Acta agriculturae Slovenica, 118/1, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.1.2132 Original research article / izvirni znanstveni članek Morphological and molecular characterization and new distributional record of Tetrastichus miser (Nees, 1834) (Hymenoptera: Chalcidoidea: Eulophidae) from Kashmir Ajaz RASOOL 1, Bashir A. GANAI 2, Shreevihar SANTHOSH 3 and Tariq AHMAD 1, 4 Received March 12, 2021; accepted January 22, 2022. Delo je prispelo 12. marca 2021, sprejeto 12. januarja 2022 1 Entomology Research Laboratory, Department of Zoology, University of Kashmir, Srinagar, India 2 Centre of Research for Development (CORD), University of Kashmir, Srinagar, India 3 Systematic Entomology Laboratory, Malabar Christian College – Calicut, Kozhikode, India 4 Corresponding author, e-mail: drtariqento@kashmiruniversity.ac.in Morphological and molecular characterization and new dis- tributional record of Tetrastichus miser (Nees, 1834) (Hyme- noptera: Chalcidoidea: Eulophidae) from Kashmir Abstract: Tetrastichus miser (Nees, 1834) (Hymenoptera: Eulophidae: Tetrastichinae) is a parasitoid of Curculioninae and Scolytinae infesting various trees of economic importance. In the present study, it was collected from dried Cedrus deodara (Roxb.) G. Don (Pinaceae) infested with Scolytus beetles using sweep net and aspirator. The species is reported first time from Kashmir valley. Identification of a parasitoid is of paramount significance for studying its behavior, ecology, life cycle and usage in various biological control programmes. In addition to morphological description, molecular analysis using Cyto- chrome C Oxidase Subunit I was carried out to complement morphotaxonomy and to facilitate its easier identification for future studies. Phylogenetic analysis by Bayesian inference (BI) and Maximum Likelihood (ML) method showed Isolates of Tetrastichusmiser species clustering in same clade and separat- ed from its closest match Tetrastichinae sp.Inter-specific diver- gence between Tetrastichusmiser and Tetrastichinae sp. was evi- dent and ranged from 0.09 to 0.10 % (0.05 % mean). No overlap was observed between maximum distance within species and minimum distance between species. Key words: Tetrastichus miser; Cedrus deodara; beetle; Cytochrome C Oxidase Subunit I; Clade; morphotaxonomy; molecular analysis Morfološka in molekularna določitev ter novi podatki o raz- širjenosti vrste Tetrastichus miser (Nees, 1834) (Hymenopte- ra: Chalcidoidea: Eulophidae) v Kašmirju Izvleček: Vrsta Tetrastichus miser (Nees) (Hymenoptera: Eulophidae: Tetrastichinae) je parazitoid hroščev iz podružin Curculioninae in Scolytinae, ki napadajo različne gospodarsko pomembne drevesne vrste. V raziskavi je bil parazitoid nabran s stresalnimi mrežami in aspiratorjem na posušenih himala- jskih cipresah, Cedrus deodara (Roxb.) G.Don (Pinaceae), ki so bile napadene s hrošči iz rodu Scolytus. O vrsti poročajo prvič iz doline Kashmir. Določitev parazitoida je zelo pomembna za preučevanje njegovega obnašanja, ekologije, življenskega kroga in pri njegovi uporabi v različnih programih biotičnega zatiranja škodljivcev. Poleg morfološkega opisa je bila za lažjo določitev v bodočih raziskavah uporabljena molekularna anal- iza na osnovi podenote I citohrom C oksidaze. Filogenetska analiza z metodama Bayezinove inference (Bayesian inference, BI) in največje verjetnostni (Maximum Likelihood, ML) je pokazala, da so se izolati vrste Tetrastichus miser združevali v istem kladu, ločeno od najbližjih, ki se ujemajo s predstavniki poddružine Tetrastichinae. Ločitev vrste Tetrastichus miser in predstavnikov poddružine Tetrastichinae je bila očitna in je znašala od 0,09 do 0,10 % (v povprečju 0,05 %). Opaženega ni bilo nobenega prekrivanja med maksimalno razdaljo znotraj vrste in minimalno razdaljo med vrstami. Ključne besede: Tetrastichus miser; Cedrus deodara; hrošč; podenota I citohrom C oksidaze; klad; morfotaksonomi- ja; molekularna analiza Acta agriculturae Slovenica, 118/1 – 20222 A. RASOOL et al. 1 INTRODUCTION Eulophidae (Hymenoptera: Chalcidoidea) is one of the largest