Animal and Plant Quarantine Agency, in the Republic of Korea under Grant (#Z-
Acknowledgments
ED
M
1543086-2017-21-01).
PT
We thank Dr. Jae-Kyoung Shim at Kyungpook National University in the Republic of Korea for her help with sequencing and molecular analysis. We also thank Pijush Jhan at Kyungpook
A
CC E
National University in the Republic of Korea for his help with the whitefly collection.
References Ahmed, M.Z., Barro, P.J., Greeff, J.M., Ren, S.X., Naveed, M., Qiu, B.L., 2011. Genetic identity of the Bemisia tabaci species complex and association with high cotton leaf curl disease (CLCuD)
incidence
in
Pakistan.
Pest.
Manag.
Sci.
67,
307–317.
IP T
https://doi.org/10.1002/ps.2067. Bedford, I.D., Briddon, R.W., Brown, J.K., Rosell, R.C., Markham, P.G. 1994. Geminivirus
from
different
geographic
regions. Ann.
SC R
transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes App.
https://doi.org/10.1111/j.1744-7348.1994.tb04972.x.
Biol.
125,
311–325.
U
Bellows, T.S., Perring, T.M., Gill, R.J., Headrick, D.H., 1994. Description of a Species of
N
Bemisia (Homoptera: Aleyrodidae). Ann. Entomol. Soc. Am. 87, 195–206.
A
https://doi.org/10.1093/aesa/87.2.195.
M
Boykin, L.M, Shatters, R.G, Rosell, R.C., McKenzie, C.L., Bagnall, R.A., De Barro, P., Frohlich, D.R., 2007. Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae)
ED
revealed using Bayesian analysis of mitochondrial CO1 DNA sequences. Mol.
PT
Phylogenet. Evol. 44, 1306–1319. https://doi.org/10.1016/j.ympev.2007.04.020. Boykin, L.M., De Barro, P.J., 2014. A practical guide to identifying members of the Bemisia
CC E
tabaci species complex: and other morphologically identical species. Front. Ecol. Evol. 2, 1–5. https://doi.org/10.3389/fevo.2014.00045.
A
Brown, J.K., 2010. Phylogenetic biology of the Bemisia tabaci sibling species group. In Bemisia: Bionomics and Management of a Global Pest. pp. 31-67. Springer Netherlands. https://doi: 10.1007/978-90-481-2460-2_2.
Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer program to estimate gene genealogies. Mol. 294x.2000.01020.x.
Ecol.
9,
1657–1659.
https://doi.org/10.1046/j.1365-
De Barro, P.J., Boykin, L.M., 2013. Global Bemisia dataset release version 31 Dec 2012. V1. CSIRO Data Collection. 10.4225/08/50EB54B6F1042. De Barro, P.J., Driver, F., Trueman, J.W., Curran, J., 2000. Phylogenetic relationships of world populations of Bemisia tabaci (Gennadius) using ribosomal ITS1. Mol. Phylogenet. Evol. 16, 29–36. https://doi.org/10.1006/mpev.1999.0768.
IP T
De Barro, P.J., Hidayat, S.H., Frohlich, D., Subandiyah, S., Ueda, S., 2008. A virus and its vector, pepper yellow leaf curl virus and Bemisia tabaci, two new invaders of Indonesia.
SC R
Biol. Invasions 10, 411-433. https://doi.org/10.1007/s10530-007-9141-x
De Barro, P.J., Liu, S., Boykin, L.M., Dinsdale, A.B., 2011. Bemisia tabaci: A statement of species status. Ann. Rev. Entomol. 56, 1–19. https://doi.org/10.1146/annurev-ento-
U
112408-085504.
tabaci
(Hemiptera:
Sternorrhyncha:
Aleyrodoidea:
Aleyrodidae)
A
of Bemisia
N
Dinsdale, A., Cook, L., Riginos, C., Buckley, Y.M., Barro, P.D., 2010. Refined global analysis
M
Mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries. Ann, Entomol, Soc, Am. 103, 196–208. https://doi.org/10.1603/AN09061.
ED
Elbaz, M., Lahav, N., Morin, S., 2010. Evidence for pre-Zygotic reproductive barrier between
PT
the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae). Bull. Entomol. Res. 100, 581–590. https://doi.org/10.1017/ S0007485309990630.
CC E
Ellango, R., Singh, S.T., Rana, V.S., Priya, N.G., Raina, H., Chaubey, R., Naveen, N.C., Mahmood, R., Ramamurthy, V.V., Asokan, R., 2015. Distribution of Bemisia tabaci
A
genetic
groups
in
India.
