The Northern Ireland Irish Hare Lepus timidus hibernicus Survey

2002

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
 Contents
Page
Executive Summary 1
1. Background to the Current Project 5
2. Objectives 7
3. Methods 7
3.1 Estimates of Hare Abundance 7
3.1.1 Method 1 - Night Driven Transects (NDT) 8
3.1.2 Method 2 - Day Walked Squares (DWS) 11
3.2 Results 13
3.2.1 Night Driven Transects 13
3.2.2 Day Walked Squares 16
3.3 Creation of a library of genetic markers 20
3.3.1 Scientific Background for the Genetic Work 20
3.3.2 Materials, Methods & Results 22
3.3.3 Discussion 28
4. Overall Discussion 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.

1
 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). Non-
destructive 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.

3
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.

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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

7
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.

9
10
11
 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.

12
13
 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

14
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 Total distance driven
observed (km)
Co. Armagh 17 250
Co. Londonderry 7 212
Co. Fermanagh 14 289
Co. Tyrone 4 266
Co. Antrim - Upland 110 365
Co. Antrim - Lowland 24 347
Co. Down - Lowland 9 242
Co. Down – Upland 54 310
Total 239 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.

15
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 Total distance

observed driven (km)
Co. Antrim - Upland (Spring) 96 278.9
Co. Antrim - Upland (Autumn) 20 371.4
Co. Down – Upland (Spring) 70 374
Co. Down – Upland (Autumn) 3 236.8
Combined Lowland (Spring) 13 484.3
Co. Down - Lowland (Autumn) 2 352.8

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 Lower 95% Upper 95%
(mean) confidence limit confidence
limit
Hare Density per km2 1.0 0.5 1.8
Estimated Total No. of 14,000 7,000 25,200
Hares

16
Table 5. Estimate of the density of Irish Hare in Spring and Autumn spotlight
counts in County Antrim (DISTANCE 3.5) (Looney, 2001).

Estimate Lower 95% Upper 95%

(mean) confidence limit confidence
limit
Hare Density per km2 1.76 1.32 2.3
(Feb 1997, 1998, 1999)
Hare Density per km2 1.28 0.56 2.4
(Oct 1997, 1998)

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).

17
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 No. of Squares

Gain in presence of Hares 4
Decline in presence of Hares 60
Same where Hares were found to be 41
absent
Same where Hares were found to be 30
present
Total Number of Squares Re-surveyed 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).

18
19
20
 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 Description

Group
1 Drumlin Farmland
2 Lakeland
3 Marginal Lowlands
4 Central Lowlands
5 Marginal Uplands
6 Settled Uplands
7 High Uplands
8 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 1 2 3 4 5 6 7 8 Grand Total

Gain 1 2 1 4
Loss 8 3 8 10 14 9 3 5 60
Same Absent 9 1 7 13 5 4 2 41
Same Present 5 2 3 4 3 9 4 30
Total 23 4 19 26 24 16 12 11 135

Table 10. Comparison of the percentage occurrence of Hares in 1km2 areas in different
land class types since Dingerkus (1997).

Land Class Type 1 2 3 4 5 6 7 8 Total

% Occurrence, 26 0 21 12 21 19 75 36 25
2002
% Occurrence, 57 7 53 50 75 75 10 82 67
1997 5 0

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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

22
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.

23
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 non-
destructive/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.

24
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 Extraction Volume/Root material

50 µl 100 µl
IH1 3 roots (1) 3 roots (3)
IH2 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 cross-
contamination. 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.

25
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.

26
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 Sat-5 Sat-13 Sat-16 Ssol-331 2

3 1 2 3 1 2 3 1 2 3 1 2 3 M

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

27
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.
Amplify product of Amplify product of Final Tm (anneling
Microsatellite Final Primer No. of
expected size expected size temperature) and number
locus concentration alleles
(low PCR stringency) (high PCR stringency) of cycles
D1L1 D Yes Yes 51 x 28 0.5pM 6
D1L5 G7 Yes Yes 52 x 28 3pM 2
D1L5 B11* Yes Yes 51 x 27 1pM 6
D1L2 B4* Yes Yes 51 x 27 2pM 5
OCM 1SAT Yes Yes 51 x 28 0.5pM 2
OCM SAT 5 Yes Yes 51 x 26 0.5pM 4
SOL 33 Yes Yes 55 x 25 2.5pM 6
SAT 16 Yes Yes 55 x 25 1pM 3
SAT 5 Yes Yes 52 x 28 5pM 1
SAT 13 Yes Yes - - -
SAT 3 No - - - -
OCM SAT2 Yes No/non-specific - - -
SOL 03le No - - - -
SOL 33le Yes No/non-specific - - -
SOL 44 No - - - -
D1L1 G3 No - - - -
D1L3 H7-1 No - - - -
OCM SAT3 No - - - -

28
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.

Sol-3 OCM SAT 5

D1L5 G7 D1L1 D

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.

29
 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

30
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.

32
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.

33
References

Bell, D. J. (1983). Mate choice in the European rabbit. In: Mate Choice (ed. Bateson,
P.P.), pp. 211-223. Cambridge University Press, Cambridge.

Biodiversity In Northern Ireland. Northern Ireland Species Action Plans - Irish Hare,
Chough, Culew. Environment and Heritage Service, October 2000.

