<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>24047755
                                                                                            Universiteit
               2    Universiteit
                                                                                            Antwerpen
             63
                                                    vereniging
         Onderzoeksvoorstel
         Onderzoek             naar     de   verspreiding en het voedingsgedrag
         van     grijze     wolven         (Canis lupus) in Nederland
         Studieperiode:          maart 2023      —
                                                    28 februari 2026
         Studiegebied: Gerapporteerde                 wolvenhabitat in Nederland
         Draft Research                   proposal
         A   study       on    distribution and           feeding behaviour              of grey
         wolves         (Canis lupus)           in The Netherlands
         Study period: March 2023 February 28" 2026
                                                —
         Study area: Reported wolf habitat in The Netherlands
         Dit driejarige onderzoeksvoorstel is  een samenwerking  tussen de Nederlandse Zoogdiervereniging,
         Nijmegen,   het Instituut voor Milieuwetenschappen Leiden, Nederland en de afdeling Evolutionaire
         Ecologie, Universiteit Antwerpen, Belgié.
                                                                                                           0000000026
</pre>

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<pre>24047755
         Table of Contents
             Nederlandse   samenvatting                              WwW
                                                                     NO
             Introduction
                Distribution of grey wolf  (Canis lupus) in Europe
                Feeding ecology    of grey wolf
                Conflicts between wolves and humans                  OO
                Objectives                                         —  -
             Materials and methods                                 14
                Study  area                                        14
                Assessment of diet    composition                  16
                Prey preference   and avoidance                    17
                Livestock attacks in the  study  area              17
                Data  analysis  and statistics                     17
             Proposed work schedule                                19
             Budget                                                20
             References                                            21
                                                                         0000000027
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<pre>24047755
         Nederlandse                     samenvatting
         Het voortdurende proces          van   de herkolonisatie        van  wolven in Nederland,      een   door   mensen
         sterk gedomineerd gebied,           laten de    spanningen opnieuw           opvoeren die historisch      geleid
         hebben tot de      uitroeiing   van  de wolf in het      grootste    deel   van  Europa.  De basis   van    het
         conflict is de  voedingsecologie         van   de  wolf, die mogelijk economische           en emotionele schade
         kan veroorzaken door aanvallen op              vee en     huisdieren,     en  concurrentie met de     jachtsector.
         Daarnaast    rijst  de vraag of door de        mens    gedomineerde landschappen             een  duurzame
         hoeveelheid wilde        prooien   bieden    voor   wolven of dat wolven overgaan op tamme                prooien   als
         er niet genoeg wilde        prooien   beschikbaar      zijn.   Bovendien is de invloed      van   de wolf op het
         gedrag   van  herbivoren in Nederland onbekend. Het is daarom                      van  groot belang   om
         nauwkeurige      informatie     over  de  voedingsecologie         van   de Nederlandse wolf te      verkrijgen    om
         beleidsvorming       te ondersteunen       en  de   verspreiding      van   misvattingen   onder het   grote publiek
         te voorkomen,       en  als  zodanig    het samenleven met           een   roofdier dat  aan  de top  van   de
         voedselketen staat te        vergemakkelijken.
         Om inzicht te    krijgen    in het voedingsgedrag          van   de wolven in Nederland, stellen        we een
         dieetanalyse    voor    op basis   van een      combinatie      van   DNA    gebaseerde    methoden     (i.e.
         metabarcoding)        en  mechanische       benaderingen (i.e. microscopische haaranalyse).                 Dit
         voorstel is  gebaseerd        op een   succesvolle      pilot  studie   naar  wolven diéten op de Noord
         Veluwe, die werd uitgevoerd door de universiteit Antwerpen                      en  de universiteit Leiden in
         samenwerking        met de    Zoogdiervereniging         en   het Instituut   voor  Natuur  en  Bos onderzoek in
         Antwerpen gedurende 2021. Hierbij               werden beide        genoemde      technieken   getest   en   het bleek
         dat beide methoden zwakke             en  sterke   punten hebben. Op 14 september 2022 vond                   een
         presentatie plaats      van    deze  pilot in Emma’s Oord          (Kroondomein)      voor een   groot   aantal
         beheerders    en    vertegenwoordigers         van   Provincies     en   gemeenten. Uit    de  pilot studie bleek
         dat door DNA      gebaseerde       en  mechanische         benaderingen       te combineren    en  de resultaten te
         vergelijken,  de beschreven         problemen      van    beide methoden worden          opgelost   en  de
         voedingsanalyse        wordt versterkt.
