Foraging ecology of horseshoe bats and pest consumption through molecular techniques.

FORAGING ECOLOGY OF HORSESHOE BATS
AND PEST CONSUMPTION
THROUGH MOLECULAR TECHNIQUES
PhD Thesis by
Mi en Aldaso o Lezea
Leioa, 2025
Fo aging Ecology o Ho seshoe Ba s
and Pes Consump ion h ough
Molecula Techniques
PhD Thesis by
Mi en Aldaso o Lezea
Leioa, 2025
Unde de supe ision o :
D . Joxe a Aiha za Azu za
D . Oihane Diaz de Ce io A uaba ena
(cc) 2025 Mi en Aldaso o Lecea (cc by-nc-sa 4.0)
E xekoei.
"In na u e, no hing exis s alone."
Rachel Ca son

FUNDING
This esea ch was unded by:
● G an PID2019-108123GB-I00, unded by MICIU/AEI/10.13039/501100011033 and he
Eu opean Union.
● Basque Go e nmen (Resea ch g an IT1571-22).
● Basque Go e nmen Biodi e si y Di ec o a e (Expedien No 049‐2022‐42).
● Genomic Resou ces Resea ch g oup o he Uni e si y o he Basque Coun y (UPV/EHU)
TABLE OF CONTENTS
ESKER ONAK - ACKNOWLEDGMENTS..........................................................................9
I. LABURPENA....................................................................................................................... 1
I. SUMMARY........................................................................................................................... 5
II. SARRERA OROKORRA.................................................................................................. 9
II. GENERAL INTRODUCTION........................................................................................21
III. STATE OF THE ART..................................................................................................... 43
III.1. HYPOTHESIS.......................................................................................................... 43
III.2. OBJECTIVES........................................................................................................... 44
IV. RESULTS.......................................................................................................................... 45
CHAPTER 1: Op imise p ime s combina ion o analysing he die o ho seshoe ba s
by me aba coding............................................................................................................... 47
LABURPENA................................................................................................................ 49
ABSTRACT....................................................................................................................49
1.1. INTRODUCTION................................................................................................... 50
1.2. MATERIALS AND METHODS.............................................................................52
1.3. RESULTS................................................................................................................ 54
1.4. DISCUSSION..........................................................................................................59
1.5. REFERENCES........................................................................................................ 62
1.6. SUPPORTING INFORMATION............................................................................ 65
1.I. Addendum o CHAPTER 1:....................................................................................67
1.I.1. REASON FOR THE ADDENDUM..................................................................... 67
1.I.2. MATERIALS AND METHODS...........................................................................68
1.I.3. RESULTS..............................................................................................................69
1.I.4. DISCUSSION AND CONCLUSIONS.................................................................73
1.I.5. REFERENCES......................................................................................................75
1.I.6. SUPPORTING INFORMATION..........................................................................77
CHAPTER 2: Quan i a i e pes de ec ion in ba aeces: i s a emp wi h mul iplex
liga ion-dependen p obe ampli ica ion (MLPA)............................................................... 79
LABURPENA................................................................................................................ 81
ABSTRACT....................................................................................................................81
2.1. INTRODUCTION................................................................................................... 82
2.2. MATERIALS AND METHODS.............................................................................85
2.3. RESULTS................................................................................................................ 91
2.4. DISCUSSION..........................................................................................................96
2.5. REFERENCES........................................................................................................ 99
CHAPTER 3: Seasonal and geog aphic a ia ion in he ophic ecology and habi a
dependence o Rhinolophus hipposide os........................................................................103
LABURPENA.............................................................................................................. 104
ABSTRACT..................................................................................................................105
3.1. INTRODUCTION................................................................................................. 106

Summa y
I. SUMMARY
This disse a ion p esen s a signi ican and imely con ibu ion o he unde s anding o ho seshoe ba s'
o aging ecology. I adop s a comp ehensi e app oach ha conside s seasonal and egional die a y
a ia ions, en i onmen al in e ac ions, and he in luence o p ey a ailabili y and habi a s uc u e.
In a- and in e speci ic compe i ion also play a c ucial ole in shaping o aging s a egies and esou ce
pa i ioning among coexis ing species. The use o DNA me aba coding, a powe ul ool o
in es iga ing ophic ecology, p o ides high- esolu ion axonomic iden i ica ion o p ey i ems. The
speci ic ocus is on he ophic ecology o h ee ho seshoe ba species, wi h a pa icula emphasis on
he lesse (Rhinolophus hipposide os) and he g ea e (Rhinolophus e umequinum) ho seshoe ba s.
The s udy has been pe o med in he Basque Coun y (No he n Ibe ian Peninsula), a co e a ea o he
dis ibu ion o hese wo species.
In he i s pa o he hesis, we ocused on he me hodological pa o he s udy, aiming o op imise
he me aba coding app oach and es a new semi-quan i a i e assay o he mul iplex iden i ica ion o
speci ic p ey species.
On he one hand, since using a single p ime se can in oduce biases in he die a y composi ion
analyses by me aba coding, we i s e alua ed whe he combining wo di e en p ime se s imp o ed
he co e age o iden i ied p ey i ems. Each p ime se e ealed a dis inc subse o p ey, wi h only a
small p opo ion de ec ed by bo h. Consequen ly, each p ime p o ided a di e en pe spec i e on he
ophic niche o he s udied species. Ou inding con i med ha using complemen a y p ime s
signi ican ly enhances he accu acy o die cha ac e isa ion in gene alis insec i o ous ba s. This
e alua ion demons a ed ha he combina ion o ANML and FWH1 was he mos e ec i e,
maximising p ey di e si y co e age, p ey quan i y, and p eda o iden i ica ion. This p ime
combina ion was, he e o e, selec ed o he subsequen s udies.
On he o he hand, we sough o de elop a eliable molecula assay o he simul aneous de ec ion and
semi-quan i ica ion o mul iple pes species in ba aeces using a mul iplex liga ion-dependen p obe
ampli ica ion (MLPA) assay. This s udy ep esen s he i s a emp o adap he MLPA app oach o
de ec ing p ey DNA in aecal samples. We designed speci ic p obes a ge ing ou key mo h pes
species—Thaume opoea pi yocampa, Cydia pomonella, Au og apha gamma, and Lobesia bo ana.
While we success ully es ed h ee p obes on pu e mo h DNA and de ec ed T. pi yocampa in ba
aeces, u he op imisa ion is needed o de e mine de ec ion limi s and alida e mul iplexing
capabili ies. The e o e, we le his echnique aside and ocused on me aba coding.
