Checklist of free-living marine nematodes from intertidal sites along the central west coast of India

The present study provides a checklist of free-living marine nematode species from nine intertidal sites located along the central west coast of India covering an area between latitudes 16°33′08.47″ N and 16°38′50.75″ N and longitudes 073°19′30.13″ E and 073°23′34.97″ E. The list includes 33 species of marine nematodes belonging to 20 genera and 13 families. The occurrence of nematode species identified appears to be correlated to character of the sediments across sampled intertidal sites. Ten families were encountered in sandy sites while in muddy sites the number was ten. Three families, namely Camacolaimidae, Ironidae, and Microlaimidae, were exclusive to the sandy sites, while Anoplostomatidae, Comesomatidae, and Linhomoeidae were only found across muddy sites. Some of the dominant free-living marine nematode groups encountered across all sites was represented by the genera Ptycholaimellus , Viscosia , Oncholaimus , Halalaimus , and species such as Sphaerolaimus balticus and Metachromadora suecica .


INTRODUCTION
In estuarine and coastal sediments, free-living marine nematodes are usually dominant for abundance and diversity (e.g., Lambshead 2004). Recent estimates have shown that marine nematode species number could be as high as 50,000 with 86% of the existing species waiting to be discovered (e.g., Appletans et al. 2012). They are abundant in particulate shores, reaching their highest number in muddy estuaries and salt-marshes (around 20 million per m²) and lowest in very exposed sandy shores as well as in deep sea sediments (0.1 million per m²) ).
Free-living marine nematodes play key roles in decomposition of organic matter, recycling of nutrients as well as serving as a food source for higher trophic groups (Austen 2004;Nascimento et al. 2012). Additionally, marine nematodes can be effectively used as bioindicators for environmental stress and pollution across the marine realms (Boyd et al. 2000;Moreno et al. 2011). Despite their ecological importance, studies of marine nematodes are largely neglected because their identification, which is predominantly based on morphological characters, requires taxonomic expertise (e.g., Platt and Warwick 1988;Bhadury et al. 2008). The understanding of an ecosystem depends not only on holistic synthesis of all components, but also on how the individual components work. Therefore, the accuracy of the identification is fundamental to our understanding of ecological attributes of any organism in its environment. Moreover, most of the studies that have focused on free-living marine nematodes systematic and biogeography are focused on European coastal ecosystems and our understanding of tropical and subtropical coastal environments is very poor (Semprucci and Balsamo 2012). One of the poorly studied tropical coastal settings is the central west coast of India. The central west coast of India represents the continental shelf region and the region facing the Arabian Sea is highly productive (Rajendran and Biddandra 1994) and known to harbour a rich marine biodiversity, including benthic macrofauna (e.g., Ramaiah et al. 1996;Joydas and Damodaran 2009). Several river mouths, creeks, small bays, cliffs and beaches make up the physical attributes of this coastal zone. The region also harbour eco-sensitive habitats, including mangroves and coral reefs (e.g., Ndaro and Ólafsson 1999;Raes et al. 2007;Ansari et al. 2012;Semprucci et al. 2013).
Ratnagiri and Vijaydurga, located along the central west coast of India, represent ecologically sensitive

LISTS OF SPECIES
Bhadury et al. | Nematodes from the central west coast of India stations during beginning and end of summer 2012 at low tide. In situ environmental parameters such as surface water temperature, salinity, and pH were measured using hand-held instruments at the time of sampling.
Immediately after collection, Rose Bengal stain (1g/L) was added to sediment samples, which were subsequently preserved based on established protocol (e.g., Somerfield and Warwick 1996). As part of this study, sediment sub-sample from core weighing 5 g representing top 3 cm were sieved from each station in 63 µm sieve and the meiofauna extraction was undertaken following the method of Somerfield and Warwick (1996). The extracted meiofauna was transferred to a petridish and nematodes were handpicked using a fine needle under a stereomicroscope (Zeiss Stemi DV4). Handpicked meiofauna were transferred to pure glycerin (Seinhorst 1959) and mounted on microscopic glass slides for taxonomic identification. Taxonomic identification of free-living marine nematodes was undertaken based on available pictorial keys Warwick 1983, 1988;Warwick et al. 1998) and the NeMys online identification key (Steyaert et al. 2005). Identified marine nematodes were subsequently photographed using an Olympus BX53 microscope equipped with XC50 CCD camera and CellSens Standard software. The voucher nematodes in the form of slides have been submitted to Zoological Survey of India and additionally slide of same specimens have been submitted to Marine Meiofauna Laboratory of Indian Institute of Science Education and Research Kolkata.

