The current occurrence , habitat and historical change in the distribution range of an endemic tiger beetle species Cicindela ( Ifasina ) willeyi Horn ( Coleoptera : Cicindelidae ) of Sri Lanka

Tiger Beetles (Coleoptera: Cicindelidae) are a group of attractive, fastflying and fast-running insect predators that occur in many diverse habitats around the world (Pearson & Cassola 2007). The ease with which most species can be found and identified in the field, their habitat specificity and their value as indicators of habitat health and biodiversity have generated considerable interest among amateurs and professional biologists alike (Pearson & Cassola 2007). As a result, a total of 2,559 species have been described world wide since the first eight cicindelid species were identified by Carl Von Linné in the 18th century (Pearson & Cassola 2005). Most tiger beetle species (29% of described species) are found in the Oriental (Indo-Malaysian) region of the world, while northeastern India has the highest recorded number of species in a small area which is an 80km stretch between Siliguri and Darjeeling of West Bengal (Pearson 1988; Pearson & Cassola 2005). Sri Lanka is also attributed with a high number of cicindelid species and ranks amongst the top 30 countries of the world with the highest number of species (Cassola & Pearson 2000). The existing literature (Tennent 1860; Horn 1904; Fowler 1912; Naviaux 1984; Acciavatti & Pearson 1989), and collections available at the National Museum of Colombo and Natural History Museum of London, documents 54 species of tiger beetles from Sri Lanka belonging to five genera (Table 1), Cicindela, Tricondyla, Derocrania, OPEN ACCESS | FREE DOWNLOAD JoTT CommuniCation 3(2): 1493-1505


INTRODUCTION
Tiger Beetles (Coleoptera: Cicindelidae) are a group of attractive, fastflying and fast-running insect predators that occur in many diverse habitats around the world (Pearson & Cassola 2007).The ease with which most species can be found and identified in the field, their habitat specificity and their value as indicators of habitat health and biodiversity have generated considerable interest among amateurs and professional biologists alike (Pearson & Cassola 2007).As a result, a total of 2,559 species have been described world wide since the first eight cicindelid species were identified by Carl Von Linné in the 18 th century (Pearson & Cassola 2005).Most tiger beetle species (29% of described species) are found in the Oriental (Indo-Malaysian) region of the world, while northeastern India has the highest recorded number of species in a small area which is an 80km stretch between Siliguri and Darjeeling of West Bengal (Pearson 1988;Pearson & Cassola 2005).Sri Lanka is also attributed with a high number of cicindelid species and ranks amongst the top 30 countries of the world with the highest number of species (Cassola & Pearson 2000).
We report in this paper the first recorded occurrence of an endemic tiger beetle species, Cicindela (Ifasina) willeyi, from two locations of Sri Lanka and the habitat preferences of the species.

