Genetic Diversity and DNA Barcoding of Thrips in Bangladesh

Simple Summary Thrips, the notorious sap-sucking insects, serve as the vector of plant viruses. Their accurate species identification is essential for determining the vector species and implementing successful pest management techniques. Mitochondrial COI (DNA barcode) sequence variation has proven to be effective in identifying species in many insect pest groups. In this study, we identified 19 thrips species that were found on different host plants in Bangladesh. Among the 19 species, we identified four prominent vector species (Frankliniella intonsa, Thrips tabaci, Scirtothrips dorsalis and T. palmi) and one significant pollinator, Microcephalothrips abdominalis. The findings presented here emphasize the importance of conducting DNA barcoding, analyzing population structure, and assessing the genetic diversity of thrips species in this region. This research is crucial for comprehending their host preferences, potential for adaptation, and genetic variation at both local and regional levels. Accurate identification of pests and invasive species is essential for implementing effective control and quarantine measures. Misidentifications can lead to the application of ineffective control strategies. Abstract Thrips are economically important pests, and some species transmit plant viruses that are widely distributed and can damage vegetables and cash crops. Although few studies on thrips species have been conducted in Bangladesh, the variation and genetic diversity of thrips species remain unknown. In this study, we collected thrips samples from 16 geographical locations throughout the country and determined the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene in 207 thrips individuals. Phylogenetic analysis revealed ten genera (Thrips, Haplothrips, Megalothrips, Scirtothrips, Frankliniella, Dendrothripoides, Astrothrips, Microcephalothrips, Ayyaria, and Bathrips) and 19 species of thrips to inhabit Bangladesh. Among these, ten species had not been previously reported in Bangladesh. Intraspecific genetic variation was diverse for each species. Notably, Thrips palmi was the most genetically diverse species, containing 14 haplotypes. The Mantel test revealed no correlation between genetic and geographical distances. This study revealed that thrips species are expanding their host ranges and geographical distributions, which provides valuable insights into monitoring the diversity of and control strategies for these pests.