Environ.
Entomol.
44,
1258–1264.
https://doi.org/10.1093/ee/nvv062.
Excoffier, L., Laval, G., Schneider, S., 2005. ARLEQUIN version 3.0: An integrated software package for population genetics data analysis. Evol. Bioinfo. online. 1, 47–50. https://doi.org/10.1177/117693430500100003. Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from
metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 131, 479–491. http://www.genetics.org/content/131/2/479. Fauquet, C.M., Briddon, R.W., Brown, J.K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X., 2008. Geminivirus strain demarcation and nomenclature. Arch. Virol. 153, 783–821. https://doi.org/10.1007/s00705-008-0037-6.
bootstrap. Evolution. 39, 783–791. https://doi.org/10.2307/2408678.
IP T
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the
SC R
Firdaus, S., Vosman, B., Hidayati, N., Supena, E.D., Visser, R.G., Heusden, A.W., 2013. The
Bemisia tabaci species complex: Additions from different parts of the world. Insect Sci. 20, 723–733. https://doi.org/10.1111/1744-7917.12001.
U
Frohlich, D.R., Torres-Jerez, I.I., Bedford, I.D., Markham, P.G., Brown, J.K., 1999. A
N
phylogeographical analysis of the Bemisia tabaci species complex based on
A
mitochondrial DNA markers. Mol. Ecol. 8, 1683–1691. https://doi.org/10.1046/j.1365-
M
294x.1999.00754.x.
Götz, M., Winter, S., 2016. Diversity of Bemisia tabaci in Thailand and Vietnam and of
species
replacement. J.
ED
indications
Asia. Pac.
Entomol.
19,
537–543.
PT
https://doi.org/10.1016/j.aspen.2016.04.017. Guo, T., Guo, Q., Cui, X.Y., Liu, Y.Q., Hu, J., Liu, S.S., 2015. Comparison of transmission of
CC E
papaya leaf curl China virus among four cryptic species of the whitefly Bemisia tabaci complex. Sci. Rep. 5, 15432. https://doi.org/10.1038/srep15432.
A
Haider, S.M., Bedford, I.D., Evans, A.A.F., Markham, P.G., 2003. Geminivirus transmission by different biotypes of the whitefly Bemisia tabaci (Gennadius). Pakistan. J. Zool. 35, 343–351. https://doi.org/00309923.
Hameed, S., Hameed, S., Sadia, M., Malik, S.A., 2012. Genetic diversity analysis of Bemisia tabaci populations in Pakistan using RAPD markers. Electron. J. Biotechn. 15. https://doi.org/10.2225/vol15-issue6-fulltext-5.
Hogenhout, S.A., Ammar, E., Whitfield, A.E., Redinbaugh, M.G., 2008. Insect vector interactions with persistently transmitted viruses. Annu. Rev. Phytopathol. 46, 327–359. https://doi.org/10.1146/annurev.phyto.022508.092135. Horowitz, A.R., Kontsedalov, S., Khasdan, V., Ishaaya, I., 2005. Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance. Arch. Insect
IP T
Biochem. Physiol. 58, 216–225. https://doi.org/10.1002/arch.20044. Hu, J., Barro, P.D., Zhao, H., Wang, J., Nardi, F., Liu, S.S., 2011. An extensive field survey
alien
whiteflies
in
SC R
combined with a phylogenetic analysis reveals rapid and widespread invasion of two China. PLoS
One.
https://doi.org/10.1371/journal.pone.0016061.
6,
016061.
U
Hu, J., Jiang, Z.L., Nardi, F., Liu, Y.Y., Luo, X.R., Li, H.X., Zhang, Z.K., 2014. Members of
species
in
the
Yunnan
China.
J
Insect
Sci
14:1-8.
M
https://doi.org/10.1093/jisesa/ieu143.
province
A
alien
N
Bemisia tabaci (Hemiptera: Alerodidae) cryptic species and the status of two invasive
Hu, J., Chen, Y.D., Jiang, Z.L., Nardi, F., Yang, T.Y., Jin, J., Zhang, Z.K., 2015. Global
ED
haplotype analysis of the whitefly Bemisia tabaci cryptic species Asia I in Asia.