         Het hoofddoel      van   dit onderzoek is het       bepalen     van   de  voedingssamenstelling       van   grijze
         wolven in Nederland         en  grensregio's,     met   behulp    van   de twee    complementaire     vormen van
         voedingsanalyse (mechanische              en   op DNA      gebaseerde benaderingen)          uit wolven drollen,     en
         het opzetten    van een      monitoringprogramma           voor   de   lange termijn.   Wolven drollen zullen
         worden verzameld         gedurende      drie  jaar  door    vrijwilligers  van  het  wolvenmeldpunt,      door MSc
         studenten uit Leiden        en  Antwerpen,      door onderzoekers          en  door lokale  parkmanagers.       Het
         zwaartepunt     van    deze studie omvat de         vijf regio's   in Nederland     waar  wolven   zijn gemeld: 1)
                                                                                                                              3
                                                                                                                                 0000000028
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<pre>24047755
         Noord-Veluwe, 2) Midden-Veluwe, 3) Zuidwest-Veluwe, 4) Provincie Drenthe,                        en   5)  Provincie
         Zuidoost-Brabant. Het hoofddoel is           opgesplitst in de   volgende onderzoeksvragen:
         1. Wat   zijn de   belangrijkste componenten        van het dieet   van   grijze wolven in Nederland?
         2. Wat is de    prooidichtheid     in elk  van de studiegebieden     waar    wolven in Nederland
         voorkomen      en   worden deze      prooidierdichtheden weerspiegelen         in het wolven dieet?
         3. Wat is de    prooivoorkeur     van   wolven in Nederland?
         4. Wat is de    bijdrage   van vee en     andere huisdieren     aan  hun dieet?
         5. Is het  mogelijk    om  individuele wolven diéten te      bepalen (i.s.m. WENR),         en zo   ja,  is er een
         verschil in dieet tussen     vrij levende individuele wolven       en  wolven die in roedels leven?
         Output   voor   beleid  en  beheers     ondersteuning   van   de  provincies   zal zijn;
              =»  Inzicht in de variatie     van  wolven diéten   gedurende      verschillende seizoenen         over
                  verschillende     jaren  in verschillende   gebieden.
              =>» Inzicht in de    bijdrage   van  gedomesticeerde      dieren  aan   het wolven dieet      gedurende
                  verschillende seizoenen         over  verschillende  jaren.
              =»  Het   aanwijzen    van   probleem gebieden,     waar    het aandeel    van  gedomesticeerde
                  dieren in het wolven dieet relatief       hoog  is.
              =»  Genereren      van  wetenschappelijke      informatie   over  wolven    om  het  publieke
                  maatschappelijke       debat te voeden met      objectieve    informatie
              =»  Een aanzet      voor een    langdurig monitoring    programma       van  wolven diéten in
                  Nederland.
         Daarnaast     zijn  we  bezig  om een     uitwisselingsprogramma       met de    onderzoeksgroep         van   dr.
                              van  WENR, Wageningen op te starten. Verder is de codrdinatie                 van    het
         project  in handen     van  de  Zoogdiervereniging.     De eerste    zes   maanden     van   het  project    zullen
         tevens worden       gebruikt  om    het netwerk te versterken     en  te optimaliseren     via de
         terreinbeheerders in de verschillende studielocaties.            Uiteindelijk  zal na  dit  driejarige project
         een   protocol   voor een   jaarlijkse dieetmonitoring     via de  verkregen     methoden     en
         gestandaardiseerde procedures             worden  aangeleverd     aan  de   Zoogdiervereniging.
                                                                                                                             0000000029
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<pre>24047755
         Introduction
         Scientific research    on the  feeding ecology     of wolves in relation to      population structure, spatial
         movements and habitat        use   is essential for  a  better  understanding     of the role this top
         predator plays    in shaping   the structure and function of human-dominated              ecosystems.      Data
         on  diet composition    could   significantly  enhance     our  understanding of the prey species (both
         wild and   domestic) that    are  selected   by the wolves and the related        cascading effects      on  such
         ecosystems     and its functions     (Ripple, Larsen,    Renkin & Smith      2001).
         Distribution of grey wolf                 (Canis lupus)          in   Europe
         Grey wolve populations       are  known to exist in 28      European     countries.  Chapron     et al. (2014)
         suggest   that  Europe’s wolf population (more         than 12,000 individuals in      total) can  be divided
         into 10  subpopulations     which are, in    descending order,      the  following:  Dinaric-Balkan     (~3,900
         individuals), Carpathian (>3,000 individuals),          NW Iberian    (> 2,500 individuals),     Baltic  (> 1,400
         individuals),  Italian peninsula (> 800 individuals), Scandinavian (260-330 individuals),
         Karelian   (150-165 individuals), Alpine (160       individuals    grouped    in 32  packs), Central
         European     Lowlands    (150 individuals),    Sierra Morena      (probably extinct) (Figure 1,      upper
         graph).  The estimates     are  for the years 2010 to 2012. The Scandinavian, Central                European
         Lowlands and      Alpine subpopulations       were  entirely  extinct  according    to estimates from the
         1950-1970s     (Chapron    et al., 2014).
         The   population of wolves     in the Netherlands until 2020 consists of          one  pack (Veluwe)      and
         four territorial individuals  (2  females at the Veluwe,       one   male in Drenthe and      one   male in the
         Eindhoven     region) (Fig 1,  lower    graph).  Since 2015, 22 individual wolves have been
         genetically identified   in the Netherlands. Most of these wolves            originate from Germany.        At
         least 3 individuals crossed the border into        Belgium     and several wolves returned to         Germany.
