New species of the genus Bionychiurus Pomorski, 1996 (Collembola: Onychiuridae) from India

The genus Bionychiurus Pomorski, 1996 is not recorded from India so far. A new species nominated as Bionychiurus tamilensis sp. n. is described from the Nilgiri Hills in the Western Ghats. Bionychiurus tamilensis sp. n. possess a dorsal pso formula as 32/133/33,343 and a ventral pso formula as 11/011/11,121. The dorsal pso formula is similar to Bionychiurus changbaiensis, B. normalis, and B. oblongatus reported from China and Korea. The 658 bp mitochondrial cytochrome oxidase subunit I DNA region (COX1) of the new species was sequenced for its molecular identification. A modified key to genus Bionychiurus is provided.


Introduction
The genus Bionychiurus (family Onychiuridae) was established by Pomorski (1996) based on morphological characters of the 1 st larval instar of Onychiurus normalis Gisin, 1949. Weiner (1996 similarly established a new genus Bagnallophorus based on O. normalis. Later Pomorski (1998) synonymized Bagnallophorus with Bionychiurus. Based on the characters described by Pomorski (1998), and Sun and Wu (2012) the diagnostic characters of the genus are as follows: posterior cephalic pseudocelli present, welldeveloped post antennal organ with compound vesicles, chaeta d0 on the head present, and the furca reduced to cuticular pocket with 2 + 2 setulae. The distal whorl of setae on tibiotarsi has 11 pointed setae. Only six species are included in the genus Bionychiurus (Bellinger et al. 1996(Bellinger et al. -2020. The representatives of the genus were recorded from Europe (B. normalis and B. orghidani), South Korea (B. oblongatus and B. yongyeonensis), and China (B. qinglongensis and B. changbaiensis) (Gisin 1949;Gruia 1967;Lee and Park 1986;Wu 2012, 2014). We report the first Indian record of Bionychiurus from the Nilgiri Hills, a mountain peak (~2500 m a.s.l) in the Western Ghats Mountain Chain. The Nilgiri Biosphere Reserve is a World Heritage Site (UNESCO) with a high level of endemism is a hot spot of biodiversity (Ramachandra and Suja 2006;Raman et al. 2020). The paper describes the morphological and molecular traits of the new species.

Materials and methods
We obtained the specimens of the new species initially from the grasslands of Udhagamandalam (Ootacamund), the Nilgiris, Tamilnadu, India. Our subsequent surveys recorded the species from the shola-grasslands and humus-rich soils from the higher elevations of the Nilgiris (~above 1900 m a.s.l.). The species was obtained by extraction in Tullgren's funnels 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. (1996Bellinger et al. ( -2020 for the discrimination of morphological characters of the genus. The new species was identified and described following Sun and Wu (2012;. DNA was isolated from the three different complete specimens retrieved from the temporary mount in glycerol after identification, and preserved in absolute ethanol. We used Qiagen DNA easy Blood and Tissue kit (Cat No: 69504) for DNA extraction. We digested the specimen in ATL buffer, 0.5 mM EDTA (pH 8), and proteinase K for 48 h before extraction. The 658 bp fragment of mitochondrial cytochrome oxidase I (COX1) gene, referred to as the barcoding (Folmer) region described by Hebert et al. (2003), was amplified for molecular identification. We used the primers designed by Greenslade et al. (2011) for amplification of the COX1 region.
We performed the PCR amplification in ABI StepOne™ Real-Time PCR (RT-PCR) using 10X standard Taq reaction buffer, dNTPs, 1 μM forward and reverse primers, Taq DNA polymerase, and nuclease-free millipore water. The 20 µl final assay concentration includes 1U 2X Taq (Taq DNA polymerase), PCR buffer with 1.5 mM MgCl 2 and 200 µM of each dNTP, 1μl 1% bovine serum albumin, 1 µl each of the primers, and 5 µl DNA template. The thermocycling conditions were as follows: initial denaturation at 95°C for 180 s, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension 72°C for 30 s. The final extension temperature was 72°C for 10 minutes, followed by an infinite hold at 4°C. The amplified products were visualized in 2 % agarose gel for confirmation. The amplified PCR products were purified using the Qiagen PCR purification kit (Cat No. 28104). The purified products were sequenced by Sanger method after following the 'Snap chill' protocol, bidirectionally in the ABI 3730 Genetic Analyser. We deposited the sequences in NCBI Genbank with Accession Number MW712716. The sequence was searched for similarities in the BLAST and BOLD platforms. The available published sequences of Onychiuridae 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. 2017). The maximum Abbreviations used in descriptions. Ant -antennal segments, Th -thoracic segments, Abdabdominal segments, PAO -postantennal organ, psxparapseudocellus, ms -microsensillum, pso -pseudocellus, AIIIO -sensory organ of Ant III segment.
Description. Colour white in alcohol. Body length (excluding antennae and spines) 0.96-1.21 mm (Refer to Online resource 1 for detailed morphometric analysis).
Type material. Holotype male, seven paratypes: 3 males and 4 females, India, Nilgiri Hills, Tamil  Etymology. The name of the new species is Bionychiurus tamilensis sp. n., denoting the state of Tamil Nadu in India from where the specimen was collected.
Remarks. Comparison with other reported species is given in Table 1.
Key to species of the genus Bionychiurus (modified after Sun and Wu 2012)