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For: The Environment and Heritage Service (DoE, N.I.). By: The Queen's University of Belfast, School of Biology & Biochemistry. Authors: Dr Jane Preston, Dr Paulo Prodöhl, Dr Alex Portig & Professor Ian Montgomery. Date: February, 2003
Page Executive Summary 1. Background to the Current Project 2. Objectives 3. Methods 3.1 Estimates of Hare Abundance 3.1.1 Method 1 - Night Driven Transects (NDT) 3.1.2 Method 2 - Day Walked Squares (DWS) 3.2 Results 3.2.1 Night Driven Transects 3.2.2 Day Walked Squares 3.3 Creation of a library of genetic markers 3.3.1 Scientific Background for the Genetic Work 3.3.2 Materials, Methods & Results 3.3.3 Discussion 4. Overall Discussion 1 5 7 7 7 8 11 13 13 16 20 20 22 28 29
Executive Summary The Irish hare Lepus timidus hibernicus, (Linnaeus 1759) is an endemic subspecies now believed to be genetically distinct from the Scottish Mountain hare and more closely related to mainland European populations of Lepus timidus (Hamill, 2001). The best available evidence suggests a major decline in Hare numbers in Northern Ireland from the beginning to the latter part of the 20th Century. This decline has been attributed to environmental changes, notably loss of plant species richness, associated with intensification of agriculture (Dingerkus & Montgomery, 1997). The only comprehensive survey of the population of the Irish Hare throughout Northern Ireland was carried out in the mid-1990s by Dingerkus (Queen's University Belfast). Surveys were conducted by walking diagonally across 1km squares and counting the number of Hares observed as they were flushed from cover and searching intensively throughout each square. This survey demonstrated that although widespread throughout Northern Ireland, the Irish Hare occurred at low densities of around 1-2 per km2. These observations prompted a Biodiversity Species Action Plan (EHS, 2000) to set as targets:
“to maintain the existing range and demonstrate a population increase by 2005; double present population by 2010 over as much of the range as possible and, maintain and increase the area and quality of suitable hare habitat.”
The Irish Hare still appears on the quarry list and may be hunted legally in Northern Ireland at certain times of the year and by certain methods including coursing by dogs (Wildlife (NI) Order, 1985). The Northern Ireland Assembly in 2001 debated Hare coursing and introduced a new amendment to the Game Preservation Act (NI) 1928 governing the issue of permits to net Hares - Game Preservation (Amendment) Act (NI) 2002. This requires that the Department be satisfied that the trapping of Hares for the purposes of coursing has no impact on the wild population.
This change, together with the targets of the Species Action Plan, highlighted not only the necessity for current information on the distribution and abundance of Irish Hares in Northern Ireland but also the need for a system of regular, cost effective monitoring. The current investigation aimed to provide this information and to assess any changes that had occurred since Dingerkus (1997). It also aimed to develop protocols necessary to investigate the occurrence of sub-structuring and levels of genetic diversity of Irish Hare populations in Northern Ireland. It is impossible to count every individual during surveying. Therefore, surveys are frequently used to obtain estimates of abundance. They do not provide an absolute census but provide a population estimate with an upper and lower confidence limit. Two methods were used to estimate Hare abundance. The first method - Night Driven Transects - has already been used by Q.U.B. to estimate numbers of Foxes in both upland and lowland areas in Counties Antrim and Down. For the purposes of the current investigation these established routes were re-used and the method extended to cover all 6 counties. Using this methodology a transect of approximately 100km is driven repeatedly at night and spot lamps are used to locate Hares. The second method used to estimate Hare abundance followed the survey technique used by Dingerkus (1997) but was carried out in summer as opposed to late winter / spring. This was due to the urgency of obtaining estimates of Hare density for the purposes of issuing coursing licences. It was originally planned to carry out the surveys between the first and second silage cuts thus alleviating potential problems with vegetation height. However, the summer of 2002 was the wettest summer in Northern Ireland since 1961 and silage remained uncut in many fields causing problems with survey technique. The difficulties experienced in the field with the Day Walked Transects indicate that it is not possible to use the information gathered as a reliable estimate of current Hare abundance. Results from the Night Driven Transect surveys allow a reliable estimate of Hare abundance to be calculated. These surveys support the Dingerkus estimate of 1 Hare km2 and give a population estimate ranging between 7,000 and 25,200 Hares in Northern Ireland. Observations made in the field suggest that Hare surveys may significantly underestimate overall numbers since Irish Hares have two main responses to 2
disturbance, to flee and outrun the chasing predator or to 'sit tight' and hope they will not be detected. Surveys only count the fleeing Hares. However, this does not invalidate the survey techniques used as a means of detecting population change in the Irish Hare. A panel of microsatellite DNA markers (i.e. forensic based genetic markers) originally developed for other lagomorph species have been implemented for population genetic based studies of the Irish hare. The usefulness of these markers for future Irish hare studies has been confirmed by the genetic screening of specimens provided from colleagues in University College Dublin (N = 171) and from road killed individual Hares (N = 6). Nondestructive sampling procedures for obtaining sufficient DNA for genetic analysis have been successfully implemented. A microsatellite genomic library has been successfully developed for the Irish Hare. This will allow for any future requirement of additional markers for population based studies in this species. The research team recommends that future monitoring of the Irish Hare population should be carried out using the Night Driven Transect methodology in order to detect future changes in Hare numbers. A re-survey should be carried out in spring 2004 and repeated at 2-yearly intervals. This will provide data in support of the Species Action Plan targets for 2005 and 2010.
PRACTICAL RECOMMENDATIONS (1-5 as per Dingerkus, 1997). • Improvements in agricultural practice to allow for:
1. The production of a more species rich grass sward to allow Hares a more varied diet and promote survival of leverets. 2. Controls on livestock grazing levels to prevent disturbance to 'nesting' Hares. 3. Controls on the timing of silage cutting to prevent leveret and adult Hare deaths during the breeding season. 4. Changes in the method of silage cutting - working from the inside out would allow 'nesting' Hares and leverets to escape. 5. The provision of Hare refuge sites or buffer areas within farmland that allow rough vegetation to flourish. This would promote wildlife in the countryside in general. • • • Hare friendly agricultural practice in ESAs and CMS schemes. Removal of the Irish Hare from the quarry list and protection given under the Wildlife Order. Increased awareness of the plight of the Irish Hare by liaison with farming groups, the rural community and the Department of Agriculture (DARD).