Introduction
Thrips are serious sap-sucking insect pests and vectors in many economically important crops worldwide [1,2].Many species of thrips are polyphagous, launching assaults both on perennial and annual crops, thereby enhancing their survival and propagation capabilities [3].Thrips can drastically undermine crop productivity and nutritional security by either directly feeding on crops or indirectly transmitting pathogenic plant viruses.Orthotospoviruses are transmitted and spread in nature by vector insects belonging to the family Thripidae (Thysanoptera), genus Frankliniella, and Thrips.Fourteen species of thrips (Thripidae) are vectors of orthotospoviruses, and nearly 1% are pests of agricultural crops [4].Thrips are the insect vectors of orthotospoviruses (genus Orthotospovirus and family Tospoviridae, and order Bunyavirales), which affect cultivated and wild plants in several unrelated plant families worldwide [5,6].Among the known species of thrips (Thripidae); Ceratothripoides claratris, Frankliniella bispinosa, F. cephalica, F. fusca, F. gemina, F. intonsa, F. occidentalis, F. schultzei, F. zucchini, Thrips flavus, T. palmi, T. setosus, T. tabaci, Scirtothrips dorsalis, and Dictyothrips betae have been reported as vectors of plant pathogens [1,[7][8][9][10][11][12].Orthotospoviruses, including the tomato spotted wilt virus (TSWV, Orthotospovirus tomatomaculae), have become significant contributors to global economic losses in food and ornamental crops, with losses potentially reaching up to 100% [9,13,14].At present, TSWV has an infection rate of 50-90% and is considered one of the top ten most economically detrimental plant viruses.It causes annual worldwide losses exceeding one billion dollars [15].
Identifying thrips at the species level is challenging because of several factors.These include their minuscule size, slight morphological differences, intraspecific polymorphisms, and sexual dimorphisms.This situation is further complicated by the presence of multiple species on the same host plant, significant intraspecific variations within thrips populations, and the requirement for specialized taxonomic knowledge [16][17][18][19].Molecular identification systems are attractive because thrips are difficult to identify morphologically [20].The molecular identification of thrips offers substantial benefits over morphology-based analysis.It effectively addresses the complexities arising from morphological variations across different life stages and the subtle morphological differences between species [16,17].DNA barcodes have been providing accurate and rapid species identification since their inception.Barcode data on economically significant taxa, like Thysanoptera, can establish a common platform for diverse biological studies.These studies can encompass taxonomy, ecology, behavior, life history, pest management, and vector-virus relationships.Moreover, DNA barcode data can enhance taxonomic research by uncovering cryptic species, resolving species complexes, and assisting in the identification of new species [21,22].The DNA barcoding technique provides molecular identification of thrips species [3,[23][24][25], sex and polymorphisms [26], and the ability to discriminate between cryptic species [27,28], biotypes [29], haplotypes [30], and host and geographical genetic differences [6,17].The mitochondrial cytochrome c oxidase subunit I gene (mtCOI) is the "DNA barcode", showing 2% sequence divergence within species and >2% between species [31,32].In addition, DNA barcoding can be used in insect pest management programs to determine the selection and timing of management practices based on polymorphisms and host adaptations [17,28].
According to previous studies, different genetic lineages and populations have varying abilities to transmit plant viruses [33][34][35][36][37][38], host plant preferences [35,39,40], and insecticide resistance [41].In the natural environment, ecological and evolutionary dynamics are primarily influenced by two key factors: genetic diversity and population structure [42].To ensure biodiversity conservation, all aspects of genetic diversity, including those of wild animals, insects, and agricultural crops, must be considered.Several parameters can be used to assess the degree to which an animal has a high level of genetic diversity efficiency and the subdivision of its population, such as the expected heterozygous number (He), observed heterozygosity (Ho), nucleic acid diversity (Pi), and polymorphism information content (PIC) [43].
In Bangladesh, it is common to grow vegetables, pulses, and flowers in both commercial fields and backyard gardens.However, crop production is severely hampered by insect infestation, which is one of the main causes of reduced yields [44].Among the various insect pests that attack these crops, thrips are especially problematic.Thrips are widely distributed throughout Bangladesh and cause damage to many crops, such as onions, brinjals, ornamental flowers, chili, cotton, sweet potato, pumpkin, rose, marigold, gourds, beans, and tomatoes.Thrips can also transmit viral diseases to crops and cause significant yield losses.According to a survey conducted by the Asian Vegetable Research and Development Center in Asia, aphids, moths, and thrips are the most common vegetable pests.Particularly, during dry weather conditions, thrips exhibit heightened multiplication rates, contributing to yield losses ranging from approximately 50% to 90% [45].In Bangladesh, production per unit area is relatively low due to insect pests, which generally cause 30-40% losses and sometimes 100% losses if no control measures are taken.Nevertheless, it varied from place to place and over time.In Bangladesh, farmers use synthetic insecticides extensively to combat thrips, spraying them more than once a week throughout the growing season.As a result of the indiscriminate use of pesticides, natural enemies are destroyed, beneficial arthropods are harmed, and eventually, they become resistant to those pesticides [46,47].
Few studies were done by other researchers [48] using Bangladeshi thrips samples from only bean host plants, but the genetic structure and variation of thrips populations in different host plants in Bangladesh remain unclear.In Bangladesh, the taxonomy and molecular identification of thrips species are not clearly defined; therefore, we conducted a study to identify thrips species in various host plants and gain some preliminary insight into their genetic and geographic diversity and their potential damage to crops.The objective of this study was to investigate the genetic variation and DNA barcoding of thrips species inhabiting various hosts across different regions in Bangladesh, utilizing mitochondrial DNA.The necessity to survey the DNA barcode, population structure, and genetic diversity of thrips species in this region stems from the need to comprehend host preferences, potential for adaptation, and genetic variation on both local and regional scales.This understanding is crucial for limiting the development and execution of new control strategies.

