The modification DNA isolation technique demonstrated considerable success in extracting high-quality DNA from thrips specimens collected in Türkiye. Furthermore, the DNA isolated through this method facilitated successful PCR amplification, underscoring its compatibility with subsequent molecular analyses. This method was pivotal in acquiring dependable genetic material essential for species-specific identification via DNA barcoding methods, thereby illustrating its significant potential to advance molecular research within the realm of thrips entomology.
In this study, two prevalent methodologies were applied to analyze DNA barcoding results. The first method involved leveraging BLAST (Basic Local Alignment Search Tool) for comparing the query sequences against a reference database to identify the closest matches. The second approach utilized taxonomic assignment algorithms, specifically MEGA, to assign species identities and to construct phylogenetic trees based on the alignments of sequences. Both methods were employed for the analysis of the query sequences and those pertaining to thrips. The COI analysis yielded positive results through both methodologies, suggesting that this gene region is suitable for the identification of thrips species.
The study successfully identified 1 species from the Aeolothripidae family (Rhipidothrips gratiosus Uzel, 1895), eight species from the Thripidae family (Neohydatothrips gracilicornis (Williams 1916), two samples of Sitothrips arabicus Priesner 1931, Anaphothrips obscurus (Müller 1776), Kakothrips priesneri Pelikan 1965, two samples of Pezothrips nigriventris (Pelikan 1956), and Stenothrips graminum Uzel 1895, Chirothrips aculeatus (Bagnall 1927), Chirothrips kurdistanus zur Strassen 1967) and three species from the Phlaeothripidae family (Haplothrips andresi Priesner 1931; Haplothrips distinguendus (Uzel 1895); Haplothrips reuteri (Karny 1907)) collected individually. This diverse collection of thrips specimens underlines the efficacy of the employed DNA barcoding methods in accurately identifying species within the order Thysanoptera, contributing valuable insights to the field of entomology.
Neohydatothrips gracilicornis (Williams 1916) (Thysanoptera: Thripidae) is characterized by a simple dark brown body, featuring microtrichia between the two discal campaniform sensilla and along the anterior margin of abdominal tergites II-IV. A complete microtrichia comb is present on the posterior margin of tergite VII, as detailed by zur Strassen (2003). Sitothrips arabicus Priesner 1931 (Thysanoptera: Thripidae) exhibits a slender and elongated body shape, with a distinct ventrad-directed tooth on the apical edge of the first segment of the front tarsi, as described by zur Strassen (2003).
Species within the Anaphothrips genus typically have five vein setae on the fore wing clavus, provided they are macropterous. Anaphothrips obscurus (Müller 1776) (Thysanoptera: Thripidae), one of the 86 species in this genus, possesses an 8-segmented antenna, which appears to be 9-segmented, according to zur Strassen (2003). Kakothrips priesneri Pelikan 1965 (Thysanoptera: Thripidae) is distinguished by the presence of a small or large tooth on the fore tarsi and sternite VII S1 setae notably distanced from the posterior margin, as noted by zur Strassen (2003).
Pezothrips nigriventris (Pelikan 1956) (Thysanoptera: Thripidae), a species within the Pezothrips genus, features a head with dorsally long interocellular setae and two dorsal setae closely positioned at antennal segment I, as described by zur Strassen (2003). Stenothrips graminum Uzel 1895 (Thysanoptera: Thripidae) possesses a slim, elongated body and abdominal tergite VIII with a long, slightly curved linear ctenidium. Its microtrichia comb, partly arising individually and partly in twos or threes from a triangular basal platelet, is complete, as detailed by zur Strassen (2003). Contrary to zur Strassen's (2003) suggestions for the habitat of Sitothrips arabicus Priesner, 1931 (Thysanoptera: Thripidae), this study found it in wheat (Triticum), indicating a potential variance in host plant preference or distribution.