RESEARCH INITIATIVES • Detailed genetic studies to establish the status and population dynamics of the Irish Hare. Re-description to species status would afford greater protection and conservation merit. • Research into the ecology of the Irish Hare concentrating primarily on:
1. Investigations into optimal Hare habitat - reasons for the occurrence of Hare 'hot-spots'. 2. Investigations into optimal Hare diet and foraging strategies. 3. Investigations into the major causes of adult and leveret mortality. 4. Investigations into the current reproductive success of Hares. 5. Investigations into the behaviour and reproductive strategies of the Irish Hare.
1. Background to the Current Project The Irish hare Lepus timidus hibernicus, (Linnaeus, 1759) is an endemic subspecies which is believed to be genetically distinct from the Scottish Mountain Hare and more closely related to mainland European populations of the Mountain Hare Lepus timidus (Hamill, 2001). The Irish Hare was formally recognised in 1833 (Fairley, 2001). The fur of the Irish Hare is more russet coloured than the introduced Brown Hare (Fairley, 2001). In Ireland, the Irish Hare inhabits not only upland areas, but lowland areas normally occupied by the Brown Hare in the rest of Europe (Wolfe & Hayden, 1996). The mean weight of the Irish Hare is between 2.3 to 4.3kg and average lengths range between 521-559mm (Fairley, 2001). Irish Hares have an estimated running speed of between 32-48 kmph (Fairley, 2001). Hares are primarily nocturnal creatures. In studies on Hare behaviour it has been calculated that Hares rest for 40% of their time (primarily during the day) and feed for 30% (mostly at night) with other activities such as grooming and scent marking taking up smaller periods of time (Fairley, 2001). Breeding extends from December to October with an average litter size consisting of 2/3 leverets (Fairley, 2001). However, large females may produce more young (Dingerkus, 1997). Hares have a maximum life span of around 9 years (Dingerkus, 1997). In 1979 the Irish Biological Records Centre recorded the Irish hare as "common and widespread, occurring both on lowlands and mountains" (Fairley, 2001). In the 1970s, the mean density on Co. Mayo bog was estimated to be 1/km2 and 40-50/km2 on grassland (Fairley, 2001). On farmland in Co. Kildare in 1982, mean density was estimated to be 4/km2 (Fairley, 2001). In Co. Meath in 1992/93, average densities of 12.4/km2 were observed (Fairley, 2001). On North Bull Island, Dublin Bay, mean density estimates ranged from 17/km2 in the 1970’s to between 5-7/km2 in 1994 with a peak in the estimated population density of 51/km2 in the 1980s (Fairley, 2001). The decline in population density over the twenty year study period was attributed to disturbance (Fairley, 2001). Densities of 46/km2 and 3/km2 were recorded for Lepus timidus scoticus in Scotland on heather moors over alkaline and acidic rocks respectively. In comparison with Hare populations elsewhere, densities of the Irish Hare in Northern Ireland are considered extremely low being up to an order of magnitude lower than that found in other places (The Decline of the European 5
Hares, 2001). Numbers of Irish Hares in the latter part of the 20th century were considered well below peak abundance in the first part of the century and had even declined from the 1980s to the 1990s. During the mid 1990s Dingerkus demonstrated that although widespread, the Irish Hare occurred at low densities in Northern Ireland with total population estimates ranging between 8250 (min) and 21000 (max) with Hares being more widespread on upland areas (Dingerkus, 1997). Hare densities are probably related to habitat diversity, with lower body weights and high mortality rates in areas of low habitat diversity (Dingerkus, 1997). Available evidence regarding land class and habitat affiliations and diet suggest that environmental change associated with more intensive systems of agriculture are the major cause of declines in Irish Hare abundance (Dingerkus and Montgomery, 1997). Adverse effects of agriculture on Hares include; reduction in diversity of grass species, removal of cover, increased cutting of grass for silage and herbicide/pesticide pollution (Fairley, 2001). Increased stocking densities may also adversely affect hares due to trampling of young leverets by livestock (Dingerkus & Montgomery, 2002). Increased agricultural mechanisation may also increase leveret mortality. Predation also affects survival rates (Dingerkus & Montgomery, 2002). Foxes are the biggest predatory threat but Badgers and birds of prey will also take Hares, especially leverets (Fairley, 2001). There is evidence to suggest that Foxes play a major role in the population dynamics of the Brown Hare in many environments (Reynolds & Tapper, 1995). In addition, the Game Conservancy in the UK estimate that a family group of Foxes can consume 33 adult Brown Hares annually (cited in Edwards, Fletcher, & Berny, 2000). However, Looney (2001) found a very low frequency of Hare remains in faecal analysis of Fox scats in County Antrim, Northern Ireland. Grass is the preferred foodstuff of the Irish Hare (Fairley, 2001). In analyses of faecal pellets collected from Hares in Northern Ireland, 64-75% of faecal remains were found to consist of a wide variety of grass species (Dingerkus, 1997). Hares require a diverse sward to maintain an adequate diet especially during lactation. The diversity of grass species has also been affected by agricultural intensification. Indeed, fast growing grass species may be unpalatable to Hares (Dingerkus & Montgomery, 2002). Mortality of leverets due 6
to inadequate maternal food supply rather than predation is regarded to be the major cause of population decline in European Hares (The Decline of the European Hares, 2001). If Hares require a varied diet one may assume that they may avoid intensively farmed areas that also lack refuge areas (Dingerkus & Montgomery, 2001). Two of the plausible hypotheses for the decline of the Irish Hare in Northern Ireland relate to changes in land use. First, Hares cannot survive at high densities due to poor and unvaried diet and second, there is insufficient cover to survive extremes in weather and avoid predation (Dingerkus & Montgomery, 2001). Given the recent declines that have been demonstrated in Hare populations throughout Ireland, it is vital that the current status of the Irish Hare in Northern Ireland is regularly reviewed. The last detailed survey by Dingerkus (1997) suggested that estimates of population density were extremely low. The current investigation aimed to re-assess the status of the Irish Hare and provide a bench-mark against which future monitoring programmes could be established and conservation recommendations made.
