Specimens of the new species were collected in the grassland of Udhagamandalam (Ootacamund), the Nilgiris, Tamilnadu, India. Material was obtained by extraction in Tullgren’s funnel and preserved in absolute ethanol. The specimens for morphological investigation were sorted using Olympus stereomicroscope, and mounted in Hoyer’s medium. Morphological characters were studied using NLCD–307B Lawrence and Mayo microscope. We followed Massoud (1967), Deharveng (1983), Yoshii (1996), Weiner (1996), Fjellberg (1999), D’Haese (2003), and Bellinger et al. (1996–2020) for the description up to genus. The descriptions of Sun and Wu (2012; 2014) were followed for the identification of new species.
DNA was isolated from the three different complete specimens retrieved from the mount after identification and preserved in 99.9% alcohol. The DNA extraction was done using Qiagen DNA easy Blood and Tissue kit (Catalogue No: 69504) with 48hr initial digestion in ATL buffer (QIAGEN) and proteinase K. The 658 base pair mitochondrial cytochrome oxidase I (COI), referred to as the barcoding region described by Hebert et al. (2003) was amplified for molecular identification. The primers designed by Greenslade et al. (2011) were used for amplification of the COI region.
Forward primer: 5’WYTCDACWAAYCRYAARGAYATYGG-3’
Reverse primer: 5’-TANACYTCNGGRTGNCCRAARAATCA-3’
The PCR assay was carried out using ABI step one Reverse Transcription PCR (RT-PCR) in a 20µl assay using 10X Standard Taq Reaction Buffer mMdNTPs, 10 µM Forward Primer, ten µM Reverse Primer, Taq DNA Polymerase, and Nuclease-free Millipore water. The assay concentration is finally 1U 2X Taq (Taq DNA polymerase, PCR buffer with 3Mm MgCl2 and 400 µM of each dNTP), 1.0 mM MgCl2, 1x BSA, 1 µl each of the primers and 5 µl DNA template. Thermocycling conditions were as follows: initial denaturation at 950C for 60 seconds, followed by 35 cycles of denaturation at 940C for 30s, 500C for 30s, and 720C for 30 sec. The final extension was 720C for 10 minutes, followed by the hold at 40C. PCR products were purified using the Qiagen PCR purification kit (Cat No. 28104). The purified products were sequenced by the Sanger method after following the snapchil protocol, bidirectionally in the ABI 3730 Genetic Analyser. We deposited the sequences in NCBI Genbank with Accession Number (Will be provided). The sequence were searched for similarities in the BLAST and BoLD platforms. The available published sequences of collembola were downloaded from NCBI for phylogenetic analysis. The maximum likelihood tree was constructed using the downloaded sequences and the query sequence in R studio using the phangorn 2.5.5 package (Schliep 2011, Schliep et al. 2011). The maximum likelihood was estimated following Felsenstein (1981) with 1000 bootstraps. The branch length was optimized in ‘GTR+I+G’ evolutionary model based on the Bayesian Index Criterion (BIC) values. The phylogenetic trees were visualized in FigTree v1.4.3 software.
Abbreviations used in descriptions
Ant - antennal segments, Th - thoracic segments, Abd - abdominal segments, PAO - post antennal organ, psx - parapseudocellus, ms - microsensillum, pso - pseudocellus, AIIIO – sensory organ of Ant III segment.
The pseudocellar formula: anterior head, posterior head / Th.I, Th.II, Th.III/ Abd.I, Abd.II, Abd.III, Abd.IV, Abd.V (eg: 32/133/33343).
Description of species
Bionychiurus tamilensis sp. nov
Etymology. The name of the new species is Bionychiurus tamilensis sp. nov., denoting the state of Tamil Nadu in India from where the specimen was collected.
Type material. The specimens were obtained from the samples collected between 14 - 28 August 2018 by A.M. Thunnisa and N. Sumithra. The holotype is male, and the seven paratypes include both males and females. The specimens were extracted from the soil samples collected from the grasslands of Old Ooty (11°41'N, 76°70'E), Garden mund (11°41'N, 76°69'E), and Government Arts College campus (11°41'N, 76°71'E), Nilgiri Hills, Tamil Nadu, India. We deposited the holotype and paratypes at the ‘Apterygota Division’ of Zoological Survey of India, Kolkata, (Acc. No 2027/H14) and the ‘Collembola repository’ of Molecular Biodiversity Lab, GAC, Ooty.