         All the wolves in the Netherlands but         one are   genetically   linked to the Central    European
         Lowlands    population.   In the Eindhoven      region,   one  territorial individual  was   identified to
         originate  from the   Alpine population.
                                                                                                                           0000000030
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<pre>24047755
                                 Individuen
                                 Hl   GW1729f (vrouw)
                                 WM cwsosm (man)
                                 ¥ cwi261m (man)
                                    Gwissam (man)
                                                                        07-07-2020
                                 ),,  GW1625m  (man)
                                _Landgebruik
                                                             23.05.2020
                                                             28-05-2020
                                                             30-05-2020
         Figure 1. Upper: Distribution of grey        wolves in  Europe (EC, 2012). Lower: Wolf habitat in The
         Netherlands  (BIJ12, 2020).
                                                                                                               0000000031
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<pre>24047755
         Feeding ecology of grey                        wolf
         The  Grey wolf is      a highly adapted        carnivore       species.    Its evolution has resulted in                   a  broad
         range diet   consisting      of different prey       species of      variable size          (Peterson        & Ciucci,      2003).
         These prey    species      could be classified into six main              categories:           domestic animals, medium-
         sized wild  ungulates, large         wild   ungulates,       medium-sized mammals, small mammals and
         rodents. These      categories       have different contributions to the wolf's diet,                        depending        on the
         continent of residence         (Figure 2) (Newsome             et  al., 2016). Wolves             can    be successful         as
         predators   or  scavengers,        depending       on    the available     resources            (Stephens & Krebs, 1986)&
         Krebs  1986).
                        180.0 1
                                                                      Europe
                        160.0
                        aia
                                                                                                                      0  Rodent
                    -t
                    Q
                    5   120.0
                              1
                                                      =
                                                                                                                      B® Small mammal
                    £
                    ©
                        100.0 A
                                  —                             a   A
                                                                                   -                        m
                    A    8001
                                                                                                                         Medium-sized
                    S                                                                                                    mammal
                                                                -                                 |
                    S
                    <
                         60.0                         oN                                                              BO Medium-sized
                                                                                                                         wild ungulate
                         40.0
                                                                                                            i
                                                                                                                      ®Large wild
                              A
                                                                                                                         ungulate
                         2001
                          0.0
                                  Alps
                                         T
                                           Central
                                                   T
                                                     Greece
                                                            T
                                                              Nth Spain
                                                                        —
                                                                          Scand
                                                                                 T
                                                                                   Bn
                                                                                    Italy ~~
                                                                                             T           T
                                                                                               Sth Russia Sth Spain
                                                                                                                    1
                                                                                                                      ODomestic
         Figure 2. The average percentage of six main food categories in the diet of grey wolves in different
         bioregions in each of the three continents (see Fig. 1 for locations and abbreviations). The most
         commonly used method to express the importance of each taxon in the diet was percentage frequency
         of occurrence per sample (%FQ), but data from other methods are also included here. Since each scat
         or stomach may contain the remains of more than one prey taxon, the percentages as expressed by
         %FO may add up to >100%. From Newsome et al., 2016.
                                                                                                                                              0000000032
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<pre>24047755
         In many   regions  of the Iberian Peninsula, wild       ungulates   have    disappeared   and  as a
         consequence, wolves have become            increasingly dependent         on garbage dumps     and domestic
         animals  (Barja, 2009). Barja (2009) presented          an  example of wild ungulate preferences of
         the Iberian wolf  (Canis lupus signatus)      in  an environment with       no human-wildlife conflicts
         because wolves     rarely predate    on livestock. The     analysis   of 593 wolf scats showed that wild
         ungulates   were  consumed      preferentially   over  other prey   (i.e. domestic   ungulates,  carnivores
         and  lagomorphs).     Roe deer   (Capreolus capreolus)        was  the most    important prey species
         followed  by  red deer   (Cervus elaphus)    and wild boar      (Sus scrofa).   Domestic    ungulates  were
         poorly represented     in the wolf diet. Predation    frequencies of      domestic and wild   ungulates
         varied seasonally    and between years      (Figure 3, Barja, 2009).       The  consumption of    roe deer
         and wild boar increased      during  the birth season,     probably   due to the   higher vulnerability of
         newly  born animals. Wolves       predate mainly     on  juvenile  roe  deer and wild boar.
                                100         —=—Roe deer deer
                           w                                —s— Red
                           ©     8}        —— Wild boar ——       Sheepigoat
                           iT    9
                           Eg
                           Oo    4F
                           Oo
                           ©     2
                           o™                     1
                                  0
                                         1998         1999           2000          2001         2002
                                        MW Roe deer O Wild boar @ Red deer 0             Sheep/goat
                                100
                            LJ
                                  80
                            Ww
                            0     60
                            c
                            ac
                            3
                            Q
                            oO
                                 440
                            =     20
                                    0
                                          Spring           Summer           Autumn            Winter
         Figure 3. Top figure; Percentage (frequency of occurrence) of two domestic and three wild ungulate
         species in the Iberian wolf diet per year. Lower figure; percentage (frequency of occurrence) of two
         domestic and three wild ungulate species consumed per season. The percentage of occurrence for
         each prey species is based on the total number of scats found per season (Barja, 2009).