PT
Mitochondrial DNA. 26, 232–241. https://doi.org/10.3109/19401736.2013.830289. Hu, J., Zhang, X., Jiang, Z., Zhang, F., Liu, Y., Li, Z., Zhang, Z., 2017. New putative cryptic
CC E
species detection and genetic network analysis of Bemisia tabaci (Hemiptera: Aleyrodidae) in China based on mitochondrial COI sequences. Mitochondrial DNA Part A. 29, 474-484. https://doi.org/10.1080/24701394.2017.1307974.
A
Islam, W., Lin, W., Qasim, M., Islam, S.U., Ali, H., Adnan, M., Arif, M., Du, Z., Wu, Z., 2018. A nation-wide genetic survey revealed a complex population structure of Bemisia tabaci
in
Pakistan.
Acta
trop.
183,
119-125.
https://doi.org/10.1016/j.actatropica.2018.04.015. Jahan, S.M.H., Lee, K.Y., Howlader, M.I.A., 2015. The third genotypic cluster of Bemisia
tabaci (Gennadius) (Hemiptera: Aleyrodidae) found in Bangladesh. Bangladesh. J. Agril. Res. 40, 1–16. https://doi.org/ 10.3329/bjar.v40i1.23751. Jensen, J.L., Bohonak, A.J., Kelley, S.T., 2005. Isolation by distance, web service. BMC Genet. 6, 13. https://doi.org/10.1186/1471-2156-6-13. Jiu, M., Hu, J., Wang, L.J., Dong, J.F., Song, Y.Q., Sun, H.Z., 2017. Cryptic species
IP T
identification and composition of Bemisia tabaci (Hemiptera: Aleyrodidae) complex in Henan Province, China. J. Insect Sci. 17, 1–7. https://doi.org/10.1093/jisesa/iex048.
SC R
Jones, D.R., 2003. Plant viruses transmitted by whiteflies. Eur. J. Plant Pathol. 109, 195–219. https://doi.org/10.1023/a:1022846630513.
Jukes, T.H., Cantor, C.R., 1996. Evolution of protein molecules. In: Munro HN, editor,
U
Mammalian Protein Metabolism. New York: Academic Press. Pp. 21-132.
N
https://doi.org/10.12691/jaem-3-3-4.
A
Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions
M
through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. https://doi.org/10.1007/BF01731581.
ED
Kumar, N.R., Chang, J.C., Narayanan, M.B., Ramasamy, S., 2016. Phylogeographical structure
PT
in mitochondrial DNA of whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) in southern India and Southeast Asia. Mitochondrial DNA Part A. 28, 621–631.
CC E
https://doi.org/10.3109/24701394.2016.1160075. Lee, W., Park, J., Lee, G., Lee, S., Akimoto, S., 2013. Taxonomic status of the Bemisia tabaci
A
complex (Hemiptera: Aleyrodidae) and reassessment of the number of its constituent species. PLoS One. 8, e.63817. https://doi.org/10.1371/journal.pone.0063817.
Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism
data. Bioinfo.
https://doi.org/10.1093/bioinformatics/btp187.
25,
1451–1452.
Moya, A., Guirao, P., Cifuentes, D., Beitia, F., Cenis, J.L., 2001. Genetic diversity of Iberian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) based on random amplified polymorphic
DNA-Polymerase
chain
reaction. Mol.
Ecol.
10,
891–897.
https://doi.org/10.1046/j.1365-294X.2001.01221.x. Nei, M., Tajima, F., 1983. Maximum likelihood estimation of the number of nucleotide from
restriction
sites
data.
Genetics.
105,
207–217.
IP T
substitutions
http://www.genetics.org/content/105/1/207.
species
by
genomic
and
SC R
Perring, T., Cooper, A., Rodriguez, R., Farrar, C., Bellows, T., 1993. Identification of a whitefly behavioral
studies. Science.
https://doi.org/10.1126/science.8418497.
259,
74–77.
U
Prasanna, H.C., Kanakala, S., Archana, K., Jyothsna, P., Varma, R.K., Malathi, V.G., 2015.
N
Cryptic species composition and genetic diversity within Bemisia tabaci complex in
A
soybean in India revealed by mtCOI DNA sequence. J. Integr. Agr. 14, 1786–1795.