Thrips Collection
Thrips (adults and nymphs) were collected from various host plants (bean, garlic, rose, marigold, lemon, pumpkin, cucumber, mustard, brinjal, ash gourds, bitter gourd, sweet potato, ornamental flowers, chili, cotton, tomato, etc.) (Table 1) across 16 regions of Bangladesh from 2021 to 2023.Nymphs and adults were gathered on white paper utilizing a plant beating technique and subsequently preserved in separate vials containing 80% alcohol, categorized based on their respective locations and host plants.These vials were then stored at −20 • C pending further analysis (refer to Table S1).An estimated 20-150 thrips were collected from each site, with a minimum of three sites surveyed per crop field.

DNA Extraction
Genomic DNA from individual thrips was extracted using the PureLink TM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA).Each sample was placed into a 1.5 mL centrifuge tube containing 180 µL of digestion buffer and 20 µL of proteinase K (50 µg/mL), and then incubated at 55 • C for 4 hours.Following the manufacturer's guidelines, DNA samples were extracted.The DNA concentration was measured using a NanoPhotometer TM (Implen GmbH, Schatzbogen, Germany).

PCR Amplification
mtCOI DNA was amplified using the primer pair HCO-2198 and LCO-1490 [49].The PCR process was conducted with a total reaction volume of 20 µL, which included 10 µL SmartGene 2× Dye Mixed Taq (SmartGENE, Daejeon, Republic of Korea), 1 µL of each primer (10 pmol/µL), 5 µL of nuclease-free water and 3 µL of template DNA solution (40 ng).The reaction mixtures underwent amplification with the following parameters: an initial denaturation at 94 • C for 5 min; this was 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.This process was carried out in a T100 Thermal Cycler from Bio-Rad (Hercules, CA, USA).The PCR products were electrophoresed on a 1% agarose gel, which was then purified using Expin TM Gel SV (GeneAll, Seoul, Republic of Korea).The PCR amplicons were subcloned into a cloning vector (the pGEM-T Easy vector, Promega, Madison, WI, USA) and sequenced using Sanger sequencing (Macrogen, Seoul, Republic of Korea).

DNA Sequence and Barcoding Analysis
The GenBank database of the NCBI was searched using BLAST [50], and nucleotide sequence alignment was performed using CLUSTAL X1.83 [51].The mtCOI sequences identified from Bangladeshi thrips samples were submitted to GenBank.The mean genetic distance among the samples was determined using MEGA 11.0 [52,53].DNA barcoding [31] determines the genetic distance between samples based on the K2P distance [54] to reveal barcode gaps or breaks in the distribution of genetic distances between samples belonging to the same or different species [31,54].The DNA barcode is derived from the sequence of the mtCOI gene.This sequence typically exhibits a 2% divergence between different species [31,32].

Phylogenetic Analysis
mtCOI sequences were manually edited at 655 bp for each thrips sample using CLC Genomics Workbench software (QIAGEN, Hilden, Germany).Multiple sequence alignments were performed using Clustal X 1.83 [51].A phylogenetic tree was constructed using the Interactive Tree Of Life (iTOL v.6) software [55].To test the robustness of the phylogeny, 1000 bootstrap replicates were used [56].The p-distances were estimated using MEGA 11.0 (Table 2).