                                                                                                                     0000000033
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<pre>24047755
         Wagner     et al.   (2012) analysed              the diet  composition of the       wolves in      Germany.        Wild
         ungulates,     which made up              more      than 96% of their diet. The        dominating prey species             was   the
         roe  deer   (55.3%),       followed        by   red deer   (20.8%)    and wild boar      (17.7%).       The second        important
         food category       were      the    Leporidae (e.g.        rabbit and hare, 2.9% of           Biomass),     whereas livestock
         made up     only 0.6%         of all biomass consumed. Wolves                 clearly preferred hunting              on juvenile  to
         adult red deer;       roe    deer     were     hunted at all age classes.        Wagner        et al. (2012) found        seasonal
         differences in the diet           composition         with a  higher   amount of wild boar in           spring    and winter,
         when    a high    number of         juveniles        and weakened animals,          respectively,      were    available. In the
         first years of the       study     the    percentage of       red deer    was  much     higher,     and the     percentage of
         roe  deer therefore         was     lower than the        following  years. The amount of wild boar in the wolf
         diet fluctuated most in the first three years. Diet                   composition       remained constant             during the
         last five years. Wolves needed less than two                       generations for adapting           to the   new     conditions
         in the cultivated       landscape of eastern Germany.
         Field research       implemented             in Flanders     (Belgium) by Van        der Veken et al.        (2021) (Table 1)
         revealed that the most              important prey        items in the diet of grey wolves in              Belgium     are wild
         ungulates (90.0%), lagomorphs (13.6%)                        and livestock    (12.9%).The          prey    category of wild
         ungulates     consisted of          roe    deer     (69.3%),  fallow deer    (7.1%),    unidentified deer          (5.0%)   and
         wild boar    (22.9).
         Table 1. Wolf diet of the first settled wolves in Flanders,                Belgium, based on 140 scats             collected from
         May   2018 to   February 2021, expressed as                the  frequency of occurrence (FO). Season               of collection was
         not known for three scats. From Van der                  Veken et al. (2021).
                                                                                                    FO
                         Prey  items                                         Winter    Spring    Summer      Autumn      Total
                         Wild  ungulates                                      90.0      83.3       97.4        83.3       90.0
                         Roe  deer (Capreolus capreolus)                      78.0      50.0       76.9        54.2       69.3
                         Fallow deer (Dama dama)                               8.0      12.5        2.6         8.3        7.1
                         Unidentified deer                                       -        -
                                                                                                    77         16.7        5.0
                         Wild boar (Sus   scrofa)                             12.0      41.7       25.6        16.7       229
                         Lagomorphs                                            18.0     16.7        5.1        16.7       13.6
                         European    hare (Lepus europaeus)                    10.0      4.2        2.6        12.5        7.1
                         European rabbit (Oryctolagus cuniculus)               4.0      12.5        2.6         4.2        5.0
                         Unidentified   Lagomorph                              4.0        .          -           -
                                                                                                                           1.4
                         Livestock                                             6.0       8.3        5.1        41.7       12.9
                         Sheep (Ovis aries)                                    4.0       8.3        51         20.8        8.6
                         Goat (Capra hircus)                                   20         -          -
                                                                                                               20.8        4.3
                         Domestic   pig (Sus domesticus)
                                                                                 >        -          -
                                                                                                                4.2        0.7
                         Domestic   dog (Canis   familiaris)                     -        .
                                                                                                    2.6          -
                                                                                                                           0.7
                         Domestic cat   (Felis catus)                          2.0        -          -           -
                                                                                                                           0.7
                         Small rodents                                         16.0      8.3        2.6         4.2        8.6
                         Bank vole   (Myodes glareolus)                        4.0       4.2        2.6          -
                                                                                                                           2.9
                         Field vole (Microtus agrestis)                        6.0        2          -
                                                                                                                4.2        2.9
                         Unidentified vole   (Microtus sp.)                    6.0        -          >           -
                                                                                                                           2.1
                         Wood    mouse  (Apodemus sylvaticus)                    i
                                                                                         4.2         -           .
                                                                                                                           0.7
                         Birds                                                 4.0       4.2         -           -
                                                                                                                           2.1
                         Fruits and seeds                                        -        -          -
                                                                                                                2.5        0.7
                         Black  cherry (Prunus serotina)                                  :
                                                                                                                2.5        0.7
                         # prey items                                           34       50         40          72        202
                         # scats                                                24       39         24          50         140
                                                                                                                                              0000000034
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<pre>24047755
         We conclude that the diet of grey wolves is very variable and                 adapted    to local circumstances
         and prey    availability. So   we   expect that the diet of grey wolves in the Netherlands may show
         strong   variation between sites and         during   seasons.