Rhipidothrips gratiosus Uzel 1895 (Thysanoptera: Aeolothripidae) is distinguished by having longer postero-angular setae along the posterior margin of the pronotum. Additionally, the setae on the anterior margin of the forewing are not notably short. Its antennae segments III and IV feature a unique characteristic, where, ventrally, apart from the apical sensorium that encircles the segment akin to a shield, there is at least one other rounded sensory field. This morphological detail, as described by zur Strassen (2003), sets R. gratiosus apart within its family, highlighting the intricate diversity found within the Thysanoptera order.
The overall mean distance of 0.25 in this analysis provides an estimate of average evolutionary divergence, reflecting the number of base differences per site, which is obtained by averaging across all pairs of sequences. The dataset being analyzed includes 37 nucleotide sequences. To ensure the integrity and robustness of the results, all ambiguous positions were systematically removed for each sequence pair using the pairwise deletion option. This meticulous approach resulted in a final dataset that includes a total of 242 positions. This dataset serves as a solid foundation for conducting a comprehensive analysis of the average evolutionary divergence across all sequence pairs, offering valuable insights into the genetic variation and evolutionary relationships among the species under study.
The Neighbor-Joining (NJ) phylogenetic tree constructed using Mega 11 software highlighted a clear bifurcation among thrips samples from two different families, showcasing distinct clusters for members of the Phlaeothripidae and Aeolothripidae families in proximity to the outgroup, and separate from the branch of the Thripidae family. Rhipidothrips gratiosus specimens were aligned within the same monophyletic clade as the barcoded sequence (KR141386) of the same species, positioned near Phlaeothripidae members, indicated by pink clade branches in Fig. 1. Phlaeothripidae members themselves formed nine monophyletic clades, with species such as Haplothrips reuteri, H. andresi, H. aculeatus, and H. distinguendus being distinctively positioned.
Haplothrips andresi, which lacks a sequence match in GenBank for the same species, found its place among sequences from the same genus within a distinct branch, receiving strong support from a bootstrap value (100%). Haplothrips distinguendus matched the barcoded sequence (FN545927) of the same species, also supported by a 100% bootstrap value. Haplothrips reuteri aligned with GenBank sequences of the same species (EF468741, KX622242, KP871493) as depicted in the blue clade branches of Fig. 1. Anaphothrips obscurus sequences showed similarity to other An. obscurus sequences from GenBank within the same branches, strongly supported by a 100% bootstrap value across six monophyletic clades, under the green clade branches in Fig. 1.
Neohydatothrips gracilicornis was observed in one monophyletic clade with the GenBank sequence OP462475.1 of the same species (the purple clade branch, Fig. 1). Sitothrips arabicus specimens, obtained from two different individuals, demonstrated clustering within the same monophyletic clade (dark yellow branch, Fig. 1). Pezothrips nigriventris specimens were grouped together in the same monophyletic clade (light blue clade, Fig. 1) alongside Kakothrips genus species. Kakothrips priesneri, along with K. robustus (FN546011) from GenBank, formed a monophyletic clade. Chirothrips aculeatus established the 6th monophyletic clade with a strong bootstrap support (100%), including sequences of Chirothrips meridionalis from GenBank (FN545951, KP845831, KP845787, KP871427, KP871210, HQ991636), thus forming five additional monophyletic clades within the tree.
Chirothrips kurdistanus, demonstrating monophyly with a 28% bootstrap support, was included in the same monophyly as Chirothrips genus members, following St. graminum, which intriguingly clustered with them but without strong bootstrap support (43%). This study represents a pioneering effort in employing both the NJ tree of MEGA and BLAST for DNA barcoding analysis of species including St. graminum, P. nigriventris, K. priesneri, Si. arabicus, C. kurdistanus, C. aculeatus, and H. andresi. This inaugural investigation into the DNA barcoding specific to these species offers novel insights and contributes significantly to the enhancement of the existing knowledge base in thrips taxonomy.