Population Structure and Genetic Analysis
The determination of genetic diversity parameters was carried out using DnaSP 5.12.01.This analysis included the determination of segregation sites, haplotypes, haplotype diversity, and nucleotide diversity.These parameters encompassed sequence polymorphisms, divergence, gene flow, neutrality tests (including Fu's Fs and Tajima's D), genetic differentiation, and pairwise Fst values [57,58].The Mantel test was employed to ascertain the correlation between genetic distance (Fst) and geographic distance [59].The geographic distances between the populations studied were based on the central location of the collection sites in Bangladesh.The sequences were examined using a minimum spanning network relationship among thrips species to create a haplotype network in PopART 1.7 [60].The analysis of molecular variance (AMOVA) was substantiated with 1023 permutations [61].AMOVA analysis and F-statistics of genetic variation for thrips populations in Bangladesh were derived using the pairwise distance method in Arlequin software version 3.5 [62].The total molecular variance was categorized into 'inter-group' genetic variation (Fct), 'intra-group' genetic variation (Fsc), and 'inter-population' (Fst).

Barcode Divergence of Thrips Species
In total, 207 mtCOI sequences representing 19 species were analyzed in the curre study.The analysis revealed that the overall K2P/p-distance mean genetic distance of t dataset was 0.1719/0.1463.The intraspecific and interspecific distances (K2P/p-distanc showed substantial variation, ranging from 2.81/2.57%-47.51/35.18%,respectively (Tabl 2 and S2).Intraspecies distance could not be determined for four species (T.subnudula, M abdominalis, A. tumiceps, and B. melanicornis) due to a single representative.

Genetic Diversity and Gene Flow Analysis
Variation in mtCOI gene structure was analyzed for 15 thrips species, excludin Thrips subnudula, Microcephalothrips abdominalis, Astrothrips tumiceps, and Bathrips mela icornis because only one sample was available.Genetic distances (Fst values) between species ranged from 0 to 1.0 (Table 3).The number of segregation sites and haplotyp varied among different species, with T. palmi showing the highest diversity (14 haplotyp from 97 sequences).Other species exhibited fewer haplotypes, with some showing on one (Table 2).A population genetics study was conducted for this species.Tajima's D st tistical analysis revealed negative values in five species (T.palmi, T. parvispinus, T. h waiiensis, F. intonsa, and S. dorsalis), and Fu's Fs statistics for T. palmi and F. intonsa show negative values with significant differences (Table 2).Negative Tajima's D values, whi represent an excess of low-frequency polymorphisms relative to expectation, are typical interpreted as demographic estimates of population growth or selection.Similarly, neg tive values of Fu's Fs indicate an excess number of alleles.Geographic analysis showed that T. palmi, T. hawaiiensis, and M. usitatus were widely distributed throughout the country, whereas T. parvispinus, S. dorsalis, F. intonsa, A. chaetophora, D. innoxious, B. melanicornis and T. subnudula were found only in the northern part of Bangladesh.T. florum is distributed in the central and northern regions of Bangladesh, whereas Haplothrips sp., A. tumiceps, and T. tabaci are distributed in the central regions.H. andresi, Phlaeothripidae sp.M. abdominalis was found in Khulna and Khagrachori, located in southern Bangladesh (Figure 1).

Barcode Divergence of Thrips Species
In total, 207 mtCOI sequences representing 19 species were analyzed in the current study.The analysis revealed that the overall K2P/p-distance mean genetic distance of the dataset was 0.1719/0.1463.The intraspecific and interspecific distances (K2P/pdistance) showed substantial variation, ranging from 2.81/2.57%-47.51/35.18%,respectively (Tables 2 and S2).Intraspecies distance could not be determined for four species (T.subnudula, M. abdominalis, A. tumiceps, and B. melanicornis) due to a single representative.