         Conflicts between wolves and humans
         According     to the Habitat Directive of      Europe (1992),      under ANNEX IV grey wolves              are   strictly
         protected    in  Europe. Derogations from         this Annex    can   be  put to action   only  if certain
         conditions    are  met. Under Article 16 of the        Annex, it is specifically stated that “under strictly
         supervised conditions,       on a   selective basis and to      a limited extent, the    taking    or  keeping of
         certain   specimens of     the  species    listed in Annex IV in limited numbers          specified by     the
         competent national authorities”          must   only  be  a “last resort” solution    (Linnell  et  al., 2008).
         Despite    this strict legislation,   the protection of wolves      in  some   European     countries is     failing,
         partly  due to    complex political,    social and economic interests, but also because of                a lack of
         knowledge      of sound   management practices.
         In Romania for instance, orders for the            hunting  of wolves    (and bears)    are  published yearly         as
         a means     of  controlling  their large population.      Between 2007 and 2015, orders             proposed by
         the environmental       ministry of    Romania for the control of the        population of large      carnivores
         have resulted in the      hunting   and   killing of 5,000 protected      wild  animals, including 1,586
         wolves    (Micu, 2016). People       in Romania      generally   approve this    management         method since
         they   believe that   culling  wolves is the     only  way to protect themselves and their livestock
         against   wolve attacks     (Dale-Harris, 2017).       In addition, such    culling practices provide        an
         important    economic incentive        as part of the hunting industry.
         In Norway, according        to Harvey (2016),       the government approved          a plan  to hunt and kill 47
         of the 68 wolves left in the wild in the         Norwegian countryside,        as a means     of  reducing       the
         loss of livestock due to wolves. In 2015 the state received                more  than 11,000      applications for
         hunting    licenses to shoot 16 wolves, which may have              partly  contributed to the release of the
         mentioned      plan. Ultimately     the  plan  was    suspended temporarily        in November 2017 after
         World Wide Fund for Nature           (WWF)     sued the state at Oslo District Court         (The    Local   2017).
         In Finland the situation is not that different. With          a total  population of 235 wolves,         the
         government       authorized the    culling  of 53 wolves for the winter of 2017 while 78             were   already
         killed  during   2016   (Barkham, 2017).       In  Sweden, where according          to estimates there       are   355
         wolves in the wild, last year       a  license that allowed the      hunting of 22 (instead of 50         as  in
         previous years)      wolves   was   published (The Local, 2017).
         Up  to and    including September 2020,          66 wolf attacks have been        reported    in the Netherlands
         on  livestock    (Figure 5, BIJ12, 2020).       In the  same   period   last year  (2019),   there    were   nine
         attacks   by  a  wolf. A total of 25 attacks      were   reported throughout 2019. Especially            in North
                                                                                                                               10
                                                                                                                                   0000000035
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<pre>24047755
         Brabant, the increase is significant. As far is known, two wolf attacks took place there last
         year, while this year there      are   already 31        attacks   so  far.
                                Aantal aanvallen      wv                     2020 (t/m sep)
                               Noord-Brabant
                                       Drenthe
                                   Gelderland
                                       Limburg
                                      Friesland
                                   Groningen
                                     Flevoland     0
                                     Overijssel    |p
                                        Utrecht    |0
         Figure 5. Number of attacks of wolf on livestock in the Netherlands up                to and  including September
         2020.
         In the Netherlands, there is       a  strict     zoning   for red deer, fallow deer and wild boar.        They   are
         allowed in few   areas   (though regulated)             and outside these       areas  all deer and boar will be
         shot. The Veluwe    (with    one   pack       and two territorial female        wolves)   is one area  where all
         three prey   species (and     roe   deer)       live. In both the Drenthe and Eindhoven            region only  roe
         deer is allowed,  according       to  policy.      In reality there   are  low numbers of wild boar and
         sporadic   observations of red deer in both              areas.
         Objectives
         The  ongoing   recolonization process of wolves towards human-dominated                           areas  such  as the
         Netherlands, could ratchet up the tensions again which historically led                       to the extermination of
         the wolf in most of  Europe.       Basis of conflict is the        feeding ecology of the species, potentially
         causing   economical and emotional                damage    due to attacks       on livestock and   pets, and
         competition   with the  hunting      sector       (Kleiven  et al. 2004, Bergstrom et al. 2009, Kovaiik et al.
         2014).  In addition, the   question      arises whether human-dominated                 landscapes offer    a
         sustainable amount of wild prey and if wolves switch to domestic prey if not                        enough   wild prey
         are available. Moreover, the influence of the wolf                 on  herbivore behaviour in the Netherlands
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         is unknown. It is of      great importance       to obtain accurate information        on this   topic  in order to
         guide policymaking         and to avoid the      spread of misconception among           the   general public,    and
         as   such, facilitate coexistence with         a top predator (Everaert      et al.  2018). Dietary     information
         of the Dutch wolf population is thus           greatly  needed.
