First record of Tanaorhinus viridiluteata Walker, 1861 (Lepidoptera: Geometridae: Geometrinae) from Mizoram, India

: Very little work has been done to document the moth fauna of the Mizoram state in northeast India. An emerald moth collected from three localities in Aizawl District of Mizoram was identified as Tanaorhinus viridiluteata Walker, 1861 based on morphological and molecular studies. This species has been described briefly with colour photographs of male and female genitalia. Partial mitochondrial COI gene was amplified from these specimens for molecular analysis. This study represents a first record of the genus Tanaorhinus and species T. viridiluteata from Mizoram State.


INTRODUCTION
Mizoram State is situated in the southernmost tip of northeastern India, sandwiched by Myanmar in the east and Bangladesh in the west. Though the area falls within the Indo-Burma biodiversity hotspot (Mittermeier et al. 2004), the flora and fauna of the area are poorly documented. However, recent studies taken up in the area resulted in the description of several species new to science (e.g., Lalronunga et al. 2013;Giri et al. 2019;Kirti et al. 2019;Naumann & Lalhmingliani 2019). Hebert et al. (2003) proposed the use of the mitochondrial gene cytochrome c oxidase I (COI) as a reliable marker for accurate species identification, particularly in animals. Though faced with many criticisms and pitfalls (e.g., Tautz et al. 2003;Blaxter 2004), it is a useful tool for the identification of lepidopterans in general (Hajibabaei et al. 2006;Kim et al. 2020) and geometrid moths in particular (Brehm et al. 2016;Kumar et al. 2019).
Geometrinae (commonly known as emerald moths) is the fourth largest subfamily in the family Geometridae, with more than 27,006 valid species-group names, including 23,872 species and 3,123 subspecies worldwide (Rajaei et al. 2022). The genus Tanaorhinus Butler, 1879 contains 16 nominal species and five subspecies (Scoble & Hausmann 2007;Orhant 2014;Tautel 2014;Rajaei et al. 2022) all are restricted to Asia (Scoble 1999). However, Ban et al. (2018) revealed that the genus Geometra and Tanaorhinus are polyphyletic and revived the genus Loxochila Butler 1881 to accommodate G. burmensis, G. fragilis, G. sinoisaria, G. smaragdus, T. kina, and T. tibeta. They further speculated Tanaorhinus to be a junior synonym of Geometra. However, further molecular studies with the inclusion of more taxa are required for formal taxonomic action (Ban et al. 2018). Five species, viz., T. celebensis Yazaki, 1995, T. kina kina Swinhoe, 1893, Tanaorhinus kina embrithes Prout, 1934, T. rafflessi (Moore, [1860), T. reciprocate reciprocata (Walker, 1861), and T. viridiluteata (Walker, 1861), were recorded from India (Kirti et al. 2019). Walker (1861) described T. viridiluteata from Darjeeling in West Bengal State of India. Apart from the type locality, the species was further recorded from Arunachal Pradesh, Assam, and Nagaland states in northeastern India, southern China, Taiwan, and Sundaland (Anonymous 2021;Holloway 2021). The species is characterized by dark green colour with two black cell specks enclosed by a bluish tinge on both sides of the forewing, ante and post medial waved lines closed together with irregular white suffusion on dorsal side of the body. The ventral side of the forewing is heavily suffused with brown and mauve and inner margin of the hindwing is excised forming heart shaped gap. This species is most similar to T. rafflesia, but can be distinguished from it by the presence of broad, uniform, rufous border in the ventral hindwing (vs. narrower and separated from the margin by a yellow zone in T. rafflesia) in males; and harpe in male genitalia more spatulated (vs. more acute in T. rafflesia). Herein, we report the first distribution records of T. viridiluteata from the state Mizoram in northeastern India.