Genetic Diversity and Gene Flow Analysis
Variation in mtCOI gene structure was analyzed for 15 thrips species, excluding Thrips subnudula, Microcephalothrips abdominalis, Astrothrips tumiceps, and Bathrips melanicornis because only one sample was available.Genetic distances (Fst values) between all species ranged from 0 to 1.0 (Table 3).The number of segregation sites and haplotypes varied among different species, with T. palmi showing the highest diversity (14 haplotypes from 97 sequences).Other species exhibited fewer haplotypes, with some showing only one (Table 2).A population genetics study was conducted for this species.Tajima's D statistical analysis revealed negative values in five species (T.palmi, T. parvispinus, T. hawaiiensis, F. intonsa, and S. dorsalis), and Fu's Fs statistics for T. palmi and F. intonsa showed negative values with significant differences (Table 2).Negative Tajima's D values, which represent an excess of low-frequency polymorphisms relative to expectation, are typically interpreted as demographic estimates of population growth or selection.Similarly, negative values of Fu's Fs indicate an excess number of alleles.
The Mantel test revealed no correlation between genetic distance and geographic distance (r = −0.105,p = 0.101) (Figure 3).The minimum spanning network analysis was used to determine the evolutionary relationships among the haplotypes of each species.Haplotype networks derived from mtDNA sequences revealed a close relationship between all haplotypes (Figure 4).Nineteen thrips species were categorized into 54 haplotypes and were highly distant from each other by many mutational steps.The haplotype distribution reveals a clear graphical pattern encompassing 19 thrips species, each genetically unique from the others (Figure 4).Among them, T. palmi exhibits the highest diversity.Notably, Hap1 holds a central position in the network and is linked to 13 low-frequency singleton haplotypes.Hap 6, Hap 12, and Hap 14 of T. palmi represented two and three individual haplotypes, respectively.Megalurothrips usitatus and T. parvispinus have three dominantly represented haplotypes.Thrips hawaiiensis, F. intonsa, S. dorsalis, T. tabaci, T. florum, A. chaetophora, D. innoxious, and H. bagrolis have a single dominant haplotype with closely related 1-3 singletons.Other species showed a linear form of haplotypes (Figure 4).The minimum spanning network of haplotypes showed a similar pattern to the phylogenetic tree of the 19 thrips species.Hierarchical AMOVA indicated that most of the genetic variation (Fst = 97.7%)occurred within the population based on geographic distance (Table 4).Genetic variation among the groups was also high (68.31).The genetic variation was distributed as follows: 69.9% among groups, 0.85% among populations within groups, and 2.25% among populations.

Dendrothripoides innoxius
Thrips 19 thrips species.Hierarchical AMOVA indicated that most of the genetic variation (Fst = 97.7%)occurred within the population based on geographic distance (Table 4).Genetic variation among the groups was also high (68.31).The genetic variation was distributed as follows: 69.9% among groups, 0.85% among populations within groups, and 2.25% among populations..   19 thrips species.Hierarchical AMOVA indicated that most of the genetic variation (Fst = 97.7%)occurred within the population based on geographic distance (Table 4).Genetic variation among the groups was also high (68.31).The genetic variation was distributed as follows: 69.9% among groups, 0.85% among populations within groups, and 2.25% among populations..