M
https://doi.org/10.1016/S2095-3119(14)60931-X. Rahayuwati, S., Hidayat, S.H., Hidayat, P., 2016. Genetic identity of Bemisia tabaci
ED
(Gennadius) (Hemiptera: Aleyrodidae) from endemic area of pepper yellow leaf curl
PT
Indonesia virus based on mitochondrial cytochrome oxidase I fragment (mtCOI). J. Entomol. Indonesia 13, 156-164. https://doi.org/10.5994/jei.13.3.156
CC E
Qin, L., Pan, L.L., Liu, S.S., 2016. Further insight into reproductive incompatibility between putative cryptic species of the Bemisia tabaci whitefly complex. Insect Sci. 23, 215– 224. https://doi.org/10.1111/1744-7917.12296.
A
Schaffer, A.A., Aravind, L., Madden,T.L., Shavirin, S., Spouge, J.L., Wolf, Y.I., Koonin, E.V., Altschul, S.F., 2001. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 29, 2994– 3005. https://doi.org/10.1093/nar/29.14.2994. Shah, S.H.J., Malik, A.H., Qazi, J., 2013. Identification of new genetic variant of Bemisia
tabaci
from
Pakistan.
Int.
J.
Entomol.
Res.
1,
16–24.
http://www.escijournals.net/index.php/IJER/article/view/217/206. Simon, B., Cenis, J.L., Demichelis, S., Rapisarda, C., Caciagli, P., Bosco, D., 2003. Survey of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Italy with the description of a new biotype (T) from Euphorbia characias. Bull. Entomol. Res. 93, 259-264.
IP T
https://doi.org/10.1079/BER2003233. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution, weighting,
SC R
and phylogenetic utility of mitochondrial gene sequences and a compilation of
conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651–701. https://doi.org/10.1093/aesa/87.6.651.
U
Srinivasan, R., Hsu, Y.-C., Kadirvel, P., Lin, M.-Y., 2013. Analysis of Bemisia tabaci
N
(Hemiptera: Aleyrodidae) species complex in Java, Indonesia based on mitochondrial
A
cytochrome oxidase I sequences. Philipp. Agric. Scientist 96, 290-295.
M
Tajima, F., 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585-595. https:/doi.org/PMC1203831.
ED
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular
PT
evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. https://doi.org/10.1093/molbev/mst197.
CC E
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided
A
by
quality
analysis
tools.
Nucleic
Acids
Res.
25,
4876–4882.
https://doi.org/10.1093/nar/25.24.4876.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. https://doi.org/10.1093/nar/22.22.4673.
Zhu, D.T., Xia, W.Q., Rao, Q., Liu, S.S., Ghanim, M., Wang, X.W., 2016. Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Asia Minor I. Insect Sci. 23, 531–542. https://doi.org/10.1111/1744-7917.12367.
Figures
IP T
Fig. 1. Distribution of four cryptic species of Bemisia tabaci in Bangladesh. The colored symbols represent these four cryptic species.
A
CC E
PT
ED
M
A
N
U
SC R
Asia I Asia II 1 Asia II 5 Asia II 10
IP T SC R U N A M ED PT CC E A Fig. 2. Phylogenetic tree based on CO1 sequences of Bemisia tabaci in Bangladesh. The tree was generated using COI sequences from collected samples in this study (indicated in four different colors) and other related reference sequences (black) searched from the GenBank database using the maximum likelihood method conducted in MEGA6.
IP T SC R U N A M A
CC E
PT
ED
Fig. 3. The relationship between the genetic distance (Fst) and geographic distance (km) of all samples of Bemisia tabaci in Bangladesh.
IP T SC R
Asia II 1
U
Asia I
N
Asia II 10
PT
ED
M
A
Asia II 5
A
CC E
Fig. 4. Minimum spanning network depicting evolutionary relationships of identified haplotypes of Bemisia tabaci in Bangladesh. Circle areas are proportional to haplotype frequency in the dataset. Genetic groups of each haplotype are color-coded.
I Table 1 Information about the collection sites and host plants.