chalcid families consisting of about 300 gen- era and 5000 speciesworldwide (Shree &Singh, 2015; Noyes, 2014). Eulophids have cosmopolitan distribution (Noyes, 2002) and are known to be entomophagous at- tacking insects and other arthropods like spiders and mites. Most eulophids are small in size (3 mm, average 1.5 mm). Family Eulophidae comprises of four subfami- lies i.e. Eulophinae, Entedoninae, Euderinae and Tetras- tichinae (Graham, 1975, Bouček & Graham, 1978). Eu- lophids are distinguished from other chalcidoid families by the presence of four segmented tarsi in both sexes and a short, straight protibial spur (as opposed to a gener- ously proportioned, curved spur in most other chalci- doids) (Schauff, 1997). Species of eulophidae are mostly primary parasitoids or hyperparasitoids, parasitizing a wide range of hosts like cotton boll weevil, beetles, cater- pillars of borers, midges, leaf miners, scale insects which are notorious pests of various horticultural, agricultural crops and forest plantations. Some eulophids are known to attack gall forming insects, mites (Bouček& Askew, 1968), eggs of spiders in silken egg sacs (LaSalle, 1990, 1994) and nematodes (van den Berg et al., 1990). Insect species belonging to more than 100 families and 10 or- ders are recorded as hosts for various eulophid chalcids (Talebi et al., 2010, 2011). Besides, most of the eulophids are parasitoids of insects hidden in plant tissue, such as wood borers, leaf miners, leaf rollers and gall makers. The genus  Tetrastichus  Haliday (Hymenoptera: Eulophidae: Tetrastichinae) is far and wide distributed worldwide containing 518 species worldwide (Noyes, 2014). Tetrastichus species virtually occur in all terres- trial habitats in all geographic realms, and constitute a vital component of terrestrial ecosystems. Taxonomic work on Tetratsichinae was started by Burks (1943), who provided key to North American species of Tetras- tichus. Tetrastichinae fauna of India includes 34 genera and 272 species (Hayat & Shah, 2004; Narendran, 2007). Still many species are yet to be explored and employed for various pest management programmes. The main di- agnostic character include a submarginal vein with one seta (rarely 2-4), propodeum with inverted Y shaped paraspiracular carina and hind coxa with strong reticula- tions. Tetrastichus miser (Nees, 1834) was first reported from India by Narasimham (1984). The specimens of the present study were collected during surveys in Botani- cal garden of University of Kashmir from dried Cedrus deodara (Roxb.) G.Don tree infested with Scolytus bee- tles. Asthe species is new faunal record from Kashmir valley, the present study provides a brief diagnosis and photographic illustration to authenticate the new record. In addition, molecular identification via DNA barcoding of cytochrome C oxidase subunit I (COI) was also car- ried to complement morphotaxonomy and to facilitate its easier identification. 2 MATERIAL AND METHODS Sampling on the prevalence of pest infestation was conducted in Botanical garden, University of Kashmir (34°08’09’’ N 74°49’14’’ E; 1590 m). Dead and dried Ce- drus deodara was found infested with Scolytid beetles. 11 parasitoids were collected by hand picking, aspirator and using sweep net with ethyl acetate used as killing agent. Collection after proper isolation and separation was preserved in vials in 70 % alcohol for further taxo- nomic studies. After morphological identification, speci- mens which were needed for molecular analysis were preserved in 90 % alcohol and then frigid at -20 oC. For morphological studies, Card mounted specimens were examined under Leica M205A stereozoom microscope (Leica Microsystems, Germany). 