MATERIALS AND METHODS
Surveys were conducted in Mizoram State (see materials examined section under results and discussion for details) using a 160 W mercury vapour bulb on a 4 ft. by 6 ft. white cloth screen with a Honda TM EP1000 portable generator as a power source. Specimens were killed in a killing jar containing petroleum ether, which were then removed and placed on butter paper with their wings folded vertically. Pinning, spreading, and labeling of specimens were done in the laboratory. Specimens were deposited in the Entomological Collections of the Systematics and Toxicology Laboratory, Mizoram University, Mizoram, India (MZUEC). Tissue (three legs each) was collected in a 2 ml centrifuge tube for genomic DNA extraction. The genitalia of each specimen were dissected following Sondhi (2020). Genomic DNA was extracted from the tissue sample using 10 µl of 20 mg/ml of Proteinase K with 56°C overnight treatment following standard Phenol: Chloroform: Isoamyl alcohol method (Sambrook & Russell 2001). We amplified a partial mitochondrial COI gene using the primer pair LCO-1490 and HCO-2198 (Folmer et al. 1994). PCR amplification was carried out in 25 μl aliquots containing 12.5 μL of EmeraldAmp® GT PCR Master Mix (2X) (TaKaRa Bio, Japan), 1 μl of each forward and reverse primer, 2 μl of genomic DNA, and 8.5 μl of molecular grade H2O using ProFlex™ 3 x 32-well PCR system (Applied Biosystems™, USA). The PCR conditions were as follows: initial denaturation was performed at 95°C for 5 min, followed by 35 or 40 cycles of 30 s at 94°C, 30 s annealing from 42°C to 50°C (Tables S3 and S4), 30 s at 72°C, with a final 5 min extension at 72°C. Amplified PCR products were ran on 1.5% agarose gel, viewed in IG-618GD (iGene Labserve, India) gel documentation system. The purified PCR products were sequenced bidirectionally by Sanger sequencing technology at geneOmbio Technologies Private Limited (Maharashtra, India). The chromatograms and raw sequences were edited using FinchTV 1.4.0 (Geospiza Inc., USA ) and the consensus  (Kumar et al. 2018). The barcode data of Chlorozancla falcatus (MG014741) was used as an outgroup in the present phylogenetic analysis. The uncorrected pairwise genetic distances (p-distances) between and within the studied species were estimated by MEGA X (Kumar et al. 2018).  Diagnosis: Wingspan 60-64 mm in male (four specimens) and 72 mm in female (one specimen). Upperside of forewing dark green in colour with two black cell specks enclosed by a bluish tinge; ante and post medial waved lines closed together with irregular white suffusion on dorsal side of the body; lunulate markings absent beyond the postmedial line; obscure white marks on submarginals. Underside of forewing green with costal area to beyond cell purplish-grey; oblique postmedial line with rufous patches at apex and outer angle; inner margin white. Upperside of hind wing dark green in colour except for costa which is white. Underside of hindwing yellowish with traces of postmedial line; outer area rufous; outer marginal areas yellowish (Image 1B).

Image 1 . A-Collection localities of
The specimens collected from the three localities in Mizoram agreed with the description of Tanaorhinus viridiluteata in possessing the following characters: dark green colour with two black cell specks enclosed by a bluish tinge on both sides of the forewing; ante and post medial waved lines closed together with irregular white suffusion on the dorsal side of the body; ventral side of forewings heavily suffused with brown and mauve; presence of a broad, uniform, rufous border in the ventral hindwing in males; slightly spatulated harpe in male genitalia. The maximum likelihood (ML) tree (Image 1E) further revealed that the sample sequences from Mizoram, India, formed a clade with the sequences of T. viridiluteata along with an undetermined species of Tanaorhinus with an uncorrected genetic distance (p-distance) of only 0.002-0.008. In the ML tree, T. viridiluteata and T. rafflesia are sister species, which is not surprising as the two species are very similar morphologically. The genetic distance between the two species ranges from 0.053-0.061.

DISCUSSION AND CONCLUSION
As far as Mizoram is concerned, little work has been done to document the moth fauna of the state (Ghosh 2007;Kirti & Singh 2014, 2016Kirti et al. 2014Kirti et al. , 2019Lalhmingliani et al. 2013Lalhmingliani et al. , 2014Lalhmingliani 2015), but recent studies have led to the description of several new species (e.g., Kirti et al. 2019, Naumann & Lalhmingliani 2019. Ghosh (2007) and Kirti et al. (2014Kirti et al. ( , 2019 reported on geometrid moths from Mizoram State but did not mention the genus Tanaorhinus. The present study on Tanaorhinus viridiluteata from Mizoram represents the first record for this genus and species in the state. Letters in bold indicates the newly generated sequences in this study.

www.threatenedtaxa.org
The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.