         In order the    gain insight    into  feeding    behavior of the wolves in the Netherlands,            we  suggest    a
         diet  analysis    based    on a  combination of DNA-based methods               (i.e. metabarcoding)       and
         mechanical      approaches (i.e. microscopic          hair  analysis).  Both   techniques      mentioned have
         their  pitfalls and   strong   suits. While mechanical         approaches    are   mostly   accurate    they  do take
         a   long time to master and also to          perform, making      mechanical     approaches      rather time
         inefficient, potentially severely limiting the         amount of     samples   that   can  be taken and
         analyzed. On       the other hand      metabarcoding approaches          are  high throughput       and follow
         standardized      protocols    that don't   depend    on expert knowledge, making them very time
         efficient tools which allow for         a much intensified     sampling   scheme in space and time.
         However, metabarcoding does not give information about the status of the consumed prey
         (e.g.  age   class)  and    can  include bias     occurring   due to artefacts such      as  territorial  marking by
         foxes and birds      foraging    on  dung    beetles.  Combining metabarcoding           and mechanical
         approaches       and   comparing     the results    can overcome      the described     problems of      both
         methods and contributes to           strengthening     the diet   analysis.
         The main      objective   of this research is to determine the diet         composition of grey        wolves in The
         Netherlands and border          regions, using      two complementary       forms of diet     analysis (e.g.
         mechanical and        metabarcoding approaches)            and to establish    a  long  term    monitoring
         programme. The         following   research     questions    will be addressed:
         14.       What    are  the main    components of their diet and how do these differ from other wolf
                   populations?
         2.        What is the prey      density    in each of the    geographic    areas    where wolves      occur   in the
                   Netherlands?
                   Are these densities reflected in the wolves’ diet?
                   What is the contribution of livestock and other domestic animals to their diet and does
                   this match the     findings from      bite mark saliva taken from killed livestock?
         B.        Is there   a  difference between the diet of wolves          living in  packs   and the diet of free
                   roaming     individual wolves?
         This three year research         project   will be  a collaboration between de         Zoogdiervereniging,
         Nijmegen,      the Institute of Environmental Sciences Leiden, and the                 Department of
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         Evolutionary Ecology, University     of Antwerp, Belgium.    An   exchange programme         will be
         established with the research group of Dr.                         at Wageningen       Environmental
         Research   (WENR), Wageningen.        De  Zoogdiervereniging     will coordinate the    project  and  sign
         an agreement    with  partner organisations. Wolf scats will be collected by volunteers of the
         wolvenmeldpunt     and   by MSc students from Leiden      University   and  University   of Antwerp, by
         researchers and    by  local managers. The first six month of the      project  will be used to
         strengthen  and  optimise   the methodologies     and the network with terrain managers in the
         different study sites.  Ultimately, after this three year  project  a protocol for   a yearly  diet
         monitoring  via the obtained methods and standardized         procedures     will be  supplied  to the
         Zoogdiervereniging.
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         Materials and methods
         Study      area
         The focal   area  of this  study  includes the five    areas   in The Netherlands, where wolves have
         been   reported: 1)    North  Veluwe, 2) Central Veluwe, 3) South West Veluwe, 4) Province of
         Drenthe, 5) Province Noord Brabant and Limburg.
         More recent data      on  the presence of grey wolves in the Netherlands is found via the weblink:
         https://www.bij12.nl/onderwerpen/faunazaken/diersoorten/wolf/verspreiding-wolf-in-
         nederland/
                1) North Veluwe
         The   pack  in the North Veluwe is monitored.         Droppings    collected in the field from this     pack
         confirmed the presence of        a  male animal    (GW893m).      In this territory wildlife cameras
         regularly capture photos      and videos of this male and of        an  unknown number of other
         individuals. The    public  also  provides   evidence of the presence of wolves. From this visual
         material it is difficult to differentiate between the       parent animals (GW893m and GW998f) and
         the pups from last year. However, the          camera    trap photos confirm that this spring       at least
         four  new  pups   were   born. The size of the     pack   in the North Veluwe is not      exactly known,     but
         could consist of 15 wolves in 2022.
                2) Central Veluwe
         A female wolf has established         a territory in Central Veluwe     (GW960f).     She is still present in
         the area, confirmed      by wildlife  cameras    end 2020. A DNA       sample    collected confirmed the
         presence of this wolf. The most         important period    to determine established       territory  holders is
         around the    mating   season, in    January-February.       There is  no  evidence, based      on tracks and
         wildlife cameras    that this she-wolf has     a  partner.
                3) South West Veluwe
         There has been      activity on  the Southwest Veluwe since 14 June 2020 when                 a wolf  was
         identified  on a  wildlife camera    trap. At various locations in this region wolf tracks         were  found.
