A contribution to the earthworm diversity (Clitellata, Moniligastridae) of Kerala, a component of the Western Ghats biodiversity hotspot, India, using integrated taxonomy

Study Extent

Kerala is a small state in the south–western tip of India. It is a narrow strip of coastal plain that borders the Arabian Sea from the north to south, next to the neighbouring states of Karnataka and Tamil Nadu. The state is recognized for its lush greenery, highly dense forests, diversified ecological habitats, topography, and the high biodiversity. It is bounded by the thickly wooded and forested hills of the Western Ghats to the east and the Arabian Sea to the west. Kerala occupies 38,863 sq. km and comprises approximately 1.18 % of India’s landmass (Sreedharan, 2004). Out of the total length of the Western Ghats, Kerala covers around 600 km. Nearly 56 % of the total geographical area of the state has an annual average temperature ranging between 31-37 oC and annual rainfall of 3,500 mm, mainly due to the windward location to the Ghats (Rao, 1976). Due to the integration and combination of different climatic conditions like warmer climate, altitudinal variations, two different rainfall patterns and seasons (Southwest monsoon and North–East monsoon), several soil types and agro–ecological zones, Kerala has a variety of macro environments that vary from tropical rain forests to hot dry deciduous forests. These diversified habitats and local ecological niches contributed to a variety of macro and micro environments conducive for a variety of flora and fauna requiring contrasting environment. Of the biota of India, the state sustains over 24 % of the plant species, 30 % of the animal species, and 35 % of the freshwater fish species (Sreedharan, 2004).

Sampling

Collection of earthworm samples Earthworm samples analysed in the present study were collected from different sampling sites in Kerala (fig. 1). The locations, species names, coordinates, and their BOLD accession numbers are provided in table 1. Samples were collected by digging and hand–sorting according to the method described by Satchell (1969).The specimens were anesthetized in 30 % (v/v) ethanol. Small pieces of muscle tissue from the tail region were then cut and preserved in 100 % (v/v) ethanol solution for molecular investigation. Next the earthworm samples were fixed in 10 % (w/v) formalin for morphological identification. 100 % ethanol preserved tissue of each sample was placed in the Museum of Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar, Madhya Pradesh, India as reference.

Method steps

1. Sample management and morphological classification Prior to applying the molecular technique for evaluation, we identified earthworms on the basis of specific diagnostic morphological characters under a stereoscopic zoom microscope (Leica Model No. M60) using the available literature (Stephenson, 1923; Aiyer, 1929; Gates, 1972; Julka, 1988; Narayanan et al., 2016, 2017). A camera lucida was used for drawings and abbreviations: sp.p, spermathecal pore; mp, male pore; atr, atrium; sp.d, spermathecal duct; amp, ampulla; ts.s, testis sac; vd, vas deferens; prs, prostate; atr.gl, atrial gland) were used in the figures. Voucher specimens are housed in the Museum, Department of Zoology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar, Madhya Pradesh, India. DNA sequencing For DNA sequencing the small pieces of muscle tissue from tail region were used. Total 28 samples of moniligastrids were sent to Barcode of Life Data Systems (BOLD System), Biodiversity Institute of Ontario, University of Guelph, Canada (Ratnasingham and Hebert, 2007) following appropriate protocol to obtain DNA sequences, accession numbers and Barcode. All the data used in present study is available on BOLD website under the project entitled (IEW) "Diversity studies in earthworms of India". In addition, 28 COI sequences were retrieved from the NCBI and BOLD free public domain for molecular analysis (see table 2 for more details).

1. Annelida
   rank: phylum
2. Clitellata
   rank: class
3. Haplotaxida
   rank: order
4. Moniligastridae
   rank: family
5. Drawida
   rank: genus
6. Moniligaster
   rank: genus

Kerala is a small state in the south-western tip of India. It is a narrow strip of coastal plain that borders the Arabian Sea from the north to south, next to the neighbouring states of Karnataka and Tamil Nadu. Kerala is known for its lush greenery, highly dense forests, diversified ecological habitats, topography, and the unabated biodiversity. It is surrounded by the thickly wooded and forested hills of the Western Ghats to the east and the Arabian Sea to the west. Kerala occupies 38,863 sq. km and comprises approximately 1.18% of India’s landmass (Sreedharan, 2004). With its gift of both sea and mountains, it is blessed with heavy rainfall, altitudinal diversity and fertile soils. , Kerala covers around 600 km of the total length of the Western Ghats. Nearly 56 % of the state has an annual average temperature ranging between 31 and 37 oC and annual rainfall of 3,500 mm, mainly due to the windward location of the Ghats