5
Summa y
Once he me hodological pa was inished, we ocused on he main objec i es o he hesis. While
signi ican ad ances ha e been made in unde s anding ba o aging habi a s, p e ious s udies based on
adio eleme y p o ide only sho - e m da a, as hey moni o indi iduals o limi ed pe iods. In
con as , me aba coding enables species-le el iden i ica ion o consumed p ey, allowing o an
indi ec assessmen o sou ce habi a s. To explo e seasonal and spa ial die a y a ia ions, we analysed
aecal samples om h ee R. hipposide os colonies ac oss di e en clima ic zones om sp ing o la e
summe . Using me aba coding, we examined he a ia ion o he lesse ho seshoe ba die , checking
how hei mos -consumed p ey a ied seasonally and ac oss landscapes. Ou esul s e ealed seasonal
die composi ion shi s, which we e dissimila in each s udied landscape, likely echoing espec i e
p ey a ailabili y. While woodlands and sh ublands we e p ima y p ey sou ce habi a s, R. hipposide os
also o aged in open habi a s mo e equen ly han expec ed, demons a ing high ophic plas ici y.
A simila s udy was conduc ed on R. e umequinum ac oss colonies in di e se landscapes,
u banisa ion le els, and clima ic condi ions. We obse ed signi ican spa ial and empo al die a y
di e ences, wi h a s onge eliance on ipa ian habi a s in Medi e anean a eas. The species exhibi ed
ema kable ecological adap abili y, adjus ing i s o aging p e e ences among o es s, ipa ian habi a s,
sh ubs, and g asslands. Ou esul s emphasise he impo ance o p ese ing hese habi a s o
conse a ion managemen pu poses. These indings unde sco e he impo ance o p ese ing a mosaic
o in e connec ed habi a s o suppo ho seshoe ba s and hei p ey.
Building on hese indings, we in es iga ed he ole o ho seshoe ba s in ag icul u al pes supp ession.
While insec i o ous ba s a e inc easingly ecognised o hei ole in con olling pes popula ions,
he e emains a need o quan i y hei impac . We eanalysed he die s o he six colonies— h ee R.
hipposide os and h ee R. e umequinum— ocusing on pes consump ion. Bo h species p eyed on a
subs an ial a ie y o pes insec s, wi h some species p eyed upon consis en ly and massi ely,
including p e iously un epo ed species, wi h p eda ion pa e ns aligning wi h pes ou b eaks. We
es ima ed he o al biomass o ag icul u al pes s consumed pe colony du ing he b eeding season and
he o e all pes consump ion by hese ba species in he s udied a ea, e ealing a signi ican
con ibu ion o na u al pes con ol. Howe e , ba s' body size is key in pes supp ession, di ec ly
in luencing nigh ly ood in ake. While R. hipposide os exhibi ed a highe ela i e incidence o pes
consump ion, he o al biomass o pes s consumed was g ea e in R. e umequinum. Fu he mo e,
bo h species p eyed on mo e pes species in highly an h opised a eas, emphasising hei po en ial ole
in in eg a ed pes managemen .
Following hese in es iga ions, we examined he impac o de elopmen al s age on die composi ion
by compa ing ju enile and adul die s o R. hipposide os, R. e umequinum, and he Medi e anean
ho seshoe ba (Rhinolophus eu yale). We analysed die a y di e ences bo h axonomically and based
on p ey ai s (e.g., size, ligh speed, ha dness). While ju eniles o R. hipposide os and R. eu yale
consumed signi ican ly di e en p ey han adul s, R. e umequinum showed less p onounced
6
Summa y
axonomic di e ences. We could only obse e disce nible die pa e ns h ough he ai analysis o
he la e . A sha ed pa e n ac oss species was ha ju eniles p e e en ially consumed smalle , so e ,
and slowe p ey, likely e lec ing de elopmen al cons ain s on p ey cap u e. These indings highligh
he impo ance o conside ing age- ela ed die a y needs in conse a ion s a egies.
Collec i ely, he indings o his disse a ion demons a e ha he o aging ecology o R. hipposide os
and R. e umequinum is a mo e complex han p e iously assumed. These species exhibi high
die a y plas ici y, adjus ing hei p ey selec ion seasonally and esponding o p ey ou b eaks. Thei
o aging s a egies ex end beyond expec ed habi a dependencies, e lec ing a dynamic in e ac ion
wi h hei landscapes. Mo eo e , ju enile die a y p e e ences di e om hose o adul s, highligh ing
de elopmen al cons ain s on o aging e iciency. Finally, ou esea ch unde sco es he c ucial ole o
ho seshoe ba s in ag icul u al pes supp ession, ein o cing he impo ance o main aining heal hy ba
popula ions in an h opised landscapes. These indings ha e p ac ical implica ions o u u e
conse a ion e o s, which should accoun o his ecological complexi y, ensu ing he p o ec ion o
di e se o aging habi a s and os e ing coexis ence be ween ba s and human-al e ed en i onmen s.
7
II. SARRERA OROKORRA

SARRERA OROKORRA
II.1 SAGUZAR INTSEKTUJALEEN BAZKA EKOLOGIA
Saguza ek (Chi op e a o dena) 1.400 espezie ingu u bil zen di uz e, e a, be az, ugaz unen biga en
o denik ani zena di a, desk iba u ako ugaz un espezie guz ien lau dena di ela ik (Wilson &
Mi e meie , 2019). Lu eko ekosis ema gehiene an pape ga an zi sua be e zen du e kon inen e
guz ie an zeha , An a ikan izan ezik (Kunz & Pie son, 1994), e a ugaz unen a ean baka ak di a
hegaldi p opul sa ua en ga apenean. Hegaldia en e a ekokokapena en eboluzioa un sezkoak izan di a
saguza ek in sek u gau a mo a ezbe dinak us ia zeko (Jones & Rydell, 2003), saguza en % 70
ingu u in sek ujaleak bai i a (Simmons, 2005).
Saguza in sek ujaleen a ean dagoen aniz asun ekologiko handia hegake a ekin e a ekokapena ekin
lo u a es ua du en hainba egoki zapenekin lo u a dago. Esa e ako, espezie handiago e a azka ago
ba zuek maiz asun baxuko ekokokapen-deiak e abil zen di uz e espazio zabale ako in sek uak
ehiza zeko (adb., Nyc alus lasiop e us; Ibañez & Jus e, 2023), e a saguza xikiago e a mo elagoak,
be iz, maniob aga iagoak di a e a be en ha apakinak den si a e al uagoko e emue an ha apa zen
di uz e (adb., Myo is bechs einii; Ke h & an Schaik, 2023). Gaine a, saguza in sek ujaleek
bu ezu -egi u a, ba aila e a ho z mo ologia ezbe dinak di uz e, be en beha die e ikoekin ba
da ozenak, e a ho ek haien hozkada-inda a i e a ha apakin mo ei e agi en die. Go pu z gogo eko
ha apakinak kon sumi zen di uz en saguza ek bu ezu e a ba aila sendoagoak di uz e, e a le agin
luzeagoak, ha apakinen hau espen biguna ja en du enekin alde a u a (F eeman, 1979, 1998;
Ramí ez-F áncel e al., 2021; San ana e al., 2011, 2022). Saguza espezie bakoi za en egoki zapen
espezi iko hauek e agina du e haien bazka-ekologian, non e a ze ehiza u dezake en baldin za uz
(adb., Swa z e al., 2003).
