Introduction

Hymenoptera is one of the four mega-diverse insect orders after Coleoptera, Lepidoptera, and Diptera, comprising up to a million undescribed extant species and 153,000 described ones^{1, 2}. The order includes ants, bees, wasps, etc. which are of great worth to all terrestrial ecosystems and of economic importance for humanity^{3, 4}. Bees and some species of wasps are the most effective pollinators of agricultural and wild plants and help sustain our food supplies^{4, 5, 6}. Various wasp species are the predators or parasitoids of crop pests and can be used as a substitute for chemical pesticides have benefits for the environment and are cost-effective^{4, 7, 8, 9}. The importance of ants in the ecosystem is also widespread due to their interactions with different plants, feeding on other insects and small invertebrates, seed dispersal, and seed-predator habit^{10, 11}.

A sharp decline in the population of these insects has been observed globally due to anthropogenic activities like deforestation, urbanisation, and application of pesticides in agricultural fields, leading to various adverse effects like habitat fragmentation, climate change and degradation of the insect’s habitat^{10, 11}. For the maintenance of biodiversity and ecological integrity and considering the spanning human needs and interests of these creatures, their conservation is the need of the hour. And the first step in conservation is the accurate identification of the species. The traditional taxonomic methods used for the identification require the expertise of the field with a thorough knowledge of taxonomic terms to avoid misidentifications. Not only this, the technique has limitations in the identification of morphologically cryptic species.

Moreover, the available keys also work for a particular life stage or sex¹². To overcome the limitations of the morphological taxonomic approach, a molecular technique involving the generation of species-specific barcodes was introduced. Hebert et al., 2003 first established the use of the Mitochondrial COI gene to generate bio-identification barcodes in eukaryotic animals. The mitochondrial COI gene is considered an effective ‘barcode’ in species identification and delimitation because of its faster rate of evolution, which helps in the study of closely related taxa that have recently diverged^{13, 14, 15}. The present study aims to generate the DNA barcodes for the Hymenoptera species from the Jammu region to study phylogenetic relations among them. The study helps in estimating hymenopteran diversity in the region and helps formulate conservation strategies for this species diversity.

Figure 1: Map depicting collection sites Click here to view figure

Materials and methods

Taxon sampling and vouchering

About 53 samples were collected from different areas of the Jammu region by using sweeping nets and handpicking. The insects were anesthetised using ethyl acetate and preserved in 70% molecular grade alcohol for both morphological and molecular examination. The samples were examined under the Olympus SZ2-ILST for morphological identification, and voucher IDs were assigned to them.

Molecular interrogations, sequence annotation and dataset preparation

For DNA isolation, 1-2 legs depending upon the size and finally chopped, are used in the case of bees and wasps, while an intersegmental abdominal cut was made in the case of ants. The tissue was lysed overnight in buffer ATL and proteinase-K at 56ºC with 150rpm (revolutions per minute) in Stvart SBS40 shaking water bath (Cole- Paramer Ltd. Stone, ST15 OSA, UK). After tissue lysis, the total genomic DNA was extracted by using DNeasy Blood & Tissue Kit (Hilden, Germany). Polymerase Chain Reaction (PCR) was performed in applied biosystems ProFlex PCR system by life technologies to amplify partial mitochondrial cytochrome oxidase subunit I (COI) gene by using primers and conditions as shown in Table 1. The PCR products were sent to Biologia Research India Pvt. Ltd. for the sanger sequencing. The generated forward and reverse chromatogram files for each specimen were checked in MEGA X¹⁶ . The generated sequences were further screened in BLASTn (Basic Local Alignment Search Tool) for the available similar sequences in NCBI (National Centre for Biological Information) and 10 similar database sequences of each species are obtained from NCBI and are used for data curation. ClustalW software¹⁷ has been used for pairwise and multiple alignments by using a Gap opening penalty of 15.00 and a Gap extension penalty of 6.66 to obtain the final sequence for submission. The final dataset of 22 sequences from 18 different species was submitted to the NCBI Genbank through BioEdit; an online submission portal and the GenBank accession numbers were generated for them, written in Table 2. The A, T, G, C, AT, and GC content of all the sequences were obtained by using science buddies, and online software and their percentages present in sequence are depicted in Table 3.

Table 1: Primer sequences used

Genetic divergence and phylogenetic tree interpretation

The pairwise genetic distance was analysed using the Kimura 2-Parameter (K2P) model in MEGA X^{16, 18}. The number of base substitutions per site was analysed between all 22 sequences. Codon positions included were 1st+ 2nd+ 3rd+non-coding. All ambiguous positions were removed for each sequence pair, and a total of 538 positions were present in the final dataset. The evolutionary relationship was inferred using the Neighbor-Joining (NJ) method and Kimura 2- parameter model with 1000 bootstrap replications in MEGAX^{16, 18, 19, 20}. The database sequence of Eristalis tenax (ON210045) under order Diptera was used as an out-group in the phylogenetic study.

Results and discussion

Morphological identification

The hymenopteran species were identified by consulting the available keys in the literature by Archer (2014), Bingham (1897), Carpenter and Thi Nguyen (2003), Das and Gupta (1989), Goulet and Huber (1993), Kumar and Carpenter (2013), Kumar and Srinivasan (2010), Niup and Dorji (2016), Saini et al. (2011), Williams (1998), other relevant literature and identification keys^21-30. After morphological identification, the collected specimens were identified as18 different species belonging to 5 families, namely Apidae, Formicidae, Vespidae, Halictidae, and Crabronid. The details of identified species, their collection date, the site of collection & their coordinates have been listed in Table 2.