2. Objectives The objectives of the current investigation were to: • • • • • establish current abundance and distribution ascertain change in distribution and abundance since the Dingerkus survey in 1996 review methods for monitoring Irish Hare and establish rapid, inexpensive methods for future monitoring at regular intervals create library of microsatellite markers for genetic determination of the population make recommendations for future monitoring of Hare numbers
3. Methods 3.1 Estimation of Hare Abundance It is almost impossible to count every individual animal during surveying. Therefore, sample surveys are frequently used to obtain estimates of abundance. They do not provide an absolute census but give a range with an upper and lower population estimate. Two methodologies were used to estimate Hare density in the current investigation and these are described individually below. Earlier studies
suggested that both methods lead to similar estimates of abundance (density) although based on entirely different premises and conducted during different parts of the daily activity of Hares. In the current investigation, both methods were used in order to compare estimates, using one to calibrate the other. This could potentially lead to the design of a rapid, cheap and effective means of future monitoring of the changes in Hare population size and distribution.
3.1.1 Method 1 - Night Driven Transect (NDT). Recent experience at Queen’s indicates that population estimates may be obtained using Distance sampling techniques (Buckland, Anderson, Burnham, & Laake, 1999). This method has been used with success to obtain estimates of Fox abundance in Counties Antrim and Down during spring and autumn months. Two transects of approximately 100km in length were driven across a predetermined route in each county - one covering upland areas and the other lowland habitat. For the purposes of the current investigation the routes previously used to survey Foxes were re-used and the method extended to all six counties of Northern Ireland. Unlike the transects in County Down and Antrim, a single transect was driven for Counties Armagh, Londonderry, Tyrone and Fermanagh. However, each new transect surveyed was largely representative of both upland and lowland land classes (Cooper et al., 1997). All land classes were covered within each county in the approximate abundance of occurrence (Tables 1a & 1b). Differences observed are due to the difficulty in creating a continuous route on secondary roads. The routes driven across each county are illustrated in Figure 1. Surveys were carried out between February and April, 2002. Surveying commenced after dusk and continued for a period of up to 5 hours during which time approximately half the route was driven each night. A period of up to seven nights was spent on each transect in order to maximise the number of Hares observed. A 4-wheel drive pick-up truck was used and was fitted with a viewing deck which allowed one person to stand upright whilst the vehicle was in motion. The vehicle was driven at approximately 15km/hour and the fields along one side of the road were scanned with a one million candle light spot lamp for the presence of Hares. 8
When an animal was observed the vehicle was stopped and a note was made of the approximate location of the animal along the transect using the Global Positioning System (GPS), distance travelled (kilometers), perpendicular distance to the animal (meters) and the reaction of the animal to the light. The weather conditions were noted at hourly periods throughout the night. This included visibility, cloud cover and phase of the moon.
These data can be used to generate population estimates (densities) using DISTANCE 3.5 software (Buckland, Anderson, Burnham, & Laake, 1999) from which overall Hare numbers in Northern Ireland can be derived.
3.1.2 Method 2 - Day Walked Squares (DWS). The second method of population estimation was a repeat of Dingerkus’s survey (Dingerkus, 1997) and was undertaken to permit insight into changes in Hare numbers from the mid 1990s to 2002, a five year period. Methods used by Dingerkus followed those used by Hutchings & Harris (1996). However, a number of changes were made in order to adapt the survey technique to smaller fields and the local topography (Dingerkus & Montgomery, 2001). A total of 150 1 km2 areas were surveyed by Dingerkus (1997). The squares were a stratified random sample with 5-8 squares chosen from each of the 23 land classes present in Northern Ireland (Cooper et al., 1997). Squares were recorded as having Hares present if Hares were seen, having evidence of Hares when faecal pellets and/or forms (Hare nest) were found and being reported if Hares were reported to be present by the local landowners (Dingerkus & Montgomery, 2001). If none of these categories was fulfilled, then the square was recorded as having Hares absent (Dingerkus & Montgomery, 2001). The methodology followed that used by Dingerkus (1997) but was carried out in summer as opposed to late winter / spring. This was due to the urgency of obtaining information on the current status of the Irish Hare population for the purpose of issuing licences to net Hares to coursing clubs. Initially it had been planned to carry out the surveys between the first and second silage cuts thus alleviating potential problems with vegetation height. Using the same 1 km squares surveyed as Dingerkus (1997), a daytime survey was conducted throughout Northern Ireland over a period of two months between June and July, 2001. A total of 135 1km2 areas originally surveyed by Dingerkus (1997) were resurveyed.
Figure 2 shows the distribution of these 1km2 areas throughout Northern Ireland. Surveys involved walking diagonally across the 1km2 area and looking for any evidence of Hare activity. This included the occurrence of Hare forms, faecal droppings, tracks or actual sightings. The survey sheet and a typical survey map are illustrated in Appendix 1. Each square was walked in a north-westerly direction (Figure 3) starting in the south-eastern corner of the square and proceeding diagonally across the area. On completion of the survey each 1km2 was traversed again, this time by zig-zagging back across the area (Figure 3) to the original starting point and noting any changes in land use or boundaries since the original survey in 1997.
Figure 3. An example of a survey square illustrating the direction of surveying.
Approximate Scale 1: 7,000
3.2 Results 3.2.1 Night Driven Transects (NDT). Originally it was hoped to carry out detailed statistical analyses on the data in respect to land usage. However, this was not possible due to the low numbers of Hares recorded. Table 2 gives a summary of the results obtained from NDT surveys. A total number of 230 Hares were observed along the driven transects totalling a distance of 2,281 km. The greatest number of Hares was observed in the upland areas of County Antrim (110 individuals) (Table 2) whilst fewest Hares were observed in County Tyrone (4 individuals) (Table 2). In addition, more Hares were observed in upland compared to lowland areas in Counties Down and Antrim (Table 2).