2.1 DNA EXTRACTION AND SEQUENCING 2.1.1 DNA extraction Before isolation, frigid samples were thoroughly washed with alcohol and formaldehyde to avoid con- tamination. The Genomic DNA was extracted from legs using DNA extraction kit (Nucleospin Insect DNA kit from Macheray Nagel, Germany) following manufac- turer’s protocol. PCR reaction mixture of 25 μl was pre- pared with following composition: 2.50 μl (10 x) of Taq assay buffer, 2.5  μl of dNTPs (each in 10  mM concen- tration), Forward primers and reverse primers each 0.2 μl (10 picomoles μl-1), MgCl2 buffer (1.5mM) 1.5 μl, Taq Polymerase 0.2 μl (1 U), DNA template 3 μl and Mili Q or sterilised water 14.9 μl. Universal primers (LCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ (for- ward) and HCO2198 5’-TAAACTTCAGGGTGAC- CAAAAAATCA-3’ (reverse)) were used to amplify the COI region (Folmer et al., 1994). Amplification was done by using a Thermal Cycler (Biorad Laboratories, Califor- nia) programmed to 98 °C for 5 minutes, followed by 30 cycles of 95 °C for 30 seconds, 45 °C for 45 seconds and 72 °C for 45 seconds and a final extension at 72 °C for 10 minutes. Amplified products were gel eluted in 0.8 % agarose stained with ethidium bromide and visualized using a UV trans-illuminator. Sequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator Acta agriculturae Slovenica, 118/1 – 2022 3 Morphological and molecular characterization and new distributional record of Tetrastichus miser ... from Kashmir v 3.1 Cycle sequencing Kit (Applied Biosystems , USA) following manufactures protocol at Rajiv Gandhi Centre for Biotechnology, Kerala, India. 2.1.2 DNA sequence analysis Trace files of sequences were subjected to qual- ity check by recording Phred score with a minimum of 20Q assigned to qualify as actual and further edited and trimmed at ends using Chromas 2.2.2 and Sequence scan- ner V2.The Phred score logarithmically represents error probabilities in base calling, hence this algorithm is used by majority of sequence analysis softwares (Ewing et al., 1998). Sequences were also checked for indels and numts using BioEdit 7.2 (Hall et al., 2011). The homology search was performed for the resulting consensus sequences us- ing Basic Local Alignment Search Tool (BLAST) (Alts- chul et al., 1990) and identification option of BOLD sys- tems (Ratnasingham and Hebert 2013) against sequences in GenBank to confirm the corresponding sequence tax- onomy. The generated COI consensus sequences were deposited in NCBI GenBank database and the corre- sponding accession numbers generated are MK419323, MT012501, MT017888, MT012523 and MT012515. 2.1.3 Phylogenetic analysis Phylogenetic analysis was done for the homolo- gous COI sequences obtained from Genbank database by performing similarity searches using BLASTn search algorithm (Altschulet al. 1990).A threshold of 3 % varia- tion between individuals of Chalcid wasps for COI gene was used for differentiating putative species (Hebert et al., 2004; Santos et al., 2011; Smith et al., 2018).Only top hits (sequences) with high similarity score and E-values in BLASTn were considered and non-redundant species sequences were retained for further analysis. Our se- quences did not reveal perfect matches, so a set of top 20 sequences were chosen for phylogenetic analysis and were aligned using Clustal W(Thompson et al., 1994) multiple alignment program inbuilt in MEGA X with the default alignment parameters (Kumar et al., 2018). Pair wise distance between each sequence was calculated us- ing distance option of MEGA software. In addition, vari- able sites analyses from the alignment of the dataset were performed in MEGA X (Kumar et al., 2018). 2.1.4 Abbreviations and Acronyms AIC: Akaike Information Criterion BI: Bayesian Inference BLAST: Basic Local Alignment Search Tool BOLD: Barcode of Life Data System CC: costal cell COI: Cytochrome c Oxidase Subunit 1 DNA: Deoxyribonucleic acid MCMC: Markov Chain Monte Carlo MEGA: Molecular Evolutionary Genetic Analysis ML: Maximum Likelihood MV: marginal vein NCBI: National Centre for Biotechnology Informa- tion NJ: Neighbor Joining SLG: sublateral groove SMG: submarginal groove SMV: submarginal vein ♀: Female 3 RESULTS 3.1 MORPHOLOGICAL IDENTIFICATION 3.1.1 Diagnosis Body black with copper bronze metallic reflections; mesocutum with median line weaker towards prono- tum, 5 long adnotaular setae on either side, in addition two rows of three setaeon either side of median line of mesonotum; eye