The group mean distance analysis elucidated here zeroes in on the base differences per site, calculated by averaging across all pairs of sequences among distinct groups. This in-depth analysis involved a dataset of 35 nucleotide sequences. To uphold precision in the results, all ambiguous positions were diligently excised for each sequence pair, employing the pairwise deletion strategy. This meticulous approach yielded a refined dataset, featuring 242 positions, as detailed in Table 1. For conducting the evolutionary analyses, MEGA11 (Tamura et al. 2021) was the software of choice. Utilizing this tool enabled an extensive investigation into the genetic variances and affiliations among the sequences, thus enriching the comprehension of evolutionary dynamics within the dataset under study.
Table 1
Estimates of Evolutionary Divergence over Sequence Pairs between Groups.
| Sitothrips | Pezothrips | Anaphothrips | Kakothrips | Haplothrips | Rhipidothrips | Chirothrips | Neohydatothrips |
Sitothrips | | | | | | | | |
Pezothrips | 0.260 | | | | | | | |
Anaphothrips | 0.219 | 0.267 | | | | | | |
Kakothrips | 0.213 | 0.174 | 0.248 | | | | | |
Haplothrips | 0.355 | 0.321 | 0.319 | 0.322 | | | | |
Rhipidothrips | 0.281 | 0.334 | 0.277 | 0.303 | 0.308 | | | |
Chirothrips | 0.214 | 0.199 | 0.235 | 0.210 | 0.349 | 0.277 | | |
Neohydatothrips | 0.243 | 0.250 | 0.205 | 0.204 | 0.346 | 0.282 | 0.220 | |
Pairwise genetic distances among the eight genera were meticulously documented in Table 1, with the average genetic distance spanning from 17.4–34.9%. The findings in Table 1 spotlight the narrowest genetic distance between Kakothrips and Pezothrips at 0.174, juxtaposed with the widest genetic gap observed between Haplothrips and Chirothrips, marked at 0.349. A noteworthy revelation from this analysis is the significant genetic divergence of Haplothrips species, belonging to the Phlaeothripidae family, from those within the Thripidae family. The closest genetic affiliation was observed at 0.308 with Rhipidothrips species of the Aeolothripidae family, underscoring the substantial genetic distinctiveness among these familial groupings.
For the Bayesian phylogenetic tree analysis conducted using the BEAST v1.10.4 software, the default settings were applied for both the nucleotide substitution model and the Clock model. The tree's prior configuration utilized the Yule process for modeling speciation (Gernhard 2008; Yule 1925). Subsequently, the TreeAnnotator v1.10.4 tool was employed, setting the burn-in at 100,000 states, and selecting Maximum Clade Credibility as the desired tree type.
The Bayesian phylogenetic tree generated through this methodology displayed only minor discrepancies in the placement of GenBank species and the species identified in the study within the same clades, compared to the previously constructed NJ tree. Within the Bayesian framework, Rhipidothrips was situated adjacent to the outgroup, akin to its positioning in the NJ tree, and was categorized within the same clade as a sister group alongside Haplothrips species. Similar to the NJ tree, all species were aligned within their respective clades, with the genus Anaphothrips demonstrating monophyly and Chirothrips positioned further inside. Unlike most species of the Thripidae family, Sitothrips exhibited paraphyletic branching relative to other species. Additionally, the genera Pezothrips and Kakothrips were shown to exhibit monophyly under the same node, consistent with their grouping in the NJ phylogenetic tree. This consistency across both Bayesian and NJ phylogenetic analyses underscores the robustness of the phylogenetic relationships inferred, offering insightful perspectives on the evolutionary dynamics within these thrips’ species.
This study represents a groundbreaking endeavor, especially remarkable for its thorough examination of species by simultaneously employing both morphological and molecular barcode systems on individual samples. This dual approach marks a significant leap forward in the field of thrips taxonomy, setting a new benchmark for the integration of morphological and molecular methodologies in species identification. By blending traditional morphological analyses with modern molecular barcode techniques, this research not only enhances the accuracy and efficiency of species identification but also lays the groundwork for future studies in thrips taxonomy and beyond. The innovative use of both methods from the same individual samples offers a holistic view of species characteristics, facilitating a deeper understanding of biodiversity and evolutionary relationships within the Thysanoptera order.