Animal Biodiversity and Conservation 44.1 (2021) 117 A contribution to the earthworm diversity (Clitellata, Moniligastridae) of Kerala, a component of the Western Ghats biodiversity hotspot, India, using integrated taxonomy S. S. Thakur, A. R. Lone, N. Tiwari, S. K. Jain, S. W. James, S. Yadav Thakur, S. S., Lone, A. R., Tiwari, N., Jain, S. K., James, S. W., Yadav, S., 2021. A contribution to the earthworm diversity (Clitellata, Moniligastridae) of Kerala, a component of the Western Ghats biodiversity hotspot, India, using integrated taxonomy. Animal Biodiversity and Conservation, 44.1 117–137, Doi: https://doi.org/10.32800/ abc.2021.44.0117 Abstract A contribution to the earthworm diversity (Clitellata, Moniligastridae) of Kerala, a component of the Western Ghats biodiversity hotspot, India, using integrated taxonomy. Earthworms (Clitellata, Moniligastridae) of Chaliyar River Malappuram, Eravikulam National Park, Neyyar Wildlife Sanctuary, Parambikulam Tiger Reserve, Peppara Wildlife Sanctuary, Periyar National Park, Shendurney Wildlife Sanctuary and Wayanad Forest, Kerala, a component of the hotspot of Western Ghats, India, were studied by the standard method of taxonomy, and their DNA barcode signatures using the mitochondrial gene cytochrome c oxidase I (COI) were generated for the first time. This study represents eleven species of earthworms of the family Moniligastridae: Drawida brunnea Stephenson, Drawida circumpapillata Aiyer, Drawida ghatensis Michaelsen, Drawida impertusa Stephenson, Drawida nilamburensis (Bourne), Drawida robusta (Bourne), Drawida scandens Rao, Drawida travancorense Michaelsen, Moniligaster aiyeri Gates, Moniligaster deshayesi Perrier, and Moniligaster gravelyi (Stephenson). In the phylogenetic analysis all the species were recovered in both neighbour–joining (NJ) and maximum likelihood (ML) trees with high clade support. The average K2P distance within and between species was 1.2 % and 22 %, whereas the clear barcode gap of 2–5 % was suggested by barcode gap analysis (BGA) of studied species, reflecting the accuracy of characterization. The study presents the first step in the molecular characterization of the native earthworm family Moniligastridae of India. Data published through GBIF (Doi: 10.15470/l2nlhz) Key words: COI, Genomic signature, DNA barcoding, Earthworms biodiversity, Moniligastridae, Western Ghats, Kerala Resumen Una contribución a la diversidad de lombrices (Clitellata, Moniligastridae) de Kerala, un componente de la gran diversidad en Western Ghats, en la India, utilizando taxonomía integrada. Las lombrices (Clitellata, Moniligas- tridae) del río Chaliyar de Malappuram, el Parque Nacional de Eravikulam, el refugio de especies silvestres de Neyyar, la reserva de tigres de Parambikulam, el refugio de especies silvestres de Peppara, el Parque Nacional de Periyar, el refugio de especies silvestres de Shendurney y el bosque de Wayanad, en Kerala, que son una parte de la elevada diversidad de Western Ghats, en la India, se estudiaron mediante el método convencional de taxonomía, y su código de barras del ADN utilizando el gen de la oxidasa I del citrocromo c (COI). El estudio representa 11 especies de lombrices de la familia Moniligastridae, a saber: Drawida brunnea Stephenson, Drawida circumpapillata Aiyer, Drawida ghatensis Michaelsen, Drawida impertusa Stephenson, Drawida nilamburensis (Bourne), Drawida robusta (Bourne), Drawida scandens Rao, Drawida travancorense Michaelsen, Moniligaster aiyeri Gates, Moniligaster deshayesi Perrier y Moniligaster gravelyi (Stephenson). En el análisis filogenético, todas las especies se recuperaron tanto en los árboles producidos mediante el método de unión de vcinos como en los árboles basados en la máxima verosimilitud, con un elevado apoyo de los clados. La distancia media calculada con el modelo K2P dentro de una misma especie y entre especies fue del 1,2 % y el 22 %, respectivamente, mientras que el análisis de deficiencias del código de barras (BGA) de las especies ISSN: 1578–665 X © [2021] Copyright belongs to the authors, who license the eISSN: 2014–928 X journal Animal Biodiversity and Conservation to publish the paper under a Creative Commons Attribution 4.0 License. 118 Thakur et al. estudiadas sugirió una brecha del 2–5 %, lo que refleja la precisión de la caracterización. En el estudio se presenta el primer paso en la caracterización molecular de la familia de lombrices Moniligastridae, autóctona de la India. Datos publicados en GBIF (Doi: 10.15470/l2nlhz) Palabras clave: COI, Estructura genómica, Código de barras del ADN, Biodiversidad de lombrices, Moniligastridae, Western Ghats, Kerala Received: 18 I 21; Conditional acceptance: 23 II 21; Final acceptance: 23 III 21 Samrendra Singh Thakur, Department of Biotechnology, School of Biological Sciences, Dr. Harisingh Gour, Vishwavidyalaya (A Central University), Sagar, 470003, Madhya Pradesh, India.