Bazka un sezko ja due a da animalien bizi aupene ako. Saguza in sek ujaleen kasuan, ha apakin
po en zialak bila zea, de ek a zea e a ezagu zean da za. E a be ean, ha apakina en a ze ik ibili,
ha apa u e a azkenean kon sumi u edo ez e abaki beha da (S ephens & K ebs, 1986). Ho ela,
ha apa i-ha apakin elka ekin za ha apakinen e en aga i asuna en e a de en sa-es a egien
e aginko asuna en, hala nola kamu lajea, mime iza ze gai asuna edo ihes-po ae a en mende daude,
(S ephens & K ebs, 1986; Spi z e al., 2014). Ha apakina en amaina, gogo asuna edo ihesko asuna
bezalako ak o eek e en aga i asuna i e agi en dio e, e a ha apakin esku aga i asunak e a
ha apa ien ni xo malgu asunak, be iz, die a baldin za zen du e (A aújo e al., 2011).
Saguza in sek ujaleen bazka-ekologia en ike ke a alo konplexua e a aldeaniz una da, e a asko a iko
ike ke a-me odoe an oina i u da. Me odo ho iek, analisi die e iko ik hasi e a soinu-analisia en e a
i a i- eleme ia en bidez bazka zeko po ae ak moni o iza ze aino, saguza en ekologia ule zeko
balio izan du e. Saguza en mo ologia i bu uzko ike ke ek haien elika ze-e eduei e a baliabideen
banake a i bu uzko hainba i aga pen eka i di uz e, in e en zia ekomo ologiko hauek inda zen
di uz en die a az e ke ek babes u a (adb., Fen on, 1982). Ike ke a-me odoen aniz asun ho ek
10
SARRERA OROKORRA
saguza ak alde desbe dine an sailka zea eka i du, bazka zeko es a egie an, habi a ean edo die an
oina i u a (Allen, 1939; Denzinge e al., 2018; Fen on, 1990, hu enez hu en).
II.2 EKOMORFOLOGIA ETA BAZKA-TALDEAK
Saguza en hegalen o ma e a ekokokapen deiak e abil zen di uz en bazka-ni xo espezi ikoe a a
egoki u a daude. Ko elazio honek saguza ak aldee an sailka zeko auke a ema en digu. Adibidez,
espazio i ekien ehiza zen du enek maiz asun baxuko ekokokapen dei luze e a ekuen zia banda es ua
e abil zen di uz e, dis an zia luzee a a i i si dai ezkeenak (No be g & Rayne , 1987). Hegal zo o zak,
hego-zama e a i xu a- a io handiekin ba e a, azka hegan egi eko auke a ema en die e, nahiz e a honek
maniob abili a e xikiagoa daka en. Aldiz, landa edia ik ge u hegan egi en du en saguza ek,
no malean, hego-zama xikiagoa e a hegal bo obilduagoak iza en di uz e, be en maniob abili a e
handi zen du enak (Vaughan, 1959). Saguza ho iek, gaine a, ekokokapen deie an aniz asun
ika aga ia e akus en du e, ehiza eknika en a abe a. In sek u hegala iak dis an zia xikie an a zema en
di uz en saguza ek, ekuen zia kons an ea e abil zen du e (CF), maiz asun al uagoko maiz asun
modula uko (FM) luzapen labu ekin ba e a (FM) (Schni zle , 1984). Dei luze ba edo dei labu en
so a ba igo i dezake e Dopple e ek ua konpen sa zeko, be en helbu uak de ek a zeko e a
iden i ika zeko gai izanez (Schni zle , 1968). Pa oi hau hegan ha apa zen du en ( lyca ching)
saguza en ipikoa da (Schni zle e al., 1985). Landa edia en a ean ehiza zen di en bes e saguza
ba zuek, aldiz, maiz asun al u edo baxuko konbinazio mo z edo luzeak izan di zake en FM deiak
e abil zen di uz e ingu uan ezku a u ako ha apakinak au ki zeko (Simmons & S ein, 1980).
Azale e a ik ehiza zen du en saguza ek (gleaning), bes e ik, FM dei labu ak e a in en si a e
xikikoak e abil zen di uz e be en ha apakinen es u a e a egi u ak be eiz eko (Habe se ze & Vogle ,
1983).
E edu o oko ho ie an oina i u a, Fen onek (1990) bazka-habi a a en a abe ako hi u alde handi egin
zi uen : espazio zabaleko ehiz a iak, e z e a hu sunee ako ehiz a iak e a espazio es uko ehiz a iak.
Schni zle ek e a Kalkok (2001) an zeko sailkapena egin zu en habi a a en a abe a (zabalak, e di-i xia,
oso i xiak (unclu e ed, backg ound-clu e ed, highly clu e ed), e a habi a bakoi ze ako
azpika ego iak be eizi zi uz en animalien ehiza eknike an oina i u a, hala nola saguza ai ekoak edo
azale akoak. O ain suago, Denzinge e a kideek (2018) bazka-modue an oina i u ako sailkapena egin
zu en, e a ho iek e abil zen du en habi a a ekin e e lo u a daude, hala nola, ai eko ha apa i ibil a iak
(hawke s), a as e ha apa iak ( awle s), azale e ako ha apa i ak iboak, pasiboak e a landa edia
inkoan (clu e ) de ek a zen du en ha apa iak ( lu e -de ec o s). T adizionalki, alde ho iek habi a
espezi ikoekin lo u izan di a. Ho ela, i xu ako- a io baxuak di uz en espezieak baso inkoekin e a
zuhaixka-e emuekin lo u di a, e a i xu a- a io handiak di uz enak, be iz, ingu une i ekiekin lo u di a,
hala nola bela diekin, landazabalekin edo al i ude handiekin (Findley e al., 1972).
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SARRERA OROKORRA
II.3. BAZKALEKUAK
Azken hama kade an, saguza in sek ujaleen bazka-ekologia i bu uz dugun jakin za naba men
au e a u da, ba ez e e habi a a i e a eskakizun o ikoei dagokienez. Au e apen ho i,
ike ke a-me odologien e a be ikun za eknologikoen hobekun zei dagokie, ba ez e e, hala nola
i a i- eleme ia i (adb., Fen on, 1997; Bon adina e al., 2002; Bu schkoski, 2005; D essle e al.,
2016; Lage eld e al., 2017) e a ja aipen akus ikoa en hobekun za i (Vaughan e al., 1997; Russo &
Jones, 2003; F ick, 2013; Ancillo o e al., 2023). Ike ke a hauek saguza en jokabidea i e a habi a a en
e abile a i bu uzko ezagu za sakonagoak eman di uz e. Habi a ani z e a kali a e handikoen ga an zia
azpima a zen du e, u ean zeha saguza in sek ujaleen za in sek uen uga i asun e a aniz asun
abe a sa be ma zen du enak (Nicholls & Racey, 2006; Jung & Kalko, 2010; Hagen & Sabo, 2016;
S aka e al., 2016).