METHODS AND MATERIALS
Tiger beetles were surveyed in 94 localities of Sri Lanka from May 2002 to December 2005 (Fig. 1, Table 2).The localities for collecting tiger beetles were selected using information based on previous publications of cicindelid species of Sri Lanka and information based on the different habitat types of the family on a global scale.
Field studies were conducted at Maha Oya (Dehi Owita) in August 2003and 2004, and at Handapangoda in July 2003and 2004.Study Area: Maha Oya is a stream located at Dehi Owita (6 0 57'91"N & 80 0 16'44"E, elevation 6.7m) in the Kegalle District, Sabaragamuwa Province of Sri Lanka.It covers an area of 17km 2 and encompasses   the Grama Niladhari divisions of Dehi Owita, Debagama and Thimbiripola.The stream is a branch of the much larger Kelani River which starts from the Sri Pada Mountain range and flows in to the ocean at Colombo.The water of Maha Oya is slow flowing and runs parallel to a large sandy bank with 167m elevation (Fig. 2 & Image 1).The stream at Handapangoda (6 0 47'05N & 80 0 08'03E; elevation 23m) is located near a rubber estate in the Kalutara District, Western Province of Sri Lanka.It covers an area of 11km 2 and encompasses the Grama Niladhari divisions of Handapangoda, Arakawila, Menerigama and Kurana.It is a branch of Kalu Ganga, which is the second largest river of Sri Lanka and is restricted entirely to the wet zone of the country.The water of the stream is slow flowing and runs parallel to a narrow sandy bank that consists mostly of rocks (Fig. 2 & Image 2).
Collection of Beetles: Four visits were made to each site for surveying adult tiger beetles.Three belts of land of 100 to 150 m were selected on the sandy bank of Maha Oya, while two belts were selected on the bank of Handapangoda.All sites were sampled over five hours each day between 1000 to 1400 hr.
Beetles were searched for in specific habitats and their immediate surroundings e.g.shrub area near the sandy sections of the stream, rocky substrata, pathways and trails.When encountered tiger beetles were rapidly counted at a given site using a hand tally counter (Wagtech, UK) and a sample was collected using a standard insect net.The number of beetles and the sex ratio of those that could be observed under field conditions and from collected specimens were recorded.
Specimens were collected and preserved in 96% ethanol and stored at -20 0 C for subsequent identification.
Measuring Habitat Variables: The following habitat variables of the study sites were recorded.
(i) Weather variables: The ambient temperature, degree of solar radiation, relative humidity and wind speed of the habitat were recorded using a portable integrated weather station (Health EnviroMonitor, Davis Instrument Corp., Hayward, CA, USA) with optional sensors.
(ii) Soil characteristics: These included the soil group (determined by using the generalized soil map of Sri Lanka by Moorman & Panabokke 1961); soil type / texture (determined by the sedimentation technique using the "soil textural triangle" as the reference (Bierman 2007)); soil colour (measured by comparison with a Munsell soil colour chart); soil temperature (determined by using an insert soil thermometer (SG 680-10) ranging from -10 to 110 0 C); soil pH (determined by using a portable soil pH meter (Westminster, No.259)); soil moisture (determined by selecting five random spots of a locality and collecting samples down to a depth of 10cm and estimating the difference in weight before and after oven drying to 107 to 120 0 C in the laboratory.);soil salinity (determined by a YSI model 30 hand-held salinity meter).
Determination of morphological characteristics of tiger beetles: Morphological characters important in identification and for the purpose of establishing characteristics of this species were also noted as follows.
(i) Body weight: Each beetle was weighed to the nearest mg on an analytical balance (Chyo JL180, Chyo Balance Corp., Japan).
(ii) Body length: Estimated by measuring the length from the frons of the head to the elytral apex when the head was in the normal feeding position.The spines on the caudal end of the elytra were disregarded.Measurements were taken using a dissecting microscope (Nikon Corporation SE., Japan) with the aid of an eyepiece graticule (Nikon, Tokyo, Japan) calibrated by an objective micrometer (Olympus, Japan).
Body lengths of beetles were categorized as follows, based on size classes relevant for this group of insects (Acciavatti & Pearson 1989).
1. very small -less than 8mm 2. small -8 to 10 mm 3. medium -10 to 15 mm 4. large -15 to 20 mm 5. very large -more than 20mm (iii) Mandible length / chord: The distance from the articulation point of the left mandible to the tip were measured under a dissecting microscope (Nikon Corporation SE, Japan) with the aid of an eyepiece graticule (Nikon, Tokyo, Japan) calibrated by an objective micrometer (Olympus, Japan).Only specimens with undamaged or not noticeably worn mandibles were used (Pearson & Juliano 1993;Satoh & Hori 2004).
(iv) Other characters: In addition to the above characters the following features were also recorded for each specimen.1. Colouration -Dorsal and ventral colouration of the body and its metallic or iridescent appearance was noted as was the colour of the elytral maculae, pits, eyes, antennae, mandibles, labrum and legs.
2. Number of rugae between eye and vertex.
3. Labrum -The shape of the labrum and the number of labral setae.
4. Distribution of setae on body. 5. Distribution of pits on body.The above characters were observed under a photomicrographic attachment (Microflex AFX -DX, Nikon Corporation, Tokyo, Japan) which was also used in photographing each specimen (dorsal view, ventral view, lateral view, other important features).Additional photographs of species were also taken by using the computer software programme Auto Montage (facilitated with a SMC Pentax -FA macro camera) available at the entomology laboratory of the Natural History Museum, London.
Taxonomic keys of the Cicindela of the Indian subcontinent by Acciavatti & Pearson (1989), descriptions of Horn, (1904) and Fowler (1912) were used to identify the species and confirmation of identification was done through comparisons with specimens available at the National Museum of Colombo.
DNA Sequencing: DNA analysis was carried out to determine the DNA barcode of the species.Eight specimens from the two known populations (Maha Oya and Handapangoda) were used for DNA extraction and sequencing.DNA was extracted from the abdominal region of the beetle using the DNeasy protocol (July 2003).Voucher specimens were deposited in the Entomology Collection of the Department of Zoology, University of Colombo.
Standard PCR amplifications included 5µl of NH 4 buffer, 0.5µl of each dNTP, 1µl of each primer, 0.1µl of TAQ polymerase and 2.5µl of MgCl 2 in a 50µl reaction volume.PCR amplifications were carried out on a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, California, USA) for one cycle of (94 0 C, 2.5 mins.; 47 0 C, 0.5 mins.; 72ºC, 11 mins.; 4 0 C, α) for 40 cycles.The PCR products were purified using the UltraClean PCR clean-up DNA purification kit (MoBio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer's protocol.Sequencing was performed for 10µl of cleaned PCR product using the ABI Prism Big Dye Terminator Cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA).PCR primers were used as sequencing primers and each fragment was sequenced on both strands.The reaction products were purified by ethanol precipitation and sequenced on ABI 373 (version 3.0) automated DNA sequencer.Sequence files were edited using SEQUED version 1.0.3 (Applied Biosystems) and a consensus of bidirectional sequencing was determined.