Table 2. Results from the Night Driven Transect surveys in each county. County No. of Hares observed Co. Armagh Co. Londonderry Co. Fermanagh Co. Tyrone Co. Antrim - Upland Co. Antrim - Lowland Co. Down - Lowland Co. Down – Upland Total 17 7 14 4 110 24 9 54 239 Total distance driven (km) 250 212 289 266 365 347 242 310 2281 km
These results can be compared with a previous survey for Hares in Counties Antrim and Down (Table 3). This survey covered the same transect routes but was carried out in both spring and autumn 2000 (O' Mahoney, 2001) (Table 3). As with the current findings (Table 2), the greatest number of Hares was found in upland areas of both County Down and Antrim (Table 3). Numbers also vary with time of year. In County Antrim, the greatest number of Hares was observed in spring as opposed to the autumn of the same year.
Table 3. Results from the Night Driven Transect surveys in Counties Down & Antrim carried out in spring and Autumn by O' Mahoney (2001). County No. of Hares observed Co. Antrim - Upland (Spring) Co. Antrim - Upland (Autumn) Co. Down – Upland (Spring) Co. Down – Upland (Autumn) Combined Lowland (Spring) Co. Down - Lowland (Autumn) 96 20 70 3 13 2 Total distance driven (km) 278.9 371.4 374 236.8 484.3 352.8
The model produced from the current data using the DISTANCE 3.5 package allowed an overall density estimate of Hares to be calculated. The estimated density was 1.0 Hare per km2 in Northern Ireland (Table 4). This estimate is based on the complete dataset as data for individual counties / land classes was insufficient. Based on a land area of Northern Ireland of approximately 14,000km2, the estimated total number of Hares is 14,000 with a range of 7,000 to 25,200 (Table 4). It is not possible to put a more accurate estimate range on the numbers of Hares due to the low numbers detected during the current investigation. We can compare this density estimate to those produced by Looney (2001) during spotlight surveys for Foxes in County Antrim (Table 5). Although the data are not directly comparable, since Looney (2001) covered a much broader area, estimates are largely similar. In addition, the density of Hares is higher in spring in County Antrim.
Table 4. Density Estimate of the Irish Hare in Northern Ireland (DISTANCE 3.5). Estimate (mean) Hare Density per km2 Estimated Total No. of Hares Lower 95% confidence limit Upper 95% confidence limit 1.0 14,000 0.5 7,000 1.8 25,200
Table 5. Estimate of the density of Irish Hare in Spring and Autumn spotlight counts in County Antrim (DISTANCE 3.5) (Looney, 2001).
Estimate (mean) Hare Density per km2 (Feb 1997, 1998, 1999) Hare Density per km2 (Oct 1997, 1998) 1.28
Lower 95% confidence limit
Upper 95% confidence limit
3.2.2 Day Walked Squares Extreme difficulty was experienced re-surveying the 1km2 areas. This was largely due to the vegetation height and associated problems 'spotting' Hares. Initially it had been planned to carry out the survey between the first and second cuts of silage. However, due to one of the wettest summers in Northern Ireland since 1961 (Appendix 2), the majority of grass fields remained uncut during the survey period. This makes the comparison of the current results with those obtained by Dingerkus (1997) difficult. During the current survey Hares were found to have a 25% occurrence in squares surveyed compared to a 67% occurrence in the same squares surveyed by Dingerkus in 1997 (Figure 4) (Table 6). Four out of the 135 squares surveyed were found to have Hares present where there had been none observed during the previous survey (Table 7). However, Hare activity was absent in 60 out of the 135 squares previously found to have evidence of Hare activity (Table 7). The current survey produced similar results to the Dingerkus survey in 41 squares, where there was still no evidence of Hare activity and in 30 squares where Hares were present in both surveys (Table 7).
Table 6. Comparison of the percentage occurrence of Hares in 1km2 areas since Dingerkus (1997). % Occurrence in 1km2 areas Dingerkus survey (1997) 67 Q.U.B survey (2002) 25
Table 7. Number of squares showing a loss, gain or similar results in Hare observations since Dingerkus (1997). Type of Change since 1997 Gain in presence of Hares Decline in presence of Hares Same where Hares were found to be absent Same where Hares were found to be present Total Number of Squares Re-surveyed No. of Squares 4 60 41 30 135
The data were analysed with respect to land class and compared with previous survey results. Eight land class groups were used (Table 8) (Cooper et al., 1997). Results from the current survey showed an overall decline in Hare occurrence throughout each of the land class groups (Cooper et al., 1997) (Figure 5); (Tables 9 & 10). The largest decline in the percentage occurrence of Hares was in land class 2, (Lakeland) (Figure 5), (Tables 9 & 10).