Diagnosis. Pso formula dorsally: 32/133/33343 and ventrally: 11/011/11121; Subcoxa 1 of leg I–III with 1,1,1 pso and 1,1,1 psx respectively, not visible in head and body; AIIIO with five guard chaetae and five papillae, two small sensory rods, and two sensory clubs; PAO with 22–24 compound vesicle; labial type AC; Abd. IV tergum with unpaired m0 and p0. Abd. V and VI with unpaired m0; furca are reduced to cuticular fold with four small dental hairs.
Description. Colour white in alcohol. Body length (excluding antennae and spines) 1.2–1.5 mm ( Refer supplementary material S1 for detailed morphometric analysis)
Dorsal pso formula:32/133/33343 (Fig 1a), ventral pso formula–11/011/11121. Subcoxa 1 of leg I–III with 1,1,1 pso and 1,1,1 psx, respectively. Psx not visible on the head and body. Antennae shorter than the head; length up to 0.1–0.2 mm. AIIIO with five guard chaetae and five papillae, two small sensory rods, and two sensory clubs (Fig 1c). Basolateral ms placed above the second proximal row of chaetae. Ant I with 6-8 chaetae, 12–14 chaetae in Ant II, 16–18 chaetae in Ant III, and 25–30 chaetae in Ant.IV dorsally with 25-30 chaetae and ventrally with 40–45 chaetae (Fig 1b).
PAO with 22–24 compound vesicles, arranged in two rows (Fig 1d). Dorsal cephalic chaetae d0 present on the head (Fig 1a). Mandible with four apical teeth, and Maxilla with three teeth and six lamellae. Labial type AC, Labium with six proximal, four basomedian (E, F, G, f), and five basolateral chaetae (b, c, d, e, e’). The labial papillae A to E with 1, 4, 0, 3, and 2 guard chaetae, respectively (Fig 1e). 4+4 postlabial chaetae along the ventral groove (Fig 2a).
Body Chaetotaxy.. Macro, meso, and micro chaetae are well differentiated. S- chaetae poorly marked. Th. II and Th.III with tiny and blunt lateral ms. Th.I tergum with 9–11 chaetae. Thorax II - III with 4+4 axial chaetae, Abd I–III with 3+3 axial chaetae. Abd. IV tergum with unpaired m0 and p0. Abd. V and VI with unpaired m0. Th.I–III ventrally with 0+0, 2+2, 2+2 chaetae. Abd VI with two curved anal spines (Fig 1a).
Appendages. Subcoxae 1 of leg I, II, III with 5,6,6 chaetae, subcoxae 2 with 1, 6, 6 chaetae, respectively. Femora with 15,18,18 chaetae, and tibiotarsi of leg I, II, and III with 22 (3, 8, 11), 22 (3, 8, 11), and 21 (2, 8, 11) chaetae, respectively. Unguis without denticle, unguiculus pointed, and without inner basal lamella (Fig 2b).
Ventral tube with 7–9 + 7–9 chaetae and 1+1 basal chaetae (Fig 2c), MVO absent, furca reduced to cuticular fold with four small dental chaetae (Fig 2d). Female genital plate with 25–30 chaetae (Fig 2e) and male genital plate with 45–50 chaetae (Fig 2f). Anal valve with several acuminate chaetae; each lateral valve with a0, 2a1 and 2a2; upper valve with chaetae a0, 2a1, 2b1, 2b2, c0, 2c1, 2c2 (Fig 2g). The detailed photographic descriptions are provided as supplementary material S2. Comparison with other reported species is given in table 1.
Barcoding: The BLAST analysis indicates the species have the nearest similarity with the Allonychiurus kimi (Similarity: 85.53%, Accession no: MT975431.1; Query cover: 98% and E value: 0.0). No reports of the COI region of the Bionychiurus genera are available in NCBI or BoLD. The maximum likelihood analysis showed the new species is a separate distant outshoot of the Onychiuridae cluster, confirming the entity. The outgroups used for the present study are the Isotoma, Sminthurus, and Entomobrya. The details of various accessions used for the phylogenetic analysis and the ML tree are given as supplementary material S3.