Discussion
In this study, we examined the genetic diversity and performed DNA barcoding of thrips species in Bangladesh.This study provides the first DNA barcoding data to identify thrips species in Bangladesh.Additionally, this study contributed mtCOI sequences to the GenBank database and identified 19 species (Figure 2).We found that T. palmi was the dominant species, while T. florum was identified in only a few places.T. palmi and T. hawaiiensis distribution and population abundance were higher around the country, whereas only one or two individuals represented the other thrips species.For instance, A. tumiceps from Gazipur (Central), A. chaetophora from Rajshahi (West), B. melanicornis, D. innoxious and T. subnudula from Dinajpur (North), H. bagrolis from Rangpur (North), T. tabaci and Haplothrips sp. from Brahmanbaria (East), and H. andresi, M. abdominalis from Khulna (South) and Phlaeothripidae sp. from Khagrachori (Southeast).T. Parvispinus, M. distalis, S. dorsalis, and F. intonsa were found only in the northern regions of Bangladesh, where most of the thrips species were found (11 species).M. usitatus was abundant and widely distributed on the northern and southern sides.Most of the species included in this study are pests of various agricultural and horticultural crops.
An extensive survey of thrips species in Bangladesh showed that the pairwise mtCOI nucleotide sequence variation reached 35%, and 19 species were identified in samples collected from 16 different locations from 21 host plants during 2021-2023.Each species was genetically distinct.In our study, DNA sequencing of molecular studies has been conducted, and the results indicate clear genetic differences between the identified species (Figure 2 and Table 2).Species of thrips belonging to two suborders (Terebrantia and Tubulifera) and two families (Thripidae and Phlaeothripidae) were identified (Table 1).Most species were polyphagous, and only one was a pollinator [63].Among the 19 species, Dendrothripoides innoxius was found in only one host plant from one location (Table 1).Similarly, Reyes [64] listed five species within the genus Dendrothripoides: one species native to South Africa, one to Thailand, two found in the Philippines, and one species commonly found on sweet potato plants worldwide.It is noteworthy that T. palmi was found to be more abundant than the other species.However, F. occidentalis, a more common vector species of TSWV, was not detected in this study.This species is absent in Bangladesh, even though it has invaded neighboring countries.In 2015, F. occidentalis was reported for the first time in India based on specimens collected from tomatoes [65].Douglas [48] reported that M. usitatus, M. distalis, T. palmi, T. hawaiiensis, F. intonsa, Phlaeothripidae sp., and Haplothrips sp. were present only in bean host plants in Bangladesh.The BOLD database showed three species; Azaleothrips (GMBCA5994, GMBCM2508), Plicothrips apicalis (GMBCD2954), and Cephalothrips (GMBCD2987) reported from southern Bangladesh that we did not find in our study.In this study, we found most thrips species on multiple hosts, including commercial and experimental fields in Bangladesh.
T. palmi, also known as the Melon thrips, is a common insect pest that affects vegetables and ornamentals [66].There has been an increase in the prevalence of melon thrips in Southeast Asia, throughout most of the rest of Asia, Australia, North Africa, Central and South America, and the Caribbean [66,67].The sequence divergence range of T. palmi was 0.2-6.8(Table S3).The maximum K2P distance observed was 19.9% for T. palmi, 10.4% for T. tabaci [24].Rebijith et al. [25] reported that 12.3% and 13.8% barcode divergence was found in T. palmi and T. tabaci from Indian populations.Furthermore, distance analysis revealed a maximum divergence of 13% in T. palmi and 12% in T. tabaci from Pakistan.Barcode gap analysis showed the maximum intraspecies distances (>2%) of A. intermedius, H. reuteri, T. palmi and T. tabaci [23].A high maximum intraspecific distance indicates cryptic species within T. palmi populations [66].Populations of T. palmi in various regions of Bangladesh exhibited 14 haplotypes.The genetic database of T. palmi has been expanded through numerous recent studies.A DNA polymorphism analysis of T. palmi populations worldwide uncovered 29 haplotypes.Phylogenetic analysis suggests that the T. palmi population can be categorized into three distinct lineages.
T. parvispinus (Karny) is a serious pest affecting several agricultural and horticultural crops in southeast Asia.In our study, T. parvispinus was found to be distributed on the northern side of Bangladesh in different host plants.Mound and Collins [68] reported that T. parvispinus, a member of the T. orientalis group, is present in Thailand and Australia.Our haplotype analysis showed that T. parvispinus has four haplotypes.In contrast, the Indian and Indonesian populations have two and three haplotypes, respectively [69].In the case of T. hawaiiensis, it is widely distributed on various host plants in Bangladesh with four haplotypes.The thrips species T. hawaiiensis is a global pest of various plants, including the lookalike T. florum.Some of the damage attributed to T. hawaiiensis may actually be caused by T. florum [70].The interspecific distance between T. hawaiiensis and T. florum was 15.0-15.5, and the genetic distance was 0.9847 in this study (Table 3).
Through the use of mtCOI molecular identification, we were able to identify two primary species of thrips in seven host plants: M. usitatus (14%) and M. distalis (1%).This forms the basis for future studies on the potential damage caused by these pests.The genetic distance between these two species is 0.9621 (Refer to Table 3).Thrips species that belong to the Megalurothrips genus are known to breed on Fabaceae plants.Both M. usitatus and M. distalis have a wide distribution across tropical Asia [71][72][73].The distance analysis showed that the maximum divergence between these two species was <2% and six haplotypes clusters for M. usitatus and one haplotype for M. distalis.
The occurrence of other thrips species, B. melanicornis, T. tabaci, A. chaetophora, T. subnudula, Haplothrips sp., Phlaeothripidae sp., S. dorsalis, F. intonsa, and A. tumiceps are also concerning in Bangladesh.The barcode gap showed that the lowest intraspecies distance was found in Haplothrips sp., Phlaeothripidae sp., and H. andresi (Table 2).In this study, we identified M. abdominalis in marigolds.This species has been reported as a composite thrips and has been documented as an important pollinator of various asteracean plants [74,75].However, damage to the petals and collars of Asteraceae is caused by heavy infestations of M. abdominalis.The pigmentation of the petals is lost, and seed development is hindered.They are known vectors of the tobacco streak virus in Parthenium weed (Parthenium hysterophorus) [76,77], blue mink (Ageratum houstanianum), and tobacco [78].
Kadirvel et al. [24] reported that S. dorsalis and T. palmi showed the highest intraspecific genetic variation, followed by T. tabaci and F. occidentalis.This suggests that the mtCOI gene could be a useful tool for categorizing different species and genera of thrips that coexist in specific crop environments.Our study found similar results, with the highest intraspecific genetic variation in T. parvispinus and S. dorsalis, followed by T. tabaci and H. bagrolis, using the mtCOI gene (Table 2).Species delimitation is often determined by the gap between the maximum intraspecific and minimum interspecific distances in various animal groups [79,80].This method has been used to identify a complex group of snails using barcode gap analysis and several other species delimitation methods [81].
Our research revealed significant genetic differentiation among 19 thrips species in Bangladesh.However, the Mantel test results showed no significant correlation between genetic and geographic distances in thrips populations.Despite their weak flight capacity, thrips exhibit high gene flow between locations with long geographical distances, likely facilitated by human activity [82,83].Li et al. [84] found that haplotype 2 was present in all populations of T. tabaci, suggesting that the dominance of haplotype 2 could be linked to the presence of insecticide-resistance genes.In our study, haplotype 1 was dominant in T. plami populations.Further research is needed to investigate the potential relationship between insecticide resistance and the population genetics of different species.
According to the present study, the presence of haplotypes referred to among the nineteen species showed a high degree of genetic variation.The COI barcoding-based identification gives huge advantages in that it is not constrained by developmental stages, nor the physical integrity of the samples collected.To better understand thrips population dynamics, we must understand the biology of thrips populations and determine their abundance and distribution over time, including the variation of economically important traits such as vector competency within and between populations.