However, this result is affected by the limited sample size (Table 9). There were also large declines in land classes 5 and 6 (marginal & settled uplands) in which a large proportion of the squares surveyed had no evidence of current Hare activity compared to the earlier survey (Figure 5); (Table 9 & 10). The land class showing the least change was land class 7 (high uplands) in which a large proportion of sites had Hares present in both surveys (Figure 5); (Table 9 & 10). This land class is likely to be least affected by the problems of Hare detection due to increased vegetation height. Table 8. Land class groups used in the analysis of Hare survey data (Cooper et al., 1997). Land Class Group 1 2 3 4 5 6 7 8 Description Drumlin Farmland Lakeland Marginal Lowlands Central Lowlands Marginal Uplands Settled Uplands High Uplands Mountains
Table 9. Number of squares showing a loss, gain or similar results in Hare observations in different land class groups since Dingerkus (1997). Land Class Type Gain Loss Same Absent Same Present Total 1 1 8 9 5 23 2 3 1 4 3 2 8 7 2 19 4 10 13 3 26 5 1 14 5 4 24 6 9 4 3 16 7 3 9 12 8 5 2 4 11 Grand Total 4 60 41 30 135
Table 10. Comparison of the percentage occurrence of Hares in 1km2 areas in different land class types since Dingerkus (1997). Land Class Type % Occurrence, 2002 % Occurrence, 1997 1 26 57 2 0 7 5 3 21 53 4 12 50 5 21 75 6 19 75 7 75 10 0 8 36 82 Total 25 67
3.3 Creation of a library of genetic markers. 3.3.1 Scientific Background for the Genetic Work The mountain (Irish) hare (Lepus timidus hibernicus, Linnaeus 1759) is thought to be one of Ireland’s longest established mammals with the oldest dated specimen reported to be over 28,000 years old (Hayden & Harrington, 2000). The absence of a typical winter white coat, differentiate the Irish hare from other Mountain hares which occur outside Ireland. This unique morphological feature has justified a subspecies status (i.e. hibernicus) to the Irish counterpart. Despite its intrinsic conservation importance, as one of the original members of the Irish fauna (e.g. the Mountain hare is listed in the Irish Data book as internationally important and in Appendix II of the Berne Convention as a protected species), still very little information is available about this species population structure and dynamics (Dingerkus & Montgomery, 2002). Indeed even the systematic status of the species is not entirely resolved. This lack of adequate information, which is essential for the proper management and conservation of this irreplaceable native Irish mammal, has motivated preliminary genetic investigation on the species (Hayden & Harrington, 2000). Microsatellites are basically variable non-coding regions of nuclear DNA, consisting of short tandemly repeated sequences of typically 1 - 6 bp long (Tautz, 1989). Given their multi-allelic nature and therefore high information content, microsatellites (or microsatellite DNA profiling) have become the standard methodology for the genetic analysis of natural populations. Indeed, these powerful markers have been employed to address a number of important biological questions related to a number of different fields (e.g. conservation genetics, management, forensics) and including, for instance: describing population structure and levels of genetic diversity; comparing dispersal patterns and gene flow; mating system and social structure; recent immigration history; origin of particular individuals; and population census (reviewed in Estoup & Angers, 1998). A very limited number of interesting studies have been carried out in lagomorphs using microsatellites. Surridge et al., (1999a) investigated the level of population genetic structure of European wild rabbits (Oryctolagus cuniculus) from 17 sites across the East Anglian region of Britain. Their results indicated the
existence of a substantial degree of population subdivision for the species. This was attributed to the combined effects of the social structure and random genetic drift acting on bottlenecked populations after myxomatosis. In a subsequent study, Surridge et al. (1999b) have further examined the population genetic structure of a singly free-living tagged population of European wild rabbits for two consecutive years. They have shown that a specific social behaviour, namely the formation of stable breeding groups, strongly influenced the genetic structure of the population. In addition, they have demonstrated that these breeding groups constitute genetically distinct units with low levels of gene flow between them. These earlier results contrast with more recent investigations carried out in other Lagomorphs. For instance, Burton et al. (2002), also using microsatellites, examined the population genetic structure of the cyclic snowshoe hare (Lepus americanus) in south western Yukon, Canada. This survey, which comprised over 300 specimens from 12 sites separated by distances ranging from 3 to 140 km and also included samples from Montana and Alaska (US), indicated a high level of genetic diversity, but an overall low level of genetic differentiation. Thus the authors suggest that despite observed dramatic fluctuations in density, snowshoe hares in this area, have a large evolutionary effective population size and are not strongly subdivided by either physical or social barriers to gene flow. The importance of theses previous studies is obvious in that similar species, subject to distinct evolutionary and ecological pressures are likely to respond in different ways. Thus it is possible that the social system of the Irish hare, similar to what has been suggested for other lagomorphs (Bell, 1983), has probably evolved in direct response to a number of adaptive ecological pressures, which includes predation and competition for patchily distributed key survival resources such as food and shelter. This hypothesis, however, has yet to be tested. This research work, which was carried out on behalf of the Environment and Heritage Service, had as its primary goal to develop a microsatellite library for the Irish hare and to establish a panel of microsatellite markers for subsequent studies on several aspects of the Irish hare population structure and dynamics.
Specific Objectives: 1- To evaluate and optimise non-destructive/minimum invasive method of DNA extraction for the Irish hare 2- To evaluate and to optimise five to ten microsatellite markers originally developed for other lagomorph species to enable high resolution population genetic studies in the Irish hare (Lepus timidus hibernicus). 3- To develop an enriched microsatellite genomic library for L. timidus hibernicus to enable additional species specific microsatellite markers to be readily isolated. 4- To gather preliminary descriptive information on genetic diversity of the Irish hare. 3.3.2 Materials, Methods & Results Sampling Samples of both hair and tissue were used to obtain DNA for this investigation. A collection of hair samples from 171 Irish Hares specimens were kindly provided from colleagues in University College Dublin. These were samples that had been collected by a previous postgraduate student (Dr. R. Hamill). The samples had been obtained from live Hares at coursing meetings in southern Ireland. In addition to this material, further samples of hair and tissue from road killed Hares was also collected (N = 6). These samples came from various sources and included the general public and members of staff from both the Queen’s University of Belfast and the Environment and Heritage Service. 1. To evaluate and optimise non-destructive/minimum invasive method of DNA extraction for the Irish hare A very important practical feature of microsatellites is that only small amounts of DNA are required for genetic screening. Thus, both nondestructive/minimum intrusion sampling (e.g. hair-follicle; blood, etc.) and analyses of old archival samples (e.g. preserved skins, teeth, dried tissue) are readily feasible.
DNA extraction protocol for hair follicle samples To extract DNA from hair follicle samples we employed a protocol initially suggested by Dr. R. Hamill (UCD) with minor modifications. The DNA extraction buffer consisted of 100µl 10X Bioline reaction buffer, 100 µl 25mM MgCl2, 5 µl Tween 20, 6 µl Tergitol type NP-40 (liquid form-melt at 60oC for 5 min on a heating block), 765 µl sterile distilled water, and 24 µl 20mg/ml Proteinase K (to be added to the samples prior to incubation). In order to determine the optimum ratio between buffer/tissue to yield suitable DNA for PCR, a number of combinations were carried out as follows: From each of three Irish hare specimens, three and six hair root follicles were extracted in 50 µl and 100 µl of the extraction buffer following the layout displayed in Table 2.