with long pubescence; clava (0.72 x) distinctly shorter than scape; antennal formula 1 : 1 : 1 : 3 : 3; ratio of length of antennal segments 0.79: 0.32: 0.35 : 0.32 : 0.25 : 0.57. CC subequal to MV; SMV with one dorsal seta; malar space O.62 x length of eye; SMG of scutellum 1.7x from each other than from SLG, en- closed space 1.9 x as long as broad; callus with 4-7 setae arranged in two groups, one near the spiracle and the other near hind corner of propodeum; metasoma exclud- ing ovipositor sheath a little shorter than mesosoma (2.5 : 2.6); hypopygium reaching 0.6 x length of gaster. The specimens slightly varies in the number and pattern of setae on mesonotum, the other characters are matching the redescription by Graham, 1991 (Fig. 1). 3.2 MATERIAL EXAMINED AND HOST ASSOCIA- TION Card mounted ♀, INDIA: Jammu & Kashmir, Bo- tanical garden, University of Kashmir (34°08’50.5’’N, 74°52’00.9’’E), elevation 1600 m), collected by Ajaz Rasool, May 2018, Graham (1991) reported it from Rhynchaenus alni (Linnaeus, 1758), Rhynchaenus fagi Acta agriculturae Slovenica, 118/1 – 20224 A. RASOOL et al. (L., 1758), Rhynchaenus pilosus (J.C. Fabricius, 1781), Rhynchaenus quercus (L., 1758), Rhynchaenus salicis (L., 1758), and Rhynchaenus oxyacantha (Curculonidae). In the present study, it was reported from Cedrus deodara (Roxb.) G.Don trunk infested by Scolytidae (Coleoptera: Scolytidae). 3.3 DISTRIBUTION Austria, Czech republic, Slovakia, Denmark, Fin- land, France, Germany, Hungary, India (Bangalore and J&K), Italy, Netherlands, Spain, Sweden, Ukraine, UK. Host records are in Universal Chalcidoidea Database (www.universalchalcidoideadatabase) (Noyes, 2014). 3.4 GENETIC DATA AND PHYLOGENETIC ANALYSIS The accession numbers of sequences so generated areMK419323, MT012501, MT017888, MT012523 and MT012515. Barcoding of Tetrastichus miser from Kash- mir has been carried for the very first time. Our sequenc- es did not reveal perfect hits and hence were submitted to GenBank only after morphological identification. Nu- clear copies of mitochondrial DNA (NUMT) contamina- tion were fortified by performing amino acid translation by checking for the stop codons in the sequences. Trans- lation of frame 3 of amino acid sequence containing 180 amino acids was performed using ExPASy bioinformat- ics resource portal. No haplotype diversity was seen in the Tetrastichusmiser isolates. All 5 isolates were collected from same host tree (Cedrus deodara) at same location. The final alignment of the data set resulted in 567 nucleo- tide sites having 488 conserved sites, 121 variable sites, 97 parsim-info sites and 24 singleton sites. The mean A+T content was revealed as 74.69 %. After receiving accession numbers, final alignment of top 20 hits was done each roughly 567bp long of which 5 sequences represent the current study, 6 represent Bar- yscapus sp., 2 represent Aprostocetus sp., 4 Tetrastichinae sp. and 3 eulophid species. The nucleotide composition revealed high A-T content (74.69  %) which is com- mon for arthropods. The nucleotide frequencies include 34.25 % (A), 40.44 % (T/U), 12.61 % (C), and 12.70 % (G). Phylogenetic trees for Tetrastichus miser species were constructed by Bayesian inference (BI) and Maximum Likelihood (ML) methods. Both methods were used to confirm the evolutionary history of Tetrastichus species. For BI method, model selection was based on the Akaike information criterion (AIC) computed by Partitionfinder version 2.1.1 software (Lanfear, 2012). The subset parti- tions with positions 1, 2 and 3 were done and the best fit substitution models were predicted. The BI analyses was performed using MrBayes version 3.2.2 (Ronquist et al., 2012), a stop rule convergence value of 0.01 was set, which occurred on the 1140000 Markov chain Monte Carlo (MCMC) generation and two incrementally heat- ed chains. MCMC started from a random tree, sampling Figure 1: Tetrastichus miser, female: A infested Cedrus deodara tree, B Habitus, lateral view; C Mesosoma; D Head, Antennae; E Abdomen Acta agriculturae Slovenica, 118/1 – 2022 5 Morphological and molecular characterization and new distributional record of Tetrastichus miser ... from Kashmir one of every 500 generations, with the first 550 (25 %) of the trees discarded as burn-in out of 2200. The resulting tree was imported, edited and visualized using TreeDyne (Chevenet et al., 2006) inbuilt in Phylogeny.fr (Dereeper et al., 2008) (Fig. 2).For ML method, evolutionary his- tory was inferred based on the Kimura 2-parameter model (Kimura 1980) in MEGA X (Kumar et al., 2018). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 repli- cates) is shown next to the branches (Felsenstein 1985) (Supplementary file). In both trees, similar typology was found and Tetrastichus miser isolates clustered in a single clade with strong bootstrap value (100 %) and separated from other species. 3.5 INTRA- AND INTER- SPECIFIC EVOLUTION- ARY DIVERGENCE Pairwise Kimura-2-parameter (K2P) distance was calculated to comprehend the evolutionary divergence rate among the species based on COI gene (Kimura 1980). Datasets which were used for phylogenetic analy- sis were used for estimation of Pairwise intra and inter species K2P distances among the species using option implemented in MEGA X (Kumar et al., 2018). Among Tetrastichus miser isolates, no divergence was reported as such with intraspecific nucleotide difference of 0.00  %. Tetrastichinae sp. showed intraspecific divergence of 0.01 to 0.09 % whereas among Baryscapus coerulescens (Ash- mead, 1898) species it ranged between 0.00 to 0.03  %. Interspecific divergence between Tetrastichus miser and Tetrastichinae sp. ranged from 0.09 to 0.10  % (0.05  % mean). Between Baryscapus coerulescens and Tetrastichus miser species interspecfic divergence of 0.08 to 0.09  % (0.05 % mean) was reported. 4 DISCUSSION Identification of a parasitoid is of paramount signifi- cance for studying its behavior, ecology, life cycle and us- age in various biological programmes. Tetrastichus miser parasitizes a wide range of Curculionid beetles infesting various deciduous trees (Graham, 1991). Management of various bark beetles therefore requires information of natural enemies associated with them for future biologi- cal control programs. Morphologically, it has been de- scribed in India but owing to new record from Kashmir, current study was complemented by molecular analysis Figure 2: Bayesian inference phylogenetic tree of COI gene sequences of Tetrastichus spp. The scale bar indicates the number of substitutions per nucleotide position Acta agriculturae Slovenica, 118/1 – 20226 A. RASOOL et al. for future taxonomic purposes. There have been only few molecular studies on chalcid wasps from India (Rasool et al., 2018). Employing both morphological and molecular analysis for identification and characterization of T.miser species has been carried out for the first time. COI gene was used as marker gene for barcoding purposes for this species. Card pointed specimens were observed under LeicaM205A stereozoom microscopefor morphological studies. Key morphological characters like single SMV dorsal setae, black body with metallic reflections, were consonance with studies of Narasimham (1984).The specimens slightly varies in the number and pattern of setae on mesonotum, the other characters are matching the re-description by Graham,(1991). The final alignment of the data set resulted in 567 nucleotide sites having 488 conserved sites, 121 vari- able sites, 97 parsim-info sites and 24 singleton sites. The mean A+T content was revealed as 74.69 %. The data was analyzed for sequence divergence at different taxonomic levels in MEGA X software. Pairwise distance using K2P parameter in MEGA X was used to calculate the distance matrix (Kimura, 1980). Interspecific divergence between Tetrastichus miser and Tetrastichinae sp. was noticeable and ranged from 0.09 to 0.10 % (0.05 % mean). No over- lap was observed between maximum K2P distance with- in species and minimum distance between species. High nucleotide variations indicate geographical isolation and hence limited gene flow between species (Santos et al., 2011; Powell et