RESULTS
At the time of sediment sampling, average values for surface water temperature, salinity and pH for the sites sampled were 30°C, 35 ppt and 8.4 respectively.
A total of 160 free-living marine nematode specimens were collected in the study area and the specimens coastal areas based on their rich marine biodiversity (Apte et al. 2012). To date, the benthic community and diversity, including especially the free-living marine nematodes, from these two areas have been poorly studied. A single study although from subtidal environments, reported presence of several nematode species along the central west coast of India (Nanajkar and Ingole 2010). It is important to know the structure of the free-living marine nematode community from these two regions, since these organisms can be used in the future as an "environment proxy" to monitor ecologically sensitive coastal zones located along the central west coast of India.
This work aims to provide a list of free-living marine nematode species found in the Ratnagiri and Vijaydurga coastal stretches along the central west coast of India, which will help in expanding our knowledge on marine benthic faunal biodiversity of Indian coastlines.

MATERIALS AND METHODS Study sites
Nine coastal stretches of Ratnagiri and Vijaydurg along the central west coast of India were selected for analysis of the free-living marine nematode community structure: Ansure, Vijaydurg, Madban, Shindewadi, Waghran, Jaitapur, Ghodepol, Aagarwadi, and Ambolgad. All these sites were intertidal habitats. The sites Ambolgad, Jaitapur, Madban, and Vijayadurg were characterized by sandy beach sediments while the remaining sites are characterized by muddy estuarine sediments and contain mangrove patches. All these coastal stations were highly uneven in nature, usually narrow riverine plains that fringe along the coastline and with extensive lateritic cover. GPS points of the studied sites are detailed in Table 1 and depicted in Figure 1.

Sediment sample collection
Sediment samples were collected by hand held corer (5 cm length and 5.5 cm diameter) from these nine   (Table 2). For muddy sedimentary sites, ten families were encountered, and three of them, Anoplostomatidae, Comesomatidae, and Linhomoeidae, were exclusive of this habitat ( Table 2). Many of the nematode families were found in both sandy and muddy sites which is also evident in Table 2. Fourteen genera were identified from sites represented by sandy sediments, while 16 genera were encountered in the muddy sites. Twenty-one species were encountered in the sandy sediments, while for muddy sediments, the number of species were 24 (  (Vitiello, 1967), and Rhynchonema sp. 1 were found only in the muddy sites. Morphometric details of six nematode species encountered frequently from study area and accurately identifiable based on available taxonomic keys have been detailed in Table 3. Some of the observable morphological characters for these six species have been shown in Figure 2.