N U SC R
Tables
Collection sites*
Location coordinates
Host plants
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Panchagarh Panchagarh Thakurgaon Thakurgaon Domar Domar Dinajpur Birganj Khanshama, Nilphamari Nilphamari sadar Kaharole Saidpur Rajshahi Rajshahi BARI, Gazipur BARI, Gazipur BARI, Gazipur BARI, Gazipur BARI, Gazipur BARI, Gazipur BSMRAU, Gazipur
26°20′7.35″N 26°20′7.35″N 26°2′30.62″N 26°2′30.62″N 26°9′16.78″N 26°9′16.78″N 25°37′40.48″N 25°53′36.41″N 23°7′55.83″N 25°50′53.81″N 25°47′37.41″N 25°46′58.64″N 24°21′48.92″N 24°21′48.92″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 23°59′35.07″N 24°1′43.72″N
BSMRAU, Gazipur BSMRAU, Gazipur
A 22 23
Accession numbers
Solanum lycopersicum Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum lycopersicum Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum melongena Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Gossypium hirsutum Solanum tuberosum Ipomoea batatas Ipomoea batatas Solanum tuberosum Phaseolus vulgaris
Cryptic species Asia II 1 Asia 1 Asia II 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia 1 Asia II 5 Asia 1 Asia II 1 Asia II 10 Asia II 1 Asia II 1 Asia II 5 Asia 1 Asia II 5
24°1′43.72″N 90°23′10.57″E
Solanum tuberosum
160103
Asia II 1
MF497060
24°1′43.72″N 90°23′10.57″E
Ipomoea batatas
160103
Asia II 1
MF497061
M
ED
PT
CC E
Panc-To-281 Panc-Br-283 Thak-Br-4 Thak-Br-5 Doma-Br-2 Doma-To-7 Dina-Br-2 Birg-Br-3 Khan-Br-4 Nilp-Br-1 Kaha-Br-1 Said-Br-1 Rajs-To-341 Rajs-To-342 BARI-To-15 BARI-Co-17 BARI-Po-18 BARI-Sp-19 BARI-Sp-20 BARI-Po-21 BSMRAUBe-11 BSMRAUPo-13 BSMRAUSp-33
Collection dates 160602 160602 160611 160611 160617 160617 170119 170124 170124 170121 170125 170120 160723 160723 160118 160111 160104 160110 160110 160118 151229
A
No Samples
88°33′6.11″E 88°33′6.11″E 88°25′41.74″E 88°25′41.74″E 88°48′25.52″E 88°48′25.52″E 88°37′59.43″E 88°40′20.49″E 91°56′56.48″E 88°56′29.09″E 88°36′4.36″E 88°53′53.76″E 88°37′26.89″E 88°37′26.89″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°24′18.21″E 90°23′10.57″E
MF497064 MF497042 MF497067 MF497043 MF497040 MF497041 MF497039 MF497033 MF497049 MF497050 MF497045 MF497052 MF497076 MF497051 MF497057 MF497079 MF497058 MF497059 MF497068 MF497032 MF497069
N U SC R
I PT
A
23°48′37.2″N 23°46′18.3″N 23°52′55.25″N 23°52′55.25″N 23°49′54.91″N 22°21′31.02″N 22°21′31.02″N 22°21′31.02″N 22°43′56.7″N 22°43′56.7″N 22°43′56.7″N 22°20′51.13″N 22°20′51.13″N 22°20′51.13″N 22°20′51.13″N 22°20′51.13″N 23°7′55.83″N 23°14′27.05″N 23°7′55.83″N 23°7′55.83″N 21°49′51.96″N 21°49′51.96″N 21°49′51.96″N 21°49′51.96″N 21°49′51.96″N 21°26′22.07″N 21°26′22.07″N 21°26′22.07″N
M
ED
SAU, Dhaka SAU, Dhaka Savar, Dhaka Savar, Dhaka Khustia Patuakhali Patuakhali Patuakhali Rangamati Rangamati Rangamati Chittagang Chittagang Chittagang Chittagang Chittagang Khagrachori Khagrachori Khagrachori Khagrachori Bandarban Bandarban Bandarban Bandarban Bandarban Coxsbazar Coxsbazar Coxsbazar
CC E
Dhak-Br-10 SAU-To-351 Sava-To-361 Sava-To-363 Kust-Br-2 Patu-To-1 Patu-Br-3 Patu-Br-8 Rang-To-1 Rang-Br-3 Rang-To-4 Chit-Br-1 Chit-Br-3 Chit-Po-5 Chit-Po-6 Chit-To-7 Khag-To-A Digh-To-C Khag-To-E Khag-To-F Band-Dah-1 Band-Br-4 Band-Br-5 Band-Co-8 Band-Co-10 Coxs-Sp-1 Coxs-Sp-5 Coxs-Br-9
A
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
90°24′45.07″E 90°22′37.88″E 90°16′50.83″E 90°16′50.83″E 89°4′30.9″E 90°19′54.19″E 90°19′54.19″E 90°19′54.19″E 92°17′54.65″E 92°17′54.65″E 92°17′54.65″E 91°48′44.4″E 91°48′44.4″E 91°48′44.4″E 91°48′44.4″E 91°48′44.4″E 91°56′56.48″E 92°3′50.65″E 91°56′56.48″E 91°56′56.48″E 92°22′7.08″E 92°22′7.08″E 92°22′7.08″E 92°22′7.08″E 92°22′7.08″E 92°0′27.83″E 92°0′27.83″E 92°0′27.83″E