Conclusions
Nineteen species were identified from the host plants sampled in this study.Notably, we identified ten thrips species that have not been previously reported in Bangladesh: Thrips parvispinus, Bathrips melanicornis, Microcephalothrips abdominalis, Thrips florum, Ayyaria chaetophora, Thrips subnudula, Dendrothripoides innoxius, Astrothrips tumiceps, Scirtothrips dorsalis, and Thrips tabaci.All species were polyphagous except Microcephalothrips abdominalis, an important pollinator of asteracean plants.We did not find F. occidentalis in our samples.This study contributes to a comprehensive understanding of genetic diversity and the distribution of thrips species in Bangladesh.DNA barcodes can be used to identify thrips species complexes and their genetic lineages.The consistent findings from various analyses, including DNA barcode, phylogenetic, and haplotype analyses, affirm the presence of 19 distinct thrips species within our dataset of 207 samples, providing robust support for the observed genetic differences reflecting genuine distinctions between species.In the future, the information generated here could be useful for monitoring changes in the diversity, abundance, and displacement of thrips populations.In addition, this is the first study about genetic diversity, DNA barcoding, and geographic distribution of different host plants in Bangladesh.Moreover, further research is necessary to fully comprehend the precise roles that these thrips populations play in the transmission of various orthotospoviruses and to develop effective management strategies.