DISCUSSION
During the present study, 33 species were recorded from sampled sites and the dominant genera were Ptycholaimellus Cobb, 1920, Viscosia de Man, 1890, Oncholaimus Dujardin, 1845, Sphaerolaimus Bastian 1865, Halalaimus de Man, 1888, and Metachromadora Filipjev, 1918 In the sandy beach sediment samples as part of this study, Oncholaimus and Viscosia were found to be dominant genera and the reason for their dominance could be attributed to their large size (Fleeger et al. 2011) and feeding habit (omnivores/predators) (Wieser 1953). It has been shown in various studies that the family Oncholaimidae which includes genera such as Oncholaimus and Viscosia are usually dominant in sandy environments (e.g., Maria et al. 2013;Steif et al. 2013). Additionally, we found the family Oncholaimidae to be most species rich as part of this study. Although some free-living marine nematode species are common to muddy and sandy habitats, many of them are usually mud-or sand-preferring, and occur with different dominance in either sediment type (Heip et al. 1985;Semprucci et al. 2010). Some of the families (Comesomatidae, Linhomoeidae, and Sphaerolaimidae) and genera such as Sabatieria Rouville, 1903, Sphaerolaimus Bastian 1865, Terschellingia de Man, 1888, and Daptonema Cobb, 1920 are typically dominant in muddy sediments (Boucher 1972;Vitiello 1974;Travizi 2010), and confirmed in this study. Some of them, such as Sabatieria sp. and Sphaerolaimus sp., are known to inhabit stressed and anoxic sediments (Vincx et al. 1990;Vanreusel 1990;Gyedu-Ababio et al. 1999;Boyd et al. 2000;Mirto et al. 2002). The genus Ptycholaimellus was also encountered frequently in sandy and muddy sediments as part of this study with high abundance in particular in muddy sedimentary sites, indicating that food resources might be plentiful in the study area or that there is an anoxic sediment layer relatively close to the surface layers of the sediment. Recently, the presence of genus Ptycholaimellus in high abundance has been also confirmed in intertidal regions of central west coast of India based on 18S rRNA clone library and sequencing approach (Kumar et al. 2014). It is already well established in published literature that the genus Ptycholaimellus which is an epigrowth feeder, is present in surface layers of the sediment (e.g., Commito and Tita 2002) and its abundance has been correlated strongly with the depth of anoxic sediment layer (e.g., Eskin and Hopper 1985). According to previous observations (e.g., Warwick 1983, 1988), many of the species encountered in this study are typically found in intertidal sedimentary habitats. Nanajkar and Ingole (2010) reported 94 species of free-living marine nematodes in subtidal environments located along the central west coast of India. Some of the species reported by them, e.g., Anoplostoma sp., Daptonema sp., Sabatieria sp., Terschellingia longicaudata, Viscosia abyssorum, Sphaerolaimus sp., and Microlaimus sp., were also encountered in this study across intertidal study   (Semprucci et al. 2013). In a very recent paper, Semprucci et al. (2014) studied the nematode assemblages from a lagoon in Maldives of Indian Ocean region and found that the most abundant and richest families were represented by Desmodoridae, Chromadoridae and Xyalidae. In our study we also report the presence of several nematode genera belonging to these families. In a different study, Ansari et al. (2012) reported the presence of 192 species of marine nematodes represented by members belonging to families Xyalidae, Desmodoridae, Comesomatidae and Linhomoeidae from southeast continental shelf region of India (Bay of Bengal sector) in the Indian Ocean. Most common species encountered by them included Viscosia spp., Halalaimus spp. and Sabatieria spp. As part of the present study, many of these genera were encountered by us from sampled sites. Therefore, the similarities in nematode assemblages, in terms of dominant families and genera between geographically distant areas of Indian Ocean region indicate the existence of iso-communities as observed in other studies (Ansari et al. 2012;Semprucci et al. 2014). For example, Semprucci et al. (2013) have reported the presence of sediment dwelling taxa (e.g., Richtersia, Ptycholaimellus and Molgolaimus) in Maldivian subtidal habitats and in our study area (central west coast of India), we encountered some of these sediment dwelling taxa across several sites, although both the areas are geographically separated from each other within the Indian Ocean region.
Some of the sites selected as part of this study are known to harbor mangrove patches and many of the encountered nematode genera such as Metachromadora, Viscosia and Sphaerolaimus have been also reported extensively from other mangrove sedimentary environments in the Indian subcontinent (e.g., Ansari et al. 2014).
The results of this checklist of marine nematodes from intertidal regions of central west coast of India provide a framework for undertaking future studies on biogeography of free-living marine nematodes and comparative studies of their distribution trends with respect to temperate intertidal ecosystems. In addition, data provided in this checklist will can serve as a baseline which can be used in future to monitor natural and anthropogenic changes along the central west coast of India.