RESULTS
Cicindela (Ifasina) willeyi H. was the only tiger beetle species observed at Maha Oya (Dehi Owita) and Handapangoda.Fifty beetles of Cicindela (Ifasina) willeyi.were observed from Maha Oya (Dehi Owita) and five beetles were collected for morphometric and morphological characterization.Ten specimens of the species were recorded from Handapangoda and three were collected for characterization.
Taxonomy of Cicindela (Ifasina) willeyi Horn, 1904: The description given in Horn (1904), Fowler (1912) and Acciavatti & Pearson (1989)      of body iridescent bluish-green with white setae.Labrum short, broad and brown in colour with eight or nine submarginal setae.Mandibles dark brown with left mandible having an average length of 1.90 ± 0.54 mm (n = 3).Pronotum with a medial dorsal bulge that is undivided by a medial line.Legs shiny blue-green in colour (Image 3).

Habitat of C. (I.) willeyi:
Beetles were found on a sandy bank at Maha Oya (Dehi Owita) which is a shallow stream found in the Sabaragamuwa Province.The periphery of the sandy bank consisted of shrub type vegetation of Gahala Colocasia esculenta, Bamboo Bambusa vulgaris and long grasses that provided shade to the habitat.Climatic and soil conditions of the site are given in Tables 4 and 5.
The climatic and soil conditions at Handapangoda were more or less similar to that of Maha Oya (Dehi Owita) (Tables 4 & 5).However, the beetles at Handapangoda were mainly found on the moist rocks on the sandy bank.Further, the habitat at Handapangoda was more better shaded than that at Maha Oya due to the large trees found on the bank.

Historical change in distribution of C. (I.) willeyi:
The species has been reported from the Central Province of the island and Labugama, Western Province from as far back as 1904 (Horn 1904;Fowler 1912;Acciavatti & Pearson 1989; collection of the National Museum of Colombo, Sri Lanka).However, it has not been studied thereafter, and although it's biology is well known, data on present distribution and habitat is lacking.According to the results of the current study, at present it occupies two habitats in Maha Oya (Dehi Owita) area and Handapangoda area.Investigations in the present study have further revealed that the species is absent from its former locations of Central Province and Labugama which have a geographical area of about 5583.5km 2 and is now restricted to an area of about 28km 2 in Maha Oya (Dehi Owita) and Handapangoda (Table 6).
Therefore, a loss of its present habitats where it is restricted in distribution may threaten the survival of the species and qualify it for a threatened category in the near future.