Kali a e handiko bazkalekuak iden i ika zea saguza en kon se bazioa en un sezkoa da (F ick e al.,
2024); habi a a en gale a –basoen us iake a e a nekaza i za ba ne– mundu osoko
biodibe si a ea en za meha xu nagusia bai a (F ick e al., 2020). Hala e e, eginkizun hau e onka ba
da. Izan e e, habi a ak babes eko eskala e a kon igu azio egokia zehaz ea, ba ez e e saguza en gaueko
mugimendu zabalak kon uan ha u a (McC acken e al., 2016; Goldsh ein e al., 2020; O 'Ma a e al.,
2021), lan konplexua e a zaila da.
Bes alde, i a i e a ul asoinu bidezko ike ke ek e e mugak di uz e. Ike ke a hauek neke suak di a e a
langile e a ekipamendu ga an zi suak beha di uz e, nahiz e a asko an lagin- amaina xikiak e a
ja aipen-i aupen muga uak lo zen di en (Bon adina e al., 2002; Mu ay & Ku a, 2004; Smi h &
Racey, 2005). Ho ega ik, ha apakinen esku aga i asunean e a baldin za klima ikoe an izandako
aldake ek e agindako saguza en a ean ohikoak di en habi a -e abile en ga aikako aldake ak asko an
ez di uz e e akus en. Habi a a i e a die a i bu uzko ike ke a in eg a ua un sezkoa da, saguza en
habi a -eskakizunei e a haien elikadu a i bu uzko ike ke ei zuzendu ako i a i-ja aipeneko ike ke a
gehienak ez bai i a asko an aldi be ean egi en, e a ez bai i a esku aga i asuna e a auke a zen du ena
e akus e a i is en (baina ikus Goi i e al., 2008; Flande s & Jones, 2009; Napal e al., 2013).
Gaine a, a opodoek bizi-ziklo konplexuak di uz enez, habi a ak babes eko ahaleginek o ganismo
ho ien bizi-ziklo osoa i eus en dio en habi a en oso asuna be ma u beha du e (A izabalaga-Escude o
e al., 2015). Ho ega ik, saguza en bazka-habi a espezi ikoen dependen zia e a haien
ha apakin-elemen u espezi ikoak zeha z-meha z iden i ika zea un sezkoa da saguza en populazioak
e aginko asunez babes eko.
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SARRERA OROKORRA
II.4 DIETA
Ha apa ien bazka en ike ke ak, his o ikoki, die a en osae a e a abe as asuna az e u di u. Ikuspegi
ho en ondo ioz, ha apa iak mono ipikoak espezializa u edo selek ibo gisa sailka u di a, e a ha apa i
mo a ani zez elika zen di enak, be iz, ha apa i jene alis a edo opo unis a gisa (Spi z e al., 2014).
Saguza in sek ujaleek jokabide die e iko uga i iza en di uz e, jene alis e ik oso espezializa ue a a.
Espezie gehienek die a malguak di uz e (adb., Jones & Rydell, 2003; Cla e e al., 2009; Aizpu ua e
al., 2013; Aiha za e al., 2023). Hala e e, espezie ba zuk in sek u axoi espezi ikoen espezialis a za
sailka u di a, haien po ae a i konplexu asun ge uza ba gehi uz. Adibidez, Pipis ellus na husii
el xoez elika zen da nagusiki (K üge e al., 2014), e a Ba bas ella sp. (Sie o & A le az, 1997) e a
Rhinolophus eu yale (Goi i e al., 2008; Salsamendi e al., 2012; And eas e al., 2013;
A izabalaga-Escude o e al., 2015) ezagunak di a si sak hobes en di uz elako. Gaine a, saguza
ba zuk, hala nola Sco ophilus i idis e a Myo is myo is, kakala doez elika zen espezializa u a daudela
ikusi da (Fen on e al., 1977; A le az, 1999, hu enez hu en).
Hala e a guz iz e e, espezializazio-maila sasoi edo ak o e geog a ikoen a abe a alda dai eke.
Adibidez, Sco ophilus i idis espeziean sasoiko elikadu a aldake ak hau eman di a, sasoi leho ean
die a zabalagoa izan e aku siz (Fen on & Thomas, 1980), elikagaia u ia denean esku a di uen
in sek uak kon sumi zen di uela adie aziz. Myo is ema gina us eskualdea en aldako asuna en adibide
ga bia da, nagusiki Alemaniako e a Belgikako behi ko e an ehiza zen bai u (Beck, 1995; S eck &
B inkmann, 2006; Ke yn e al., 2012), baina hegoaldeko eskualdee an a mia mek die a en za i handi
ba osa zen du e (Goi i e al., 2011; Galan e al., 2017). In e esga ia da, bai a kolonia mailan e e
Vallejo e al. (2019, 2023) eulien e a a mia men a eko sasoikako elikadu a aldake a beha u di uz ela,
ha apakinen esku aga i asuna en e a e en aga i asuna en a abe a. Gaine a, ike ke ek lehen asun
die e ikoak alda u egin dai ezkeela sexua en (Ma a e al., 2016) edo adina en (Hamil on & Ba clay,
1998; Salsamendi e al., 2008; A izabalaga-Escude o e al., 2019) a abe a.
Die a en aldake a ho iek es a egia aldake a ba isla dezake e saguza ek ha apakinen uga i asuna i
(Fen on, 1982), ene gia-eskakizunei (Ma a e al., 2016) edo bazka- ebe asunei (Hamil on & Ba clay,
1998) e an zu eko. Ho ega ik, saguza in sek ujaleak espezialis a gisa sailka zea ez da hain xamu a
(Fen on, 1982).
II.5 METABARCODING BIDEZ IKERKETA TROFIKOETAN IZANDAKO
AURRERAPENAK
Hama kada ba baino gehiago iga o da DNA me aba coding-a go ozkien ohiko analisi
mo ologikoa en al e na iba gisa so u zene ik, die a az e ke ak i auliz (adb., Cla e e al., 2009; Deagle
e al., 2009; Pega d e al., 2009). Teknika ho i eske , ingu umen-lagin konplexue an, hala nola
lu zo uan, u e an edo e aie an, espezie uga i zeha z iden i ika dai ezke, ohiko me odo mo ologikoek
baino be eizmen axonomiko hobea eskainiz (Bohmann e al., 2014). Gaine a, azka agoa da, ez da
13
SARRERA OROKORRA
i a i- eleme ia e a die a en analisia, go ozkien az e ke a mo ologikoa en e a in sek uen
esku aga i asuna en az e ke a en bidez. O ain suago, A izabalaga-Escude ok eknika molekula ak
e abili zi uen be e dok o e- esian (A izabalaga-Escude o, 2016) espezie ho en die a e a ekologia
sakonago az e zeko. Ha en au kikun zek age ian u zi zu en espeziea paisaia-elemen u espezi ikoen
menpeko asunak e a ha apakinen ja o izko habi a ak babes u beha ak ondo io naba menak di uz ela
saguza a en kon se bazioan (A izabalaga-Escude o e al., 2015). Gaine a, saguza ba en die a
ezauga i un zionalen ikuspegi ik az e zen lehena izan zen, aldake a o iko in aspeki ikoa
ha apakinen ezauga ien bidez (kokapena, u a oa, amaina, sexua e a adina) ebalua uz
(A izabalaga-Escude o e al., 2019).