– Azhar Rashid Lone, Nalini Tiwari, Subodh Kumar Jain, Shweta Yadav, Department of Zoology, School of Biological Sciences, Dr. Harisingh Gour, Vishwavidyalaya (A Central University), Sagar, 470003, Madhya Pradesh, India.– Samuel Wooster James, Department of Regenerative Agriculture, Maharishi International University, Fairfield, Iowa, 52557 United States. Animal Biodiversity and Conservation 44.1 (2021) 119 Introduction Material and methods Moniligastridae is a family of earthworms indigenous Study site to southeast and eastern Asia. It is believed that the family Moniligastridae originated in the Malaya Kerala is a small state in the south–western tip of Archipelago’s geographical region (Gates, 1972; India. It is a narrow strip of coastal plain that bor- Blakemore, 2014), but later Jamieson (1977) sug– ders the Arabian Sea from the north to south, next gested an origin near Myanmar. Its natural range to the neighbouring states of Karnataka and Tamil encompasses south, southeast and east Asia, from Nadu. The state is recognized for its lush greenery, peninsular India to Japan through Myanmar, China, highly dense forests, diversified ecological habitats, the extreme southern portion of far Eastern Rus- topography, and the high biodiversity. It is bounded sia, Korea, the Philippines, Borneo, and Sumatra by the thickly wooded and forested hills of the Wes- (Gates, 1972). Moniligastrids are dominant mem- tern Ghats to the east and the Arabian Sea to the bers of the earthworms fauna in India especially in west. Kerala occupies 38,863 sq. km and comprises the South and North East Regions. Three genera approximately 1.18 % of India’s landmass (Sreedha- Desmogaster Rosa, 1890; Drawida Michaelsen ran, 2004). Out of the total length of the Western 1900; and Moniligaster Perrier, 1872 are known Ghats, Kerala covers around 600 km. Nearly 56 % from India (www.earthwormsofindia.com). Among of the total geographical area of the state has an them Drawida is most diverse with 73 species in annual average temperature ranging between 31 to India. Earthworms of this family have drawn the 37 oC and annual rainfall of 3,500 mm, mainly due attention of earthworm biologists as they retain the to the windward location to the Ghats (Rao, 1976). single layered clitellum characteristic of Clitellata Due to the integration and combination of different other than earthworms (Crassiclitellata) yet function climatic conditions, like warmer climate, altitudinal ecologically as do the crassiclitellate earthworms. variations, two different rainfall patterns and seasons The Moniligastridae have a broad size range, just (Southwest monsoon and North–East monsoon), like earthworms sensu stricto. The family is cha- several soil types and agro–ecological zones, Kerala racterized by simple pointed setae, four pairs per has a variety of macro environments that vary from segment, a clitellum beginning on segment 9 or 10 tropical rain forests to hot dry deciduous forests. and extending over 3 to 10 segments, including These diversified habitats and local ecological ni- those bearing genital pores; male pores one pair ches contributed to a variety of macro and micro (Drawida, Moniligaster) or two pairs (Desmogaster) environments conducive for a variety of flora and in or near grooves 10/11, 11/12 or 12/13; female fauna requiring contrasting environment. Of the pores one pair in 11/12 or XII or XIV. The sper- biota of India, the state sustains over 24 % of the mathecal pores are one or two pairs in 7/8 or 8/9 plant species, 30 % of the animal species, and 35 % or 7/8 and 8/9; the oesophagus with two gizzards of the freshwater fish species (Sreedharan, 2004). anterior to X or two to ten gizzards at the beginning of the intestine. The last hearts are two segments in Collection of earthworm samples front of the ovarian segment; they are holonephri- dial. Testes and funnels one or two pairs enclosed Earthworm samples analysed in the present study in one or two pairs of testis sacs. Vasa deferentia were collected from different sampling sites in Kerala opening into prostate glands. One pair of ovaries (fig. 1; see also the dataset published through GBIF in the segment immediately in front of the groove (Doi: 10.15470/l2nlhz). The locations, species names, or segment on which the female pores are situated, coordinates, and their BOLD accession numbers one pair of ovisacs extending backwards from the are provided in table 1. Samples were collected by ovarian segment. One or two pairs of spermathe- digging and hand–sorting according to the method cae with long tubular ducts. Without typhlosole, described by Satchell (1969). The specimens