Buckland, S. T., Anderson, D. R., Burnham, K. P. & Laake, J. L. (2003). Distance

Sampling: Estimating Abundance of Biological Populations. [Online] Available
http://dolphin.mcs.st-and.ac.uk/distancebook/download.html Downloaded 20/01/03

Burton, C.; Krebs, C. J. & Taylor, E. B (2002). Population genetic structure of the cyclic
snowshoe hare (Lepus americanus) in southwestern Yukon, Canada. Molecular
Ecology 11: 1689-1701.

Cooper, A., Murray, R. and McCann, T. (1997). The Northern Ireland Countryside
Survey.

Dingerkus, S. K. (1997). The distribution and ecology of the Irish hare Lepus timidus
hibernicus in Northern Ireland. PhD thesis, unpublished. Queen’s University Belfast.

Dingerkus, S. K. and Montgomery, W. I. (1997). The distribution of the Irish Hare (Lepus
timidus hibernicus) in Northern Ireland and its relationship to land classification.
Gibier Faune Sauvage 14: 325-334.

Dingerkus, S. K. & Montgomery, W. I. (2001). The diet and landclass affinities of the
Irish Hare Lepus timidus hibernicus. Journal of Zoology 253: 233-240.

Dingerkus, S. K. & Montgomery, W. I. (2002). A review of the status and decline in

abundance of the Irish Hare (Lepus timidus hibernicus) in Northern Ireland. Mammal
Review 32: 1-11.

34
Edwards, P. J., Fletcher, M. R. & Berny, P. (2000). Review of the factors affecting the
decline of the European brown hare, Lepus europaeus (Pallas, 1778) and the use of
wildlife incident data to evaluate the significance of paraquat. Agriculture, Ecosystems
and Environment 79: 95-103.

Estoup, A. and Angers, B. (1998). Microsatellites and minisatellites for molecular

ecology: Theoretical and empirical considerations. In Advances in Molecular Ecology
(ed. Carvalho, G. R.). IOS Press, Amsterdam, pp. 55-86.

Fairley, J. (2001). A Basket of Weasels. Privately Published, Belfast.

Game Preservation Act (NI), 1928 as amended by the Game Preservation (Amendment)
Act (NI), 2002

Hutchings, M.R. & Harris, S. (1996). The current status of the brown hare (Lepus
europaeus) in Britain. Peterborough: Joint Nature Conservation Committee.

Hamill, R. (2001). A study of the genetic structure and phylogeography of Lepus timidus
L. subspecies in Europe, using microsatellite DNA and mt DNA. PhD thesis,
unpublished. University College, Dublin.

Hayden, T. & Harrington, R. (2000). Exploring Irish mammals. Town House and Country
House Ltd.

ICUN (2000). IUCN Red List of Threatened species. The World Conservation Union,
Switzerland.

Looney, D.J.P. (2001) The ecology of the Red Fox Vulpes vulpes in relation to sheep
farming in County Antrim. PhD thesis, unpublished. Queen’s University Belfast.

Mougel, F., Mounolou, J-C. & Monnerot, M. (1997). Nine polymorphic microsatellite
loci in the rabbit, Oryctolagus cuniculus. Animal Genetics 28: 58-59.

35
O'Mahoney, D. (2001). The Distribution, abundance and habitat use of the Irish Hare
(Lepus timidus hibernicus) in upland and lowland areas of Co. Antrim and Co. Down.
A report to The Environment & Heritage Service, Department of the Environment, N.I.

Reynolds & Tapper, 1995. Predation by foxes Vulpes vulpes on brown hares Lepus
europaeus in central southern England, and its potential impact on annual population
growth. Wildlife Biology.

Rico, C. Rico, I. Webb, N. et al. (1994). Four polymorphic microsatellite loci for the
European rabbit, Oryctolagus cuniculus. Animal Genetics 25: 367.

Surridge, A. K., Bell, D. J., Rico, C. & Hewitt, G. M. (1997). Polymorphic microsatellite
loci in the European rabbit, Oryctolagus cuniculus. Animal Genetics 28: 302-305.

Surridge, A. K., Bell, D. J., Ibrahim, K. M. & Hewitt, G. M. (1999a). Population structure
and genetic variation of European wild rabbits (Oryctolagus cuniculus) in East Anglia.
Heredity 82: 479-487.

Surridge, A. K., Ibrahim, K. M., Bell, D. J., Webb, N. J., Rico, C. & Hewitt, G. M.
(1999b). Fine-scale genetic structuring in a natural population of European wild rabbits
(Oryctolagus cuniculus). Molecular Ecology 8: 299-307.

The Decline of the European Hares. An interdisciplinary European Research Task. Berlin
18-22 April, 2001.

The Wildlife (Northern Ireland) Order, HMSO SI 1985/171 (NI 2).

Thulin, C.G., Jaarola, M and Tegelstrom, H. (1997). The occurrence of mountain hare
DNA in wild brown hares. Molecular Ecology 6: 463-467

Wolfe, A. & Hayden, T. J. (1996). Home range sizes of Irish mountain hares on coastal
grassland. Biology and Environment 96B: 141-146.

36
Wolfe, A., Whelan, J. & Hayden, T. J. (1996). The diet of the mountain hare (Lepus
timidus hibernicus) on coastal grassland. Journal of the Zoological Society of London
240: 804-810.

37
Appendix 1. A survey sheet and an example of a survey map used during
the 'Day Walked Squares' surveys.

38
39
40
41
42
43
Appendix 3.
General Guidelines for Tissue Sampling and Data Recording for DNA Work.
(School of Biology and Biochemistry - QUB & DOE - EHS).

44
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.

45