         Wolf  droppings    were   collected   on  the 20th of June 2020 and DNA          analysis   confirmed that it
         is indeed   a wolf, probably    a  daughter   from the Noord Veluwe         pack. Video   footage    from
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         September 3rd,       released     by  Natuurmonumenten shows the she-wolf is                    hunting.    This she-wolf
         is the third pup that has been         genetically identified from         the North Veluwe         pack   born in 2019
         (GW1729f).     This is the first pup known to have left the              parental pack      and   possibly     established
         a new   territory.  It is not  uncommon        for  a wolf to disperse at such         a young age,       though    most
         youngsters    do not leave their       pack    until their second year of life. For all         we   know she-wolf
         GW1729f is the first wolf born in the Netherlands that has started                      looking   for her    own
         territory. In the  past period,     no  Kills of livestock     were  found with DNA from this she-wolf, but
         more   data  are   needed to make        predictions      on  the likelihood of any future livestock attacks             by
         this individual. In 2020       a she-wolf entered Nationaal Park de               Hoge    Veluwe.      In 2021 also    a
         male and female wolf established             a  territory  in National    park    de  Hoge   Veluwe. This        pair got
         two to three cubs in       spring 2022.
                4) Province of Drenthe
         A male wolf    (GW1261m)          has been confirmed to reside in Drenthe. This                 was    confirmed     by
         DNA    analysis  of   droppings,     found   on   June 9, 2020. Also, three claims in Drenthe                 (period
         June 10-June 16,        2020)    and  one   in Friesland     (July 7)  could be traced back to this wolf.
         Because wolf GW1261m has               now    been active for     more     than six months in this        region,
         according    to the   Interprovincial Wolf       Plan this wolf is considered to have established                 a
         territory. The wolf has been observed in              a large   area on    both sides of the A28 between
         Emmen and Drachten,            probably looking       for one   suitable    place   to settle. That GW1261m is
         observed    regularly     on  both sides of the A28 is        quite unique       as well  as  worrying,    since
         highways    can   be   a  (fatal) barrier to wolves. The        area   he is    inspecting   for  a  possible
         settlement has been         recently enlarged by        a  part, passing the Dutch         —
                                                                                                      German border. A pair
         of wolves   was   formed in 2021. In        spring    2022 cubs    were      born from this     pair  in the Drents-
         Friese Wold.
                5) Province Noord Brabant and Limburg
                   Noord Brabant
         There   are  three   analysed     claims from the       period 23   -
                                                                                30   May    2020 that    originated     from the
         region   west of Den Bosch. All these            cases can     be traced back to       a  wolf that has     genetically
         been identified    as   GW1554m. This wolf has roamed the                   area    around Heusden         during May
         2020. Based     on    knowledge sharing         with institutes in    neighbouring       countries there       are  strong
         indications that this wolf is still     dispersing (looking       for  a  territory)  in the   region.   The presence
         of GW1554m       was     confirmed in Flanders and in           Germany       in June 2020. This individual
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         probably    moved to the     Vosges region        and there is unconfirmed information available that
         this wolf   was  killed.
                   South East Noord Brabant and Limburg
         Between      May  22 and    July 16,   in the   region  southeast of Eindhoven in         seven cases       the DNA
         of a wolf   was  found    on  attacked    sheep.   The wolf is identified with DNA         as  GW1625m and it
         comes     from the   French-Alpine population.         The wolf is   present in the     area   since 28 March
         2020 and     might  have established it's        own  territory by  this fall. The presence of GW1625m
         was   also confirmed in      Limburg.    It is not known from which         pack  the wolf   originates.
         Assessment of diet                 composition
         Diet  composition of grey       wolves will be determined         through microscopic        scat   analysis   and  e-
         DNA    analyses    of faecal   samples.
                Assessment of diet          composition with prey hair morphology
         Scats will be collected      throughout      the Netherlands      through    a volunteer network of De
         Zoogdiervereniging.        Scats will be    analysed microscopically using          a  reference collection of
         hairs and    body parts of     known prey      species.   In addition, other items in the diet will be
         analysed and, if possible, identified,          such  as  fruits, seeds and food of anthropogenic origin.
         Determination of the prey         species    is done   using   the method of Van der Veken et al.            (2021)
         using   a  reference collection. Cuticle scale patterns           provide    more  detailed identification for
         which   microscope      slides will be   prepared.    To   analyse   these    patterns, cuticle imprints will be
         made    by soaking     1.7 grams of     gelatine (one     sheet Dr. Oetker      white)  in 40 ml demi-water for 5
         minutes. The solution is then         placed    on a  hot  plate (65°C)    until the  gelatine   is  completely
         dissolved. A thin     layer  of  gelatine   solution will be    spread  on a    microscope     slide and left to   dry
         for 5 minutes. The hairs        are placed     on the  gelatine layer   and left until the    gelatine   is
         completely dry (at      least 60   minutes).     When the    gelatine  is  dry,  the hairs  are  removed with
         forceps    and stored in    zip-lock bags.