DISCUSSION
Cicindela (Ifasina) willeyi is an endemic tiger beetle species of Sri Lanka that was first described by Horn (1904) from the Central province of Sri Lanka, which was subsequently confirmed by Fowler (1912) and later by Acciavatti & Pearson (1989).The National Museum of Colombo houses a specimen of the species collected from Labugama in the Western Province of the country.However, the present distribution of this endemic species has been uncertain and habitat characteristics and preferences were completely unknown.The present study reveals the species from Maha Oya (Dehi Owita) (Sabaragamuwa Province), and Handapangoda (Western Province), localities from which it had not been previously recorded.Further, extensive field work carried out in riparian habitats of Meewathura, Kandy; Ma Oya, Mawanella; Pundalu Oya, Kotmale Oya, Ramboda and Silverfalls, Nuwara Eliya (Central Province); and Labugama (Western Province) during the study period confirms the absence of the species from its previous localities of occurrence (Fig. 1).This knowledge on the past and present distribution of C. (I.) willeyi indicates the possibility of a historical change in the distributional range of the species.
Various factors including urbanization, increased intensity of recreational use of beaches, increased off-road vehicle traffic, conversion of the habitat to a dumping ground for automobile parts and construction material, increased vegetation encroachment that eliminates open areas and inundation of habitat caused by the construction of dams have been used to explain historical range changes in tiger beetles in the United States (Knisley & Hill 1992;Kritsky et al. 1996;Knisley & Fenster 2005;Pearson et al. 2006;Simmons 2007).
Therefore, it is possible that C. (I.) willeyi extirpated from the localities in the Central Province and Labugama, and inhabited the area in Handapangoda and Maha Oya (Dehi Owita).Even though, the reason for this is not precisely known, possible events of the past can be suggested.
The Mahaweli Development Programme was initiated in Sri Lanka in 1979 to fulfill the water requirements for agriculture, industrial and domestic use, develop hydropower and reduce flood peaks.As a result of this programme, four major reservoirs and dams, namely, Kotmale, Victoria, Randenigala and Rantambe were built along the main stem of the river that lies in the Central Province (Dayawansa 2008).Building of a dam is known to reduce and corrode soil and rock along the river as massive deposits of soil are left within the reservoir.The massive cobblestones, sand and crushed stones held back by the dam and the transformation of the once tortuous riverbank and riverway to a relatively straight river course results in the loss of habitat for many invertebrates who survive in this environment (Mao & Zheng 2006).During reservoir filling the river and any associated wetland areas become inundated.Riffles, runs and pools of the river are lost beneath the rising waters, leading to the extirpation (or extinction) of habitat sensitive riverine species with tightly defined niche requirements (McAllister et al. 2001).Likewise, reservoir and dam construction along the Mahaweli River has resulted a considerable impact on river morphology and has inundated a vast area of land in the Central Province of the island.Aerial photographs obtained in 1985 and 2003, and topographical maps of the Survey Department of Sri Lanka, present encroachments towards the river across its floodplain area in Gohagoda, Central Province.Further, riparian areas in Peradeniya (Central Province) have changed to residential urban areas with considerable impacts of waste disposal (Dayawansa 2008).
It is also recorded that gold has been mined in the past from a concordant quartz reef in Central Sri Lanka (Nawaratne & Dissanayake 2001), and several gemming grounds are located in Nuwara Eliya, Horton Plains, Hatton and Kandy (Herath 1984).Therefore, it is strongly possible that C. (I.) willeyi went locally extinct from its historic sites in the Central Province of Sri Lanka and invaded the riparian habitats of Maha Oya (Dehi Owita) (Sabaragamuwa Province) and Handapangoda (Western Province) due to the unsuitability and loss of its former habitats.The presence of populations of the species at Maha Oya and Handapangoda, indicate the suitability of the habitat, climate and soil conditions of these locations.
Tiger beetles are known to prefer riverine habitats because of close proximity to food and water resources, safety from predators and reasonably low human disturbance (Bhargav & Uniyal 2008).Shade of the habitat is used as an oviposition cue for many species and clusters of larval burrows were found near the bases of plants in Cicindela cursitans Le Conte, of Ohio Valley, United States (Brust et al. 2005).
Further, their activity and density are greatly influenced by daily temperature patterns (Schultz 1983), and adult tiger beetles are known to maintain internal body temperatures that are just below their lethal limits of 39 0 C (Pearson et al. 2006).A ground temperature ranging from 32-33 0 C is known to be suitable for the activity and viability of tiger beetle populations, and a temperature of 34-35 0 C determined the greatest number of matings in Cicindela (Cephalota) circumdata leonschaeferi Cassola (Eusebi et al. 1989).
Tiger beetles prefer sandy soils with minimal vegetation, where periodic disturbance by wind and water removes encroaching vegetation (Warren & Buttner 2008).Colour patterns of adult tiger beetle species closely match the texture and hue of the soil substrate on which the species occurs (Pearson & Vogler 2001), and matching the body colour with that of the soil plays an important role in predator evasion by reducing discovery (Morgan et al. 2000).
The habitats at Maha Oya (Dehi Owita) and Handapangoda were both riparian with sparse vegetation that consisted of shrubs.In Handapangoda, ferns and bamboo were found on the stream banks providing shade to the habitat while a similar habitat was also found at Maha Oya.Ground temperature were similar at both sites and were at 32 0 C, while the soil temperature was 28 0 C at Maha Oya, and 27 0 C at Handapangoda.Both river banks consisted of soil with a sandy texture that was yellowish-brown at Maha Oya and dark yellowish-brown at Handapangoda.The colour of soil at both sites closely matched the colour of beetles, that were metallic brown dorsally with yellow-white maculations.
As C. (I.) willeyi is an endemic species with limited distribution and is consequently susceptible to local extinction, it is important that these habitats are identified and protected.At present, 99.5% of its distributional range has declined (Table 6).We believe that the most important conservation priority of a country is the protection of areas which house large numbers of endemic species and communities found nowhere else in the world.This study presents first evidence and importance of Maha Oya (Dehi Owita) and Handapangoda locations, as harbouring the only populations of an endemic species of tiger beetles.In view of the current human use of all of these sites and habitats and the development pressures exerted on the wet zone of the country, conservation of these sites are essential for future survival of this species.

Figure 1 .
Figure 1.Localities of Sri Lanka surveyed for the occurrence of tiger beetles (Place names of localities are provided in Figure 2. Study area at Maha Oya and Handapangoda with previous locations and the specimens available at the National Museum, Colombo matches the features observed in the specimens collected from Maha Oya (Dehi Owita) and Handapangoda sites.Diagnosis: Cicindela (Ifasina) willeyi H. closely resembles the other allied species C. (I.humeral spot; B -Pronotum; C -First elytral spot; D -Second elytral spot; E -Third elytral spot