al., 2019). For phylogenetic analysis, Maximum likelihood method and Bayesian Inference methods were used to infer evolutionary history so as to look for clustering of clades in different trees. Both methods resulted in some- what similar typology. In both the methods congeneric species cohesively clustered together with closely related genera. Besides, taxa belonging to a particular species more often than not formed a coherent cluster indicat- ing that CO1 gene sequences are useful in identification of species. The bootstrap consensus tree inferred from 1000 replicates is taken to correspond to the evolution- ary history of the taxa analyzed (Felsenstein, 1985). The taxa belonging to the same species, genus or family clus- tered together with healthy bootstrap support. It was also found that sequences from same country and genus or species clubbing in the same clade in both trees. Along with low support values at nodes, some high values were also reported when nodes include sister or conspecific sequences. The reason for this is that the COI gene frag- ment has been reported to best resolve shallow species- level relationships in arthropod fauna but showed poor results when family level and beyond relationships were taken into consideration (Waugh, 2007) and same was the case with our sequence data. Nevertheless, molecular analysis supported morphological results, besides add- ing its barcode to the databases for further exploration. Considering the role of T. miser in biological control programmes, molecular data of this study will serve as an elite DNA barcode for species identification, future molecular studies, better understanding of bio-control services and other related taxonomic studies in future. 5 CONCLUSION Tetrastichus miser is well known parasitoid of scolyt- id beetles infesting various tree species of economic im- portance. Morphologically, species has been defined and described in India, but molecular taxonomy was missing. There was rarely an entry in the GenBank database for this species. This study was carried to add to knowledge of chalcid wasps from Kashmir valley and also comple- ment its morphologically defined taxonomy. COI gene of mitochondrial genome was used as DNA barcode for the molecular analysis. The sequenced segment was found to be 567 bp long and was found to be AT rich in content. The sequence showed low percentage of match in NCBI and BOLD database systems with the closest match being Tetrastichinae sp. (91 %).Phylogenetic analysis inferred close match between Tetrastichus isolates, clustering into same clade with good bootstrap support. No overlap was observed between maximum K2P distance within spe- cies and minimum distance between species. Consider- ing the obscurity in identification of diverse insect fauna, this exercise will complement taxonomic analysis and sequence data will serve as an elite DNA barcode for spe- cies identification and other related taxonomic studies in future. 6 CONFLICT OF INTEREST The authors declare there is no conflict of interest. 7 ACKNOWLEDGEMENT Authors are highly grateful to Head of Department of Zoology and Director of the Centre of Research for Development (CORD) for providing working facilities. Authors, SS and TA acknowledge the financial assistance from SERB, Department of Science and Technology, Government of India, New Delhi for the financial assis- tance (File No. EMR/2017/005528 & EMR/2017/000215 respectively). Acta agriculturae Slovenica, 118/1 – 2022 7 Morphological and molecular characterization and new distributional record of Tetrastichus miser ... from Kashmir 8 REFERENCES Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389-3402. htt- ps://doi.org/10.1093/nar/25.17.3389 Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., & Sayers, E. W. (2008). GenBank.  Nucleic Acids Re- search, 37(suppl_1), D26-D31. https://doi.org/10.1093/nar/ gkn723 Boucek, Z., & Askew, R. R. (1968). Palearctic Eulophidae (excl. Tetrastichinae), Hym. Chalcidoidea. Le Francois. Boucek, Z., & Graham, M. D. V. 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