Fe a-saguza xikia e a handia, az e ke a-e emu osoan ohikoak, sakonago ike u beha ean daude.
Lu alde osoan bana u a egon e a ba zue an kolonia handiak so di zake en a en, haien
bazka-ekologia ez da e a-saguza medi e anea a ena bezain sakon az e u. Fe a-saguza handia en
die a i bu uzko ike ke a molekula baka a F an zian egin da (Tou nay e e al., 2021). Au eko
ike ke ek go o zen analisi mo ologikoan oina i u zi en (adb., Jones, 1990; Flande s & Jones, 2009;
Koselj, Schni zle & Sieme s, 2011), zehaz asun gu xiagoko emai zak emanez. Ho ela, a gi dago
espezie ho en bazka-ekologian sakon zeko beha a.
Ai zi ik, az e ke a-e emuan e a-saguza xikia en die a az e u izan da, baina mahas iekin e a haien
izu i e-kon sumoa ekin lo u a es ua izan (Ba oja e al., 2019, 2021). Espezie honi bu uzko au eko
ike ke ak mo ologikoak izan zi en e e (adb., McAney & Fai ley, 1989; Lino e al., 2014; Mi schunas
& Wagne , 2015). Gaine a, bi espezieei bu uzko ike ke a gehienak be en banake en ipa aldeko
e zean egin di a, e a, be az, bali eke ez iza ea espeziea en un sezko bazka-ekologia en guz iz
adie azga i (adb., R. e umequinum: Jones, 1990; Jones & Mo on, 1992; Du e gé, 1996; Ransome,
1996; Pi , 1994; Die z, Pi & Hillen, 2013; R. hipposide os: McAney & Fai ley, 1988; A le az, 2000;
Gody & Boye , 2002; R. hipposide os: McAney & Fai ley, 2002; Goye , 2002). Be az, e a-saguza
xiki e a handien ekologia o ikoa o aindik ike zeke dago.
20

II. GENERAL INTRODUCTION
Gene al In oduc ion
II.1. FORAGING ECOLOGY OF INSECTIVOROUS BATS
Ba s (o de Chi op e a) comp ise app oxima ely 1,400 species, making hem he second mos di e se
o de o mammals, accoun ing o nea ly a qua e o all desc ibed mammal species (Wilson &
Mi e meie , 2019). They play a signi ican ole in mos e es ial ecosys ems ac oss all con inen s
excep An a c ica (Kunz & Pie son, 1994) and a e unique among mammals in hei e olu ion o
powe ed ligh . The e olu ion o ligh and echoloca ion has been essen ial o ba s o exploi he
di e se ange o noc u nal insec s (Jones & Rydell, 2003) since abou 70% o ba s a e insec i o ous
(Simmons, 2005).
The high ecological di e si y among insec i o ous ba s is linked o a ious adap a ions closely ela ed
o ligh and echoloca ion. Fo ins ance, some la ge and as e species use low- equency
echoloca ion calls o hun ai bo ne insec s in open spaces (e.g., Nyc alus lasiop e us; Ibañez & Jus e,
2023), while smalle , slowe ba s a e mo e manoeu able and cap u e hei ood in clu e ed
en i onmen s (e.g., Myo is bechs einii; Ke h & an Schaik, 2023). Addi ionally, insec i o ous ba s
exhibi di e se skull s uc u es, mandibles, and ee h shapes ha align wi h hei die a y needs, which
a ec s hei bi e o ce and p ey. Ba s ha consume ha d-bodied p ey ha e mo e obus skulls and
mandibles, and longe canines, compa ed o hose ha ea so -bodied p ey (F eeman, 1979, 1998;
Ramí ez-F áncel e al., 2021; San ana e al., 2011, 2022). These species-speci ic adap a ions in luence
hei o aging ecology, de e mining whe e and wha hey can hun (e.g., Swa z e al., 2003).
Fo aging is a undamen al ac i i y necessa y o he su i al o animals. In he case o insec i o ous
ba s, i in ol es sea ching o , de ec ing, and ecognising po en ial p ey. I also equi es deciding
whe he o pu sue he p ey, ac ually pu sue i , ca ch i , and inally consume i (S ephens & K ebs,
1986). Thus, p eda o -p ey in e ac ions depend on p ey p o i abili y and he e ec i eness o de ensi e
s a egies, such as camou lage, mimic y, o escape beha iou (S ephens & K ebs, 1986; Spi z e al.,
2014). Fac o s like p ey size, ha dness o e asi eness a ec p o i abili y, while p ey a ailabili y and
p eda o s’ niche lexibili y shape die (A aújo e al., 2011).
The s udy o he o aging ecology o insec i o ous ba s is a complex and mul i- ace ed ield ha has
elied on a di e se ange o esea ch me hods. These me hods, anging om die a y analysis o
moni o ing eeding beha iou h ough sound analysis and adio- eleme y, ha e p o ided a
comp ehensi e unde s anding o ba ecology. Resea ch on he mo phology o ba s has led o a ious
p edic ions abou hei eeding pa e ns and esou ce pa i ioning, o en suppo ed by die a y s udies
ein o cing hese in e ences d awn om he ecomo phological analyses (e.g., Fen on, 1982). This
di e si y o esea ch me hods has led o he classi ica ion o ba s in o di e en guilds based on hei
o aging s a egies, habi a o die (Allen, 1939; Denzinge e al., 2018; Fen on, 1990, espec i ely).
23
Gene al In oduc ion
II.2. ECOMORPHOLOGY AND FORAGING GUILDS
Ba s' wing shape and echoloca ion calls a e adap ed o he speci ic o aging niches hey exploi . This
co ela ion allows us o classi y ba s in o g oups o guilds. Fo ins ance, open-space o age s p ima ily
use long, na owband echoloca ion calls a low equencies ha can a el o e long dis ances
( e iewed in No be g & Rayne , 1987). Thei poin ed wing ips, combined wi h high wing loading and
aspec a ios, enable hem o ly quickly, al hough his comes a he cos o lowe manoeu abili y. In
con as , ba s ha ly nea o among clu e ypically exhibi lowe wing loading and ounde wing ips,
which enhance hei manoeu abili y (Vaughan, 1959). These ba s also show an imp essi e di e si y
o echoloca ion calls depending on hei hun ing beha iou s. Some ba s ha de ec lu e ing insec s a
sho ange use cons an equency (CF) calls along wi h b ie equency-modula ed (FM) sweeps a
highe equencies (Schni zle , 1984). They may emi a long call o a se ies o sho e calls o
compensa e o he Dopple e ec , allowing hem o de ec and iden i y hei a ge s (Schni zle ,
1968). This calling pa e n is ypical o lyca ching ba s (Schni zle e al., 1985). Addi ionally, o he
ba s ha ly among clu e u ilise FM calls ha can include a a ie y o sho o long combina ions,
wi h s eep o shallow equencies, o loca e p ey hidden in hei su oundings (Simmons & S ein,
1980). Gleaning ba s, which hun on su aces, use sho , low-in ensi y FM calls o disc imina e hei
a ge s' ine ex u es (Habe se ze & Vogle , 1983).