Table 2: Information about studied samples

Molecular characterisati

Of the 53 collected specimens, DNA was successfully isolated from 45 samples, and successful amplification of the COI region of 35 was carried out. Of which, sequences of 22 samples have been generated, which can be accessed by the NCBI with accession numbers (ON254654; ON259314-ON259316; ON259323; ON259474; ON259531-ON259532; ON306317-ON306330). The results of the similarity search in the NCBI database showed 99-100% identical matches with the same species except for eight species (Tachytes sp., Bombus trifasciatus, Rhynchium carnaticum, Ropalidia brevita, Lasioglossum marginatum, Camponotus pennsylvanicus, Tapinoma melanocephalum, Formica rufibarbis). Hence, the present study contributed eight new sequences of hymenopteran species from the Jammu region to the database. The COI sequences were found to have less GC% (23-33%) and more AT% (67-77%), as depicted in table 3. The minimum GC% was found in Apidae and the maximum in Formicidae of the studied families of Hymenoptera. The findings are following the previous studies, which reported 22-41% GC content in the hymenopteran COI region with 22-29% in Apidae and 24-41% in formicidae^{31, 32}. The phylogenetic tree was constructed using the NJ method and the K2P model along with 1000 bootstrap support values because the maximum literature reviewed for this research work was found to have used the NJ tree with the K2P model with supporting bootstrap values. The phylogenetic tree constructed depicts eighteen distinct lineages of twenty-two hymenopteran species belonging to five clades corresponding to the five families (Figure 2). Family Formicidae is found to be more closely related to Vespidae, while the family Apidae is found to be more closely related to the family Halictidae. The family Crabronidae is distantly related to all the four studied families; namely, apidae, formicidae, halictidae and Vespidae, as clearly depicted in the phylogenetic tree. The phylogeny constructed using COI sequences has been found as per the previous studies based on the morphological as well as molecular approach. Brothers in 1999 studied the phylogeny and evolutionary relationship of the wasps, ants and bees by using a morphological system and their results depict that ants and wasps are more closely related to each other than their relatedness to bees³³. Relatedness between Formicidae and Vespidae has been reported in the studies using different molecular markers³⁴. There is much evidence from the literature which supports the use of molecular markers for species identification and phylogenetic studies. The successful use of molecular markers especially COI for the identification and delimitation of the species was seen in Vespa species identification, Trissolcus species identification, new species of Zaischnopsis, trichogrammatids, hymenopteran parasitoids, and Egyptian wasps are some of the examples from the literature^35-41. The molecular data has also been found to be useful for species delimitation as depicted by the studies of Chen and coworkers; Parslow and associates; Benavides and associates and Siddiqui and coworkers^42-45. Kwon and associates in their study constructed the phylogenetic tree for the species of genus Osmia using COI gene data and found that the results were satisfactory and in accordance with the traditional approach⁴⁶.  Similarly, molecular characters including COI, 16S rDNA, and 28S rDNA were used to study the evolutionary transitions in parasitic wasps belonging to Apocrita in recent times⁴⁷. Aman-zuki and coworkers through their work also suggested the usefulness of molecular markers in resolving the phylogeny of species belonging to Microgastrine wasps⁴⁸. The studies of Niehuis and coworkers with the Euchroeus group of tribe Chrysidini have detected the inappropriate rooting of the group with the help of molecular data. Their study provides the insights that the molecular data not only supports traditional taxonomic views but also helps in resolving the phylogeny of various groups where the morphological characters have given erroneous interpretations⁴⁹.  Hence, the study supports the use of COI gene sequences (molecular markers) for inferring the phylogenetic relationship among the species  in addition to species identification and delimitation. The genetic relatedness among the species was also calculated with the generated sequence data. The overall mean genetic distance among the species in the present dataset was found to be 26.9%. The intraspecific genetic distance ranges from 0.19% (Apis cerana) to 0.58% (Bombus trifasciatus). However, the highest inter-specific genetic distance (37.69%) was observed between Apis cerana and Monomorium indicum, and the lowest inter-specific genetic distance (8.67%) was observed between Apis cerana and Apis dorsata. The values are clearly depicted in Table

Table 3: ATGC content of all the studied Samples

Figure 2: NJ tree (kimura-2-Parameter) Click here to view figure

Table 4: Genetic distance among the studied species Click here to view Table

Summary and Conclusion

The present study was the first approach for carrying out molecular characterisation and phylogenetic analysis of Hymenoptera from the Jammu region. The study contributed 22 barcode sequences including 8 novel sequences belonging to the insects of order Hymenoptera to the database. The study also provided evidence in support of the use of molecular markers for phylogenetic analyses as the results are in concordance with the morphological available evidence. The current study also helps future workers, especially non-taxonomists, to identify the species based on the generated barcodes of the species to the online database.

Acknowledgement

The authors are grateful to the Head, Department of Zoology, the University of Jammu, for providing necessary facilities and equipment availability (purchased out of RUSA/ PURSE/ FIST grants). One of the authors, Charul, also acknowledges the financial support from The Department of Science and Technology, Government of India, INSPIRE Fellowship under sanction no.: DST/INSPIRE Fellowship/2018/IF180760.

Conflict of Interest

Charul carried out the extensive field work, lab work and data compilation; Sunali Bandral and Shivalika also performed the lab work; Mohd Feroz and Umer Bin Farooq identified the Hymenopteran insect species; Vikas Dogra and Rakesh K. Panjaliya has framed the work plan, provide guidance at every step and reviewed the article.

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