Irish hare specimen IH1 IH2
Extraction Volume/Root material 50 µl 100 µl 3 roots (1) 3 roots (3) 6 roots (2) 6 roots (4)
Table 2. Experimental layout to establish optimum extraction buffer/tissue ratio to yield sufficient good quality DNA material for genetic screening. Three replicates were carried out for each tissue/volume combination (i.e. 1, 2, 3, and 4). From each of the resulting 12 extractions, three volumes (1µl, 2µl and 4µl) were used as DNA template for genetic screening.
All extractions were carried out with great care to avoid cross contamination. Hairs were selected from the specimen (clump of Irish hare hair kindly provided by Dr Ruth Hamill - UCD) using watchmakers’ tweezers. The hair follicle was carefully dissected leaving about 2mm from the hair. The appropriate number of hairs (see Table 2) was carefully placed inside a tube containing the extraction buffer. Each specimen was extracted in duplicate both to ensure for an adequate quantity of DNA for subsequent PCR reactions, and to check for crosscontamination. Each specimen hair tissue + extraction buffer was subsequently incubated in a thermocycler at 60oC for 45 min. Tubes were briefly vortexed prior centrifugation for 1 min at 14000rpm on a microcentrifugue. Tubes were then placed in a thermocycler at 95 oC for 15 min. to inactivate the Proteinase K enzyme before new centrifugation at 14000rpm for 1 min. Results indicated that the optimum buffer/tissue ration was six hair follicles extracted with 50µl of extraction buffer. Reactions were stored at -20oC until required for PCR.
2. Evaluation of available lagomorph microsatellite primer sets. A number of microsatellites developed for the European wild rabbit (Oryctolagus cuniculus) are both currently available in the literature and/or into GeneBank online DNA database (e.g. Rico et al. 1994, Mougel et al. 1997, Surridge et al 1997). Although the cross-species potential of microsatellites is limited, it is well know that microsatellites developed for a particular species usually will work in other close related species. Indeed Surridge et al (1997) have demonstrated the potential of some of the microsatellite markers developed for the European wild rabbit for some 20 other species of lagomorphs including the European hare Lepus europeaus and the Mountain hare L. timidus. Thus, following considerable examination of all available information currently available, for the presentt study, a total of 18 European rabbit microsatellites were chosen for subsequent evaluation as genetic markers for the Irish hare. Although primer sequences for these microsatellites are available in the literature, in some instances new primer sets were re-designed to allow for better resolution. Each microsatellite primer set was individually tested on one Irish hare and two European rabbit (control) for amplification of products of expected sized and for initial optimisation of PCR conditions. PCR were carried out in 12ul reaction volume comprising 1X Promega Taq polymerase buffer, 1.5mM MgCl2, 100uM dNTPs, 10pM of each microsatellite primer, 100ng template DNA, and 0.5U of Promega Taq polymerase. Cycling conditions consisted of one cycle at 940C for 3 min followed by 30 cycles of 1min at 940C, 1 min at the optimum Tm oC, (see Table 1 for details) and 1 min at 720C. Resulting amplified products were separated and subsequently visualised on an Ethidium Bromide stained 2.5% 1X TBE agarose gel (Figure 1). Twelve microsatellite primer sets produced a clear, reliable amplification pattern and illustrated in Figure 1. Six microsatellites failed to cross amplify with the Irish hare in spite of producing a fragment of expected size in the European rabbit controls. These primer sets were tested at least three additional times under less stringent PCR conditions to ensure result consistency. Given that no positive results were obtained, these primers were not tested further.
Figure 1. Image illustrating a Ethidium Bromide stained agarose gel image with five microsatellite markers (Sat-3, Sat-5, Sat-13, Sat-16, and Ssol-33) tested in the Irish Hare. Three specimens were tested per primer, two specimens of the European rabbit (Oryctolagus cuniculus – numbers 1 & 2 for Sat-3, Sat-5, Sat-13 and Sat-16 and 2 & 3 for Ssol-33) and one Irish Hare (Lepus timidus hibernicus). Notice the products of expected size for Sat-5, Ssa-13, and Ssa-16 loci. Primers sets for these loci were subsequently used for genetic screening in the Irish hare. The Ssa-3 primer set yielded inconsistent results for the Irish hare and, as such, was not considered further. Ssol-33 failed to amplify a product in the Irish hare and thus it was also discarded. M – Marker size lane (100 base pair ladder).
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 M
300 200 100 bp Sat-3 1 2 Sat-5 1 2 Sat-13 1 2 Sat-16 1 2 Ssol-331 M 2
The 12 primer sets that yielded positive clear amplification were evaluated further. For each one of these, both primers were tested for end-labelling (32P) by manually screening 4 individual Irish hare. This was carried out to test for: (1) optimum primer to be subsequently end-labelled with fluorescent dyes (i.e. required for automated screening); (2) to assess the multiplexing (co-amplification of more than one microsatellite primer set in a same PCR reaction) likelihood of these primers sets; and (3) to gather preliminary information on the variability of the marker. Although poorly recorded in available literature, screening a given locus with alternate end-labelled primers can yield quite significant different results both in terms of resolution and band mobility. Ten microsatellites produced a positive signal (amplification of a DNA product of expected size) and thus, were further optimised on a Li-Cor™ dual laser automated DNA analyser. Two microsatellites were found give inconsistent results and thus, were eliminated from further testing. Of these 10 microsatellite markers, eight primer sets have been fully optimised and are now routinely used for screening (see Figure 2 and Table 1). Two microsatellite markers, although showing promising results, have still to be fully evaluated for automated screening. All primer sets were tested, based on the size of their amplified PCR product, for multiplexing (i.e. the possibility to amplify products from two or more loci in a single PCR reaction). This was carried out by careful manipulation of PCR conditions and strategic use of fluorescent labels. Thus far, two
microsatellite primer sets effectively multiplexed together under the same PCR conditions (D1L5 B11 and D1L2 B4) thereby reducing time and cost for screening.