18 Figure 1 .
Figure 1.Sample collection sites and distribution of thrips species in Bangladesh.The colored symbols represent nineteen species.Geographic analysis showed that T. palmi, T. hawaiiensis, and M. usitatus were widely distributed throughout the country, whereas T. parvispinus, S. dorsalis, F. intonsa, A. chaetophora, D. innoxious, B. melanicornis and T. subnudula were found only in the northern part of Bangladesh.T. florum is distributed in the central and northern regions of Bangladesh, whereas Haplothrips sp., A. tumiceps, and T. tabaci are distributed in the central regions.H.

Figure 1 .Figure 2 .
Figure 1.Sample collection sites and distribution of thrips species in Bangladesh.The colored symbols represent nineteen species.

Figure 2 .
Figure 2. Phylogenetic tree based on mtCO1 sequences of thrips species in Bangladesh.The tree was generated based on the mtCOI sequences from the collected samples, and the related reference sequences were searched from the GenBank database in this study using Interactive Tree Of Life (iTOL) software.Rhopalosiphum padi (MN320354) was used as an outgroup.

Figure 3 .
Figure 3.The relationship between the genetic distance (Fst) and geographic distance (km) of all samples of thrips species in Bangladesh.Each blue point represents population pairwise comparison Fst/(1-Fst) and histogram (Mantel test) shows the sampling distribution and orange arrow indicated the location of the observed correlation.The p-value was calculated using the r(AB) distribution estimated from 10,000 permutations.

Figure 4 .
Figure 4.The evolutionary relationships of the identified haplotypes of thrips species, collected from 16 locations in Bangladesh, are depicted through minimum spanning networks.The areas of the circles are proportional to the frequency of each haplotype in the dataset.The color-coding represents the genetic groups of each haplotype.

Figure 3 .
Figure 3.The relationship between the genetic distance (Fst) and geographic distance (km) of all samples of thrips species in Bangladesh.Each blue point represents population pairwise comparison Fst/(1-Fst) and histogram (Mantel test) shows the sampling distribution and orange arrow indicated the location of the observed correlation.The p-value was calculated using the r(AB) distribution estimated from 10,000 permutations.

Figure 3 .
Figure 3.The relationship between the genetic distance (Fst) and geographic distance (km) of all samples of thrips species in Bangladesh.Each blue point represents population pairwise comparison Fst/(1-Fst) and histogram (Mantel test) shows the sampling distribution and orange arrow indicated the location of the observed correlation.The p-value was calculated using the r(AB) distribution estimated from 10,000 permutations.

Figure 4 .
Figure 4.The evolutionary relationships of the identified haplotypes of thrips species, collected from 16 locations in Bangladesh, are depicted through minimum spanning networks.The areas of the circles are proportional to the frequency of each haplotype in the dataset.The color-coding represents the genetic groups of each haplotype.

Figure 4 .
Figure 4.The evolutionary relationships of the identified haplotypes of thrips species, collected from 16 locations in Bangladesh, are depicted through minimum spanning networks.The areas of the circles are proportional to the frequency of each haplotype in the dataset.The color-coding represents the genetic groups of each haplotype.

Table 1 .
Composition and status of all thrips species collected from 16 regions of Bangladesh on the different host plants during2021-2023.

Table 2 .
Genetic diversity and intraspecific distance of thrips species based on mtCOI gene.

Table 3 .
Pairwise genetic distance (Fst) among the thrips species from Bangladesh.

Table 4 .
Analysis of molecular variance (AMOVA) of thrips in Bangladesh at different hierarchical levels.