Based on hese gene al pa e ns, Fen on (1990) made h ee big g oups depending on hei o aging
habi a : open-space o age s, edge and gap o age s, and na ow-space o age s. Schni zle and Kalko
(2001) made a simila classi ica ion based on he habi a (unclu e ed, backg ound-clu e ed, and
highly clu e ed), and hey di e en ia ed subca ego ies o each habi a based on he animals' hun ing
echniques, such as ae ial o gleaning ba s. Mo e ecen ly, Denzinge e al. (2018) made a
classi ica ion based on he o aging modes, which a e also linked o he habi a hey use, as ae ial
hawke s, awle s, ac i e gleane s, passi e gleane s, and lu e de ec o s. T adi ionally, hese guilds
ha e been associa ed wi h speci ic habi a s. In his way, species wi h low wing aspec a ios ha e been
linked o dense o es s and sh ubby a eas, whe eas hose wi h high aspec a ios ha e been linked o
open en i onmen s such as meadows, moo lands o high al i udes (Findley e al., 1972).
II.3. FORAGING AREAS
O e he las decades, ou unde s anding o he o aging ecology o insec i o ous ba s has
signi ican ly ad anced, especially ega ding hei habi a and ophic equi emen s. This p og ess is
p ima ily a ibu ed o imp o emen s in esea ch me hodologies and echnological inno a ions, such
as adio eleme y (e.g., Fen on, 1997; Bon adina e al., 2002; Bu schkoski, 2005; D essle e al.,
2016; Lage eld e al., 2017) and imp o ed acous ic moni o ing (Vaughan e al., 1997; Russo &
Jones, 2003; F ick, 2013; Ancillo o e al., 2023; Ko ila e al., 2023). These s udies ha e p o ided
mo e p o ound insigh s in o ba beha iou and habi a use. Resea ch highligh s he impo ance o
24
Gene al In oduc ion
di e se and high-quali y habi a s o insec i o ous ba s, which suppo a ich abundance and di e si y
o insec s h oughou he yea (Nicholls & Racey, 2006; Jung & Kalko, 2010; Hagen & Sabo, 2016;
S aka e al., 2016).
Iden i ying high-quali y o aging a eas is a c ucial aspec o ba s' conse a ion (F ick e al., 2024),
gi en ha habi a loss—including logging and ag icul u e—, is he p ima y h ea o ba biodi e si y
wo ldwide (F ick e al., 2020). Howe e , his ask is no wi hou challenges. De e mining he
app op ia e scale and con igu a ion o habi a p o ec ion, especially conside ing he ex ensi e nigh ly
mo emen s o ba s (McC acken e al., 2016; Goldsh ein e al., 2020; O’Ma a e al., 2021), is a
complex and di icul ask.
On he o he hand, adio- acking and ul asound-based s udies also aced limi a ions. These s udies
a e labou and esou ce-in ensi e, equi ing signi ican ield pe sonnel and equipmen , o en esul ing
in small sample sizes and limi ed acking du a ions (Bon adina e al., 2002; Mu ay & Ku a, 2004;
Smi h & Racey, 2005). The e o e, hey ba ely e eal he seasonal shi s in habi a use, common
among ba s, d i en by changes in p ey a ailabili y and clima ic condi ions. The need o in eg a ed
esea ch on habi a and die is essen ial, as mos adio- acking s udies a ge ing he habi a
equi emen s o ba s and hei die s udies a e no o en simul aneously ca ied ou and ail o add ess
wha he animals ha e a ailable in hei o aging g ounds and wha hey selec he e (bu see Goi i e
al., 2008; Flande s & Jones, 2009; Napal e al., 2013).
Fu he mo e, because a h opods ha e complex li e cycles, habi a p o ec ion e o s mus ensu e he
in eg i y o a ious habi a s ha suppo hese o ganisms' en i e li e cycle (A izabalaga-Escude o e
al., 2015). The e o e, accu a ely iden i ying he speci ic o aging habi a s ha ba s depend on and
hei speci ic p ey i ems is c ucial o e ec i ely p o ec ing ba popula ions.
II.4. DIET
The s udy o p eda o s' o aging has his o ically ocused on analysing he species composi ion and
ichness o p ey in hei die s. This app oach has led o he classi ica ion o mono ypic p eda o s as
specialised o selec i e, while hose ha eed on a wide a ie y o p ey species a e ca ego ised as
gene alis o oppo unis ic p eda o s (Spi z e al., 2014).
Insec i o ous ba s exhibi a b oad a ay o die a y beha iou s, anging om gene alis o highly
specialised. Mos species display conside able die lexibili y (e.g., Jones & Rydell, 2003; Cla e e al.,
2009; Aizpu ua e al., 2013; Aiha za e al., 2023). Howe e , some species ha e been add essed as
specialised in consuming speci ic insec axa, adding a laye o complexi y o hei beha iou . Fo
ins ance, Pipis ellus na husii p ima ily eeds on mosqui oes (K üge e al., 2014), while Ba bas ella
sp. (Sie o & A le az, 1997) and Rhinolophus eu yale (Goi i e al., 2008; Salsamendi e al., 2012;
And eas e al., 2013; A izabalaga-Escude o e al., 2015) a e known o hei p e e ence o mo hs.
25

Gene al In oduc ion
Addi ionally, some ba s, such as Sco ophilus i idis and Myo is myo is, ha e been obse ed o
specialise in eeding on bee les (Fen on e al., 1977; A le az, 1999, espec i ely).
None heless, he specialisa ion le el can change based on seasonal o geog aphical ac o s. Fo
ins ance, seasonal die a y shi s ha e also been no ed in Sco ophilus i idis, which ends o ha e a
b oade die du ing he d y season (Fen on & Thomas, 1980), indica ing ha i consumes wha e e
a ailable insec s i can ind when ood is sca ce. Myo is ema gina us is a clea example o egional
a iabili y since i p ima ily hun s lies in cowsheds in Ge many and Belgium (Beck, 1995; S eck &
B inkmann, 2006; Ke yn e al., 2012), bu in sou he n egions, spide s make up a signi ican po ion
o hei die (Goi i e al., 2011; Galan e al., 2017). In e es ingly, e en wi hin he same colonies,
Vallejo e al. (2019, 2023) ha e obse ed seasonal shi s be ween lies and spide s depending on p ey
a ailabili y and p o i abili y. Addi ionally, s udies ha e shown ha die a y p e e ences can a y
be ween indi iduals based on ac o s such as sex (Ma a e al., 2016) o age (Hamil on & Ba clay,
1998; Salsamendi e al., 2008; A izabalaga-Escude o e al., 2019).