Table 1. Microsatellite primers tested for Irish Hare during this investigation. Eighteen primers were originally tested (see text) and eight are now ready for use in subsequent genetic studies. The initial usefulness (level of polymorphism here illustrated by the observed number of alleles per locus) of these markers was assessed by screening 171 specimens from a number of sites in the Republic of Ireland (samples kindly provided by Ruth Hamill – UCD). * indicates the primer sets that may be multiplexed together.
Microsatellite locus D1L1 D D1L5 G7 D1L5 B11* D1L2 B4* OCM 1SAT OCM SAT 5 SOL 33 SAT 16 SAT 5 SAT 13 SAT 3 OCM SAT2 SOL 03le SOL 33le SOL 44 D1L1 G3 D1L3 H7-1 OCM SAT3 Amplify product of expected size (low PCR stringency) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes No No No No Amplify product of expected size (high PCR stringency) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No/non-specific No/non-specific Final Tm (anneling Final Primer temperature) and number concentration of cycles 51 x 28 52 x 28 51 x 27 51 x 27 51 x 28 51 x 26 55 x 25 55 x 25 52 x 28 0.5pM 3pM 1pM 2pM 0.5pM 0.5pM 2.5pM 1pM 5pM No. of alleles 6 2 6 5 2 4 6 3 1 -
Figure 2 Gel images illustrating alleles at four microsatellite loci evaluated during this study screened for between 58 and 86 samples including with a size marker lane (triple banded lane) every 15 individuals. All microsatellites have been resolved on a 6% acrylamide gel run on the LiCor dual laser automated DNA analyser.
OCM SAT 5
3. To develop an enriched microsatellite genomic library for L. timidus hibernicus to enable additional species specific microsatellite markers to be readily isolated. The methodology applied to the development of an enriched microsatellite genomic library for L. timidus hibernicus follows a modified protocol based on Kijas et al. 1994 by Prodöhl (in prep). Briefly: total Irish hare genomic DNA was digested in separate reactions with HaeIII, RsaI and AluI. Following phenol/chloroform extraction and EtOH precipitation, the restricted fragments where pooled together and size fractionated by gel electrophoresis. "Slices" containing DNA fragments ranging from 150 to 800 bp were removed from gel. The resulting purified DNA fragments were ligated to EcoRV digested and dephosphorylated pBluescript II phagemid vector. This ligation was used as template for an asymmetric PCR reaction. The resulting single stranded PCR products were hybridised to biotynilated synthetic oligonucleotides (e.g. GAA, GATA & GACA) attached to magnetic beads. Following a series of low and high stringency washes, the released enriched fraction was used as template for a standard PCR reaction. The resulting products were double digested with EcoRI and HindIII, and ligated into EcoRI/HindIII digested pBluescript II phagemid vector.
The library construction is the most delicate stage involved in the isolation of microsatellite markers. Results from a series of strategic controls build into our protocol indicate that the resulting library is of good quality and that new Irish hare microsatellite could be readily isolated for subsequent genetic studies. 4. To gather preliminary descriptive information on genetic diversity of the Irish hare. In order to assess the usefulness of the microsatellite markers evaluated during this study, hair samples from 171 Irish hare specimens kindly provided by Ruth Hamill (UCD) were screened. Results (Table 1) indicate that the Irish hare possesses a comparatively low level of genetic diversity in comparison to other lagomorphs for which microsatellite data is available (i.e. 1 – 6 alleles v.s. 9 – 16 for the European rabbit and 4 – 36 for the snowshoe hare). This is likely to reflect relatively small effective population sizes and re-emphasize the urgency and need of further genetic information for the Irish hare. From a conservation viewpoint it is important to establish if this observed reduced level of genetic diversity is the result of ongoing human mediated pressure on local populations (e.g. habitat change; coursing activity) and/or historical evolutionary processes (e.g. bottleneck, founder effect) related to the phylogeographic history of the species. More recently, samples of tissue and hair were also acquired from live Hares (N = 53) collected by the Dungannon Hare Coursing Club. DNA has been extracted from all samples following methodology described above, and they are now being screened for these microsatellite markers. Results of analysis should be available within one week.
3.3.3 Discussion We have successfully evaluated and implemented a number of microsatellite markers that can be immediately used to further population genetic studies in the Irish hare. If required, additional species specific markers can be readily isolated from the microsatellite enriched library developed. These markers, in combination with non-invasive DNA extraction methodology can be used to address a number of fundamental biological questions involving the Irish hare. For instance, these markers can be used to assess current levels of
genetic diversity within the Irish hare on mainland Ireland and offshore island populations. This comparison could be carried out to investigate the possible genetic impact of the limited local genetic introductions of the Brown hare on the Irish hare (Thulin et al. 1997) locally and throughout its range; to establish likely breeding systems and population structure; and to investigate putative dispersal rates from local populations. These markers, in addition to new analytical procedures could also be used to obtain estimates of density of local populations. It is well know that the assessment of the relative abundance or density of a particular animal species is essential for the study of their population dynamics and for their proper conservation and management.