Solanum melongena Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum melongena Solanum lycopersicum Solanum melongena Solanum melongena Solanum lycopersicum Solanum melongena Solanum lycopersicum Solanum melongena Bt Solanum melongena Solanum tuberosum Solanum tuberosum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Solanum lycopersicum Dahlia pinnata Solanum melongena Solanum melongena Gossypium hirsutum Gossypium hirsutum Ipomoea batatas Ipomoea batatas Solanum melongena
160825 160825 160825 160825 160525 160721 160721 160721 170204 170204 170204 170205 170205 170205 170205 170205 170117 170117 170117 170117 170203 170203 170203 170203 170203 170202 170202 170202
Asia 1 Asia 1 Asia II 1 Asia 1 Asia II 5 Asia II 5 Asia II 5 Asia II 1 Asia II 5 Asia II 5 Asia II 1 Asia 1 Asia 1 Asia II 1 Asia II 5 Asia II 5 Asia 1 Asia 1 Asia 1 Asia II 5 Asia 1 Asia II 1 Asia 1 Asia 1 Asia II 1 Asia 1 Asia 1 Asia 1
MF497036 MF497037 MF497063 MF497038 MF497073 MF497074 MF497075 MF497065 MF497077 MF497078 MF497066 MF497034 MF497035 MF497062 MF497070 MF497071 MF497046 MF497047 MF497048 MF497072 MF497029 MF497055 MF497030 MF497031 MF497056 MF497053 MF497044 MF497054
*The full names are as follow: Bangladesh Agriculture Research Institute (BARI), Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Sher-e-Bangla Agricultural University (SAU).
Table 2 Summary of the pairwise comparison of COI nucleotide sequences among four cryptic species of Bemisia tabaci identified in Bangladesh. The full data is listed in Table S1 of Supplementary data. Asia II 10 is only one sequence obtained. Asia II 1
Asia II 5
0.12-0.49
Asia II 1
15.0-15.80
0.12-1.11
Asia II 5
17.10-18.10
10.40-11.30
0.12-0.99
Asia II 10
14.9-15.40
13.8-14.10
13.6-13.90
SC R
Asia 1
Asia II 10
IP T
Asia 1
-
Asia II 1 20 18 10 8 12 0.916 0.00396
A
M
ED
Number of sequences Number of polymorphic sites (S) Singleton variable sites (Svs) Parsimony informative sites (Pis) Number of haplotypes Haplotype diversity (Hd) Nucleotide diversity (Pi)
Asia I 67 34 33 1 28 0.664 0.00135
N
U
Table 3 Comparison of the genetic structure of COI sequences among four cryptic species of Bemisia tabaci identified in Bangladesh. Asia II 5 22 15 8 7 13 0.896 0.00424
Asia II 10 1 0 0 0 1 0 0
A
CC E
PT
Table 4 Genetic variation partitioning of Bemisia tabaci in Bangladesh at different hierarchical levels. Source of variation d.f. Sum of Variance Percentage of Fixation squares components variation indices (Fstatistics) Among groups 13 1482.73 10.44va 27.79 Fct = 0.278 (=regions) Among populations 40 1318.19 5.68vb 15.11 Fsc = 0.209 within groups Within populations 56 1200.91 21.44vc 47.10 Fst = 0.429 (=individuals) Total 109 4001.84 37.56 Highly significant at P< 0.01 level
29
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Table 5 The number of samples, number of haplotypes, Tajima’s D and Fu’s Fs of Bemisia tabaci from different districts of Bangladesh. District No. of samples No. of Tajima’s D Fu’s Fs haplotypes Dhaka 6 2 1.5455 13.695 Gazipur 14 10 0.6717 5.597 Thakurgaon 6 6 2.3335 1.427 Dinajpur 8 5 -1.8951 5.433 Nilphamari 10 7 -1.8733 4.035 Rajshahi 10 9 0.3912 1.824 Khustia 13 4 1.950 22.126 Patuakhali 5 4 -1.0767 3.745 Bandarban 10 6 0.1613 6.945 Chittagang 12 12 1.9258 0.277 Khagrachori 6 5 1.5470 3.318 Coxsbazar 10 5 -1.8391 1.430
30