These changes in die may e lec a change in s a egy by he ba s esponding o a ia ions in p ey
abundance (Fen on, 1982), di e ing ene gy demands (Ma a e al., 2016), o di e ences in o aging
skills (Hamil on & Ba clay, 1998). The e o e, i is challenging o classi y insec i o ous ba s s ic ly as
die a y specialis s (Fen on, 1982).
II.5. ADVANCES IN TROPHIC STUDIES THROUGH METABARCODING
Mo e han a decade has passed since DNA me aba coding eme ged as a g ound-b eaking al e na i e
o adi ional mo phological analysis o aeces, e olu ionising die s udies (e.g., Cla e e al., 2009;
Deagle e al., 2009; Pega d e al., 2009). This echnique enables he p ecise iden i ica ion o mul iple
species in complex en i onmen al samples, such as soil, wa e , o gu con en s, o e ing ine
axonomic esolu ion han adi ional mo phological me hods ( e iewed in Bohmann e al., 2014).
Addi ionally, i is as e , does no ely on expe axonomic skills, and can iden i y agmen s ha a e
di icul o classi y mo phologically ( e iewed in Pompanon e al., 2012). Since he pionee ing wo k
by Cla e e al. (2009), he de elopmen o a h opod-speci ic sho p ime s o die analyses (e.g.,
Zeale e al., 2011) and ad ancemen s in Nex Gene a ion Sequencing Technologies ( e iewed in
Pompanon e al., 2012) ha e enhanced he esolu ion and cos -e ec i eness o die analyses, despi e
some limi a ions (Cla e, 2014; Albe di e al., 2018). The e o e, ba die a y s udies ha e also been
en iched om his me hodology (e.g., Whi ake & Ka a aş, 2009; Cla e e al., 2011; Bohman e al.,
2011; Razgou e al., 2011; Zeale e al., 2011).
The apid ad ancemen o me aba coding has led o he de elopmen o se e al p ime se s a ge ing
a h opods (e.g., Zeale e al., 2011; Elb ech & Leese, 2017; Galan e al., 2017; Vamos e al., 2017;
Wangens een e al., 2018; Jusino e al., 2019). Howe e , esea che s ha e shown ha p ime se s o en
exhibi biases owa d ce ain a h opod o de s (Cla ke e al., 2014; Piñol e al., 2015; Jusino e al.,
26
Gene al In oduc ion
2019; K ehenwinkel e al., 2017; Elb ech e al., 2019). As a esul , he e has been a end owa d
designing mo e degene a ed p ime s (K ehenwinkel e al., 2017; Vamos e al., 2017; Jusino e al.,
2019) o enable he ampli ica ion o DNA om a b oade ange o a h opod g oups. Addi ionally,
combining di e en p ime se s may be a mo e e ec i e app oach o minimise biases associa ed wi h
indi idual p ime se s. This s a egy imp o es die a y co e age, p o iding a be e unde s anding o
p eda o s' ecology (Esnaola e al., 2018; Jusino e al., 2019; Penne e al., 2024).
Mo eo e , sequencing dep h is also c ucial o his echnique (Albe di e al., 2018). A ew yea s ago,
a ious sequencing pla o ms we e u ilised; howe e , ollowing he esul s o se e al s udies
compa ing he pe o mance o hese pla o ms (Loman e al., 2012; Mak e al., 2017; Tabe le e al.,
2018), he use o Illumina became p edominan . Recen ad ancemen s in sequencing echnology ha e
enabled us o achie e up o 400 million eads pe un wi h he No aSeq sys em (Illumina), in con as
o a maximum o 25 million eads pe un wi h he olde MiSeq sys em (Illumina). These
imp o emen s acili a e mo e comp ehensi e me aba coding s udies, o e ing mo e p o ound insigh s
in o ecology by allowing o inc eased sampling sizes and iden i ying mo e p ey i ems (Capo aso e
al., 2012; Han e al., 2024).
II.6. BEYOND TAXONOMIC ANALYSIS OF DIET
P ey-p eda o ela ionships a e o en examined p ima ily h ough a axonomic lens, ocusing on which
p eda o eeds on which axa wi hou conside ing he cha ac e is ics o he p ey (Spi z e al., 2014).
Many molecula s udies on ba die s ha e p ima ily been limi ed o p o iding ex ensi e lis s o p ey
(e.g., Bohman e al., 2011; Cla e e al., 2013; Rol e e al., 2014; Van den Bussche, 2016; Go don e al.,
2019; W ay e al., 2020; O’Rou ke e al., 2021). In con as , analysing die s wi h a b oade , mo e
ambi ious pe spec i e—inco po a ing a ocused sampling design while also conside ing p ey
cha ac e is ics, p eda o s, en i onmen , and seasonal a ia ions—can g ea ly enhance ou
unde s anding o ba ecology. Molecula s udies ha e shown ha ba s consume hund eds o species,
which a y o e ime (Razgou e al., 2011; A izabalaga‐Escude o e al., 2015; Aiha za e al., 2023);
space (Cla e e al., 2014); and among indi iduals (Ma a e al., 2016), allowing hem o adjus hei die
based on a ailabili y (e.g., McC acken e al., 2012; Almena e al., 2013; Napal e al., 2013; Aiha za
e al., 2023; Vallejo e al., 2023). Me aba coding s udies ha e also shed ligh on he ole o niche
pa i ioning among co-occu ing sibling species (e.g., A izabalaga-Escude o e al., 2018;
No ella-Fe nandez e al., 2020; And iollo e al., 2021; Blanch e al., 2023), and ha e e en iden i ied
many ag o o es y pes species, un eiling ha ba s may ac as pes supp esso s (e.g., Aizpu ua e al.,
2017; Ba oja e al., 2019; And iollo e al., 2021; Liu e al., 2023).
Howe e , accu a e assessmen s o o aging ecology in p eda o s like insec i o ous ba s equi e mo e
han a axonomic lis ing. I is essen ial o mo e beyond and add ess he ques ion: Why a p ey is a
p ey? This app oach in ol es unde s anding he unc ional ela ionships be ween p eda o s and p ey.