4. Overall Discussion Results from the night driven transect surveys produced a density estimate of 1 Hare per square kilometre in Northern Ireland. These results are similar to those obtained by Dingerkus in the late 1990s using the more laborious method of day walked squares. The greatest number of Hares observed was in County Antrim, particularly in upland areas. Lowest numbers were found in County Tyrone. In general, upland areas were found to have a higher number of observed Hares. This is probably a result of reduced disturbance in these remote areas and the less intensified nature of farming practices. Upland areas will have fewer arable and improved pasture fields and more unimproved, rough pasture grazed by cattle and sheep. These upland fields dominated by a greater variety of grass species and rushes not only provide a more varied diet but safer refuges for Hares. Due to the small number of Hares observed during the recent survey, it was not possible to carry out any meaningful statistical comparisons between the results obtained for night driven transects and day walked squares. However, due to the similarity in density estimates between the current survey and the more labour intensive study carried out by Dingerkus in the late 1990s, night driven transects may offer reliable, inexpensive and rapid means of estimating Hare abundance. Results of Hare observations from the re-survey of Dingerkus day walked squares are disappointing. In all cases they show a large decline in Hare numbers throughout Northern Ireland and within each land class type. These 31
surveys used a similar methodology to the previous surveys but, critically, were carried out at a different time of year. Dingerkus (1997) carried out surveys in the winter and early spring when vegetation height was low and Hares could be easily seen. However, the current surveys were carried out in the late spring/early summer. This resulted in huge problems with vegetation height that were exacerbated by an extremely wet early summer of 2002. In most areas surveyed field parcels were composed of either uncut silage or semi-mature arable crop. It was almost impossible to observe Hares in long grass unless they were flushed from ground and unfeasible to wade through fields of crops. Therefore, the results of the diurnal survey repeated here cannot be compared with the original survey carried out by Dingerkus (1997). There is no solid evidence to suggest that Hare numbers have declined since the mid 1990s. Results from the current investigation suggest that the population size in Northern Ireland may range between 7,000 and 25,000 individuals. Therefore, the population appears to be stable, albeit at a low density. It is recommended that future monitoring of the Irish Hare population should be carried out extensively throughout Northern Ireland using the Night Driven Transect methodology in order to detect future changes in Hare numbers. A re-survey should be carried out in spring 2004 and repeated at 2-yearly intervals. In addition, the following recommendations should be considered. They include those defined by Dingerkus (1997) which have been re-emphasised, as to date none of them have been implemented. PRACTICAL RECOMMENDATIONS (1-5 as per Dingerkus, 1997). • Improvements in agricultural practice to allow for:
1. The production of a more species rich grass sward to allow Hares a more varied diet and promote survival of leverets. 2. Controls on livestock grazing levels to prevent disturbance to 'nesting' Hares. 3. Controls on the timing of silage cutting to prevent leveret and adult Hare deaths during the breeding season. 4. Changes in the method of silage cutting - working from the inside out would allow 'nesting' Hares and leverets to escape.
5. The provision of Hare refuge sites or buffer areas within farmland that allow rough vegetation to flourish. This would promote wildlife in the countryside in general. • • • Hare friendly agricultural practice in ESAs and CMS schemes. Removal of the Irish Hare from the quarry list and protection given under the Wildlife Order. Increased awareness of the plight of the Irish Hare by liaison with farming groups, the rural community and the Department of Agriculture (DARD).
RESEARCH INITIATIVES • Detailed genetic studies to establish the status and population dynamics of the Irish Hare. Re-description to species status would afford greater protection and conservation merit. This research should be complemented by ecological research detailed below. • Research into the ecology of the Irish Hare concentrating primarily on:
1. Investigations into optimal Hare habitat - reasons for the occurrence of Hare 'hot-spots'. 2. Investigations into optimal Hare diet and foraging strategies. 3. Investigations into the major causes of adult and leveret mortality. 4. Investigations into the current reproductive success of Hares. 5. Investigations into the behaviour and reproductive strategies of the Irish Hare.
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Appendix 1. A survey sheet and an example of a survey map used during the 'Day Walked Squares' surveys.
Appendix 3. General Guidelines for Tissue Sampling and Data Recording for DNA Work. (School of Biology and Biochemistry - QUB & DOE - EHS).
IRISH HARE: - GENERAL GUIDELINES FOR TISSUE SAMPLING AND DATA RECORDING FOR DNA WORK (School of Biology and Biochemistry - QUB & DOE - EHS) The Irish hare is believed to be one of Ireland’s longest established mammals. The absence of a typical winter white coat, differentiate the Irish hare from other Mountain hares which occurs outside Ireland. Despite its intrinsic conservation importance, as one of the original members of the Irish fauna (e.g. the Mountain hare is listed in the Irish Data book as internationally important and in Appendix II of the Berne Convention as a protected species), still very little information is available about this species population structure and dynamics. This lack of adequate information, which is essential for the proper management and conservation of this irreplaceable native Irish mammal, has motivated preliminary genetic investigation on the species. Using modern DNA forensic based techniques, we hope to address a number important biological questions regarding the species including, for instance: describing population structure and levels of genetic diversity; dispersal patterns; mating system and social structure; recent immigration; specific origin of particular individuals; and population census. In order to fully achieve these objectives, however, we need to be able to sample as many Irish hare specimens as possible. Thus we would like to kindly ask for assistance from all members of the general public that might be able to help us with sampling and hence to assist with this important project. A very important practical feature of modern DNA forensic based techniques is that only small amounts of DNA is required for analysis. Thus, non-destructive sampling (hair-follicle) and old archival samples (preserved skins, teeth, and dried tissue) can be readily used for analysis. However, a number of easy to follow steps must be followed to ensure data quality/accuracy and thus meaningful biological interpretation of results. HAIR FOLLICLE:- Hair follicles offer an excellent alternative for an easy and non-destructive sampling. Good quality DNA can be readily obtained from this material for subsequent DNA work. Material required:a small plastic bag (for instance a coin bag, 3.5 x 5 in or 9x12.5cm), a pencil and a piece of paper (‘sampling kits’ can be provided upon request). Procedure: 1- from a captured/dead individual, remove a ‘clump’ of hairs (using your thumb and fore finger). Where possible select the darker/longer hairs as these have larger hair follicles, which yield DNA for better quality/quantity. Hair follicles can be easily seen by the naked eye; 2- carefully place the hair clump inside the plastic back making sure that all is inside; 3- On a piece of paper write down the specific location where the hare was initially found, date of capture, size, and sex of the individual (where possible), and include this information inside the plastic bag; 4 – place the plastic bag inside an envelop and post it to “The Irish hare Hair follicles conservation genetics project” at the School of Biology and Biochemistry, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road - Belfast BT9 7BL. Alternatively call Dr Jane Preston (02890272259) so that other arrangements can be made for collection. GENERAL COMMENTS: 1. Where more than one individual is sampled, the collector could elaborate an individual code. For instance XX1; XX2, XX3, etc, where the ‘XX’ stands for the abbreviation of a particular location or name. It is important, however, that any code is explained in the accompanying piece of paper inside individual bags and that a six figure grid reference is supplied for each sample. 2. Give 3. Any relevant information (in particular sex) referent to the sampled individual should be recorded in the piece of paper 4. Ensure that you have disposed of all hair from a given specimen prior sampling any additional individual to avoid cross-contamination.