                eDNA    analysis
         In addition to the    use   of microscopic analysis of prey         hair  morphology,      the  dietary
         composition     of wolf diets will be identified      by  a  recently developed      and tested method based
         on  next-generation sequencing (Shezad              et  al., 2012). This approach reveals           a larger
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<pre>24047755
         spectrum of prey items, since also the DNA of smaller prey species such                        as rodents,
         amphibians, reptiles        and fish    can  be identified. To minimize the collection of scat with DNA
         degraded by       ultraviolet   light,  or the collection of overabundant         epithelial cells from the
         predator’s     intestinal tract,   samples    will be taken from the center of the fresh wolf scat
         samples using plastic        hand     gloves   and  a sterilized  surgical    blade and    preserved    in DNA free
         Eppendorf tube containing            1 ml of 99.8% ethanol. The tubes will be labeled and stored at                 -
         20°C.    Prey   DNA from wolf scats will be extracted at the Leiden                University   molecular
         laboratory following previously optimized protocols (Kevin Groen, personal communication,
         10-2022) using       the  Qiagen QIAamp         DNA stool mini kit     (Qiagen Inc., Valencia, California,
         USA). Controls       will be included to check for contaminations              during  the extraction process.
         To  amplify    vertebrate prey DNA,         we  will conduct   a  PCR with the highly conserved universal
         primer binding      site COI for all vertebrates       (Reeves    et al., 2018). A PCR of the control,          as well
         as a  control PCR reaction will be included to rule out contamination. Obtained sequence data
         will be  analyzed using       a  pipeline   hosted at Naturalis     Biodiversity Center. Sequence            reads will
         be  filtered, clustered and BLASTed using scripts implemented in the galaxy platform.
         Prey preference               and avoidance
         To determine prey         preference      and avoidance the Jacob's index will be used            (Jacob’s, 1974).
         The Jacobs index         (E) is  given by equation:
                                                                  _r—p
                                                                  “r+p
         Whereas      r resembles is the        proportion of total  kills at a  site made up      by a species     and p
         resembles the       proportion availability of that species.        The   proportion of total    kills r was   taken
         from   carcass    counts and scat        analysis.  The  proportion of     Kills will be calculated
         inlcudingdomestic        animals.     Proportional species availability       will be taken from       existing   prey
         counts available with the fauna            management      units. The    resulting   values range from -1 to +1,
         where -1 indicates avoidance and +1               preference (Jacob's, 1974).
         Livestock attacks in the                     study     area
         Information about the number of livestock attacks and the                   identity of the  attacker    (i.e. wolf  vs.
         dog)   will be used from available databases            (e.g.  BIJ12 /   Provinces).     The databases contains
         information     on  location, time, type of livestock attacked and saliva analyses results.
         Data      analysis        and statistics
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         For the statistical   analysis of the  collected  samples,    R  programming language       version 3.4.1
         (R Foundation, Austria)      will be used. For all tests,  a  significance  level of p-value  <  0.05 will
         be used. To test for     normality,  a Shapiro-Wilkinson     test (W>0.9)  will be used.
         For the  analysis    of the diet, data taken from wolf scat     samples  will be fitted to linear
         regression    models and ANOVA will be used to determine which variables               are the most
         influential depending     on  the distribution. Independent     variables include   density  of prey,
         presence of     livestock, human disturbance (recreation, sport), human infrastructure (roads,
         villages).  A  Principal Component Analysis (PCA)         is used to reveal   patterns that may have
         been missed      by analyzing    each variable  separately. Through     this analysis,  we  expect   to
         reveal the main     contributing components      to the diet of wolves.
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<pre>24047755
         Proposed work schedule
         Specification                                      2023
                                            March     Apr June
                                                         -
                                                                 July Aug
                                                                     -
                                                                          Sep -
                                                                                Dec
         Literature Review & Research
         Design
         Field Visit | Preparation
         Field Visit |  Data Collection
         Data  Analysis
         Report Writing &    Submission
                                                            2024
                                          Jan  -
                                                 Mar  Apr June
                                                         -
                                                                 July Aug
                                                                     -
                                                                          Sep -
                                                                                Dec
         Field Visit | Preparation
         Field Visit  | Data Collection
         Data  Analysis
         Report Writing &    Submission
                                                            2025
                                          Jan  -
                                                 Mar  Apr June
                                                         -
                                                                 July Aug
                                                                     -
                                                                          Sep -
                                                                                Dec
         Field Visit | Preparation
         Field Visit |  Data Collection
         Data  Analysis
         Report Writing &    Submission
                                                            2026
                                        Jan -
                                              Feb 28"
         Data  Analysis
         Report Writing & Submission
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<pre>24047755
         Budget
         Specification                                                      Per year    For 3 years
         Postdoc 0.4 FTE
         Staff costs Postdoc eDNA       (scale 10,07)
         Researcher 0.35 FTE
         Staff costs researcher    —
                                     microscopic   hair analysis
         Laboratory analyses          -
                                        genetic   and   microscopic
         General lab costs    (pipets, PCRs,   lab consumables,  etc.)
         Metabarcoding                                                 MM            dM
         Extractions                                                   Mm            a
         Consumables    microscopic analyses                           Mm            a
         Fieldwork
         Travel
         Consumables    -
                           fieldwork
         Miscellaneous   -
                            fieldwork
         Subtotaal
         21% BTW
         Totaal (incl BTW)                                                137,335.00    412,005.00
                                                                                          20
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