27
Gene al In oduc ion
Insec i o ous ba s eed on a wide a ie y o insec s, which di e in size, ligh pa e ns, ha dness and
e asi e beha iou s, ai s ha likely in luence hei p o i abili y o ba s (Schni zle , 1987). This
a iabili y implies ha p eda o s mus be able o dis inguish be ween p ey ypes o a ying
p o i abili y (i.e. ene gy gained pe ime uni o handling: de ec ing, pu suing, cap u ing and
consuming) and adjus p ey a ge s when p ey a ailabili y changes o ensu e an a e age posi i e
ene gy balance. P ey p o i abili y may a y wi h en i onmen al condi ions (e.g., p ey abundance) o
p eda o -speci ic equi emen s (e.g., b eeding season, sex‐speci ic ene ge ic equi emen s o
indi idual di e ences in p ey-cap u ing skills) (S ephens & K ebs, 1986).
Fo ins ance, male and emale Tada ida enio is ba s di e in he ype o mo hs hey consume (Ma a e
al., 2016). Simila ly, di e ences be ween adul and ju eniles ha e been epo ed in Ep esicus uscus
(Hamil on & Ba clay, 1998) o Rhinolophus eu yale (A izabalaga-Escude o e al., 2019) and
sugges ed ha ligh and echoloca ion expe ience may be causing such di e ences.
Thus, wha ac o s in luence hei die a y adjus men s? The cha ac e is ics o hei p ey a e mo e
signi ican han axonomic classi ica ion. T ai -based app oaches, which connec p eda o and p ey
mo phology and ecology, o e a deepe insigh in o he ac o s ha shape p ey selec ion (Spi z e al.,
2014). Pionee ing his ield on ba s, A izabalaga-Escude o e al. (2019) applied ai -based me hods
o explo e he ophic ecology o ho seshoe ba s. The s udy examined how he ene ge ic needs o
males, emales in di e en b eeding s ages, and ju eniles e sus adul s in luenced hei choices o
p ey size o ligh cha ac e is ics. O he esea ch has also linked p ey size, habi a , seasonali y, and he
ba s' body condi ion o hei o aging beha iou (Ancillo o e al., 2023). This expanding ield o s udy
highligh s he complexi y o ba o aging s a egies and he c ucial ole o p ey ai s in shaping hei
die a y choices.
Mo eo e , he p ecise axonomic esolu ion p o ided by me aba coding enables linking p ey species
o hei habi a s (Cla e e al., 2011; Razgou e al., 2011; Albe di e al., 2012). While i canno
de e mine whe e he consumed p ey we e cap u ed (sink habi a ), i can indica e he o igin o hose
p ey (sou ce habi a ) (A izabalaga-Escude o e al., 2015). Consequen ly, molecula die analysis has
become pa amoun o de e mining he o aging dependencies o ba s by iden i ying he habi a s
whe e hey hun o he sou ces o hei p ey h oughou he yea . No ably, s udies on molecula die s
sugges ha o aging guilds a e mo e lexible han p e iously hough . Fo ins ance, open- o age ba
species also hun in —o abo e, o a ound— a ious habi a s, including o es s, in esponse o p ey
a ailabili y peaks (Ga in e al., 2019; Aiha za e al., 2023). Simila ly, clu e -specialis ba s will likely
ake ad an age o p ey ou b eaks in di e en habi a s, using hei echoloca ion and ligh skills a he
mic ohabi a le el by na iga ing nea by ege a ion.
28
Gene al In oduc ion
II.7. ECOSYSTEM SERVICES
DNA me aba coding has e ealed ha ba s, wi h hei di e se die o pes a h opods, play a c ucial
ole in esponding o ou b eaks (McC acken e al., 2012; Cha bonnie e al., 2014; Puig-Mon se a e
al., 2015; Aizpu ua e al., 2018; K auel e al., 2018; Ba oja e al., 2019, 2021; Ga in e al., 2019;
Ko ine e al., 2020; And iollo e al., 2021; Liu e al., 2023). In la ge-scale monocul u es, insec pes s
a e a signi ican pa o hei die (Symondson e al., 2002; Segoli & Rosenheim, 2012), and exclosu e
expe imen s ha e con i med hei ole in educing ag icul u al pes s (Maas e al., 2013, 2016; Linden
e al., 2019; Ancillo o e al., 2024; Tuneu-Co al e al., 2024). This unde sco es he po en ial o ba s in
sus ainable pes managemen , o e ing a ay o hope o he u u e o ag icul u e.
Howe e , mos s udies ely on p esence/absence da a (Aizpu ua e al., 2018; Ba oja e al., 2019; Weie
e al., 2019). I is impo an o no e ha me aba coding biases can lead o alse nega i es due o
di e en ial p ime a ini y and compe i ion among DNA sequences (Pompanon e al., 2012; E ans e
al., 2016). To add ess his, a ge ed app oaches, such as species-speci ic p ime s wi h PCR-based
me hods, o e g ea e p ecision. Con en ional PCR (cPCR) and quan i a i e PCR (qPCR) can de ec
minimum DNA quan i ies (Ja man e al., 2004) bu a e limi ed o single a ge sequences. Mul iplex
PCR allows ampli ica ion o mul iple sequences (Chambe lain e al., 1988), bu he use o di e en
p ime pai s may lead o ampli ica ion bias o ce ain DNA empla es (Mus o p e al., 2011). The
PCR-based mul iplex liga ion-dependen p obe ampli ica ion (MLPA) echnique enhances
mul iplexing by using a single p ime pai o ampli y speci ic p obes, imp o ing quan i ica ion
accu acy (Schou en e al., 2002).
Despi e he challenges in aecal DNA analysis (e.g., Piñol e al., 2015; Albe di e al., 2018; Deagle e
al., 2019), s udies ha e es ima ed ba consump ion a es (McC acken e al., 2012; Aiha za e al.,
2023) and hei economic alue in pes supp ession ( e iewed by Tuneu-Co al e al., 2023). Al hough
seasonal and landscape a ia ions a ec es ima es, unde s anding ba die s in ag oecosys ems is
c ucial o assessing hei ecosys em se ices (Russo e al., 2018).
II.8. FORAGING ECOLOGY OF HORSESHOE BATS
Ho seshoe ba s (Rhinolophidae G ay, 1825) a e a di e se amily o insec i o ous ba s known o hei
dis inc i e ho seshoe-shaped noselea es, which play a c ucial ole in echoloca ion. These noc u nal
mammals ely on high- equency calls o na iga e and hun in complex en i onmen s. Dis ibu ed
ac oss he sou he n Palea ic egion o he Old Wo ld, hey a e ca e-dwelling species pa icula ly
abundan in ka s a eas, whe e unde g ound oos s a e abundan (Cso ba e al., 2003). They mainly
use sub e anean oos s, ei he na u al (ca es) o a i icial (mines, unnels, e c.), bu also inhabi
buildings, especially o ma e ni y colonies (Russo e al., 2023). In Eu ope, hey a e p e y common in
a eas such as he No he n Ibe ian Peninsula (Palomo e al., 2007), F ance (A hu & Lemai e, 2021),
I aly (Agnelli e al., 2004; GIRC, 2004), and he Balkans (e.g., T ko ic, 2006; K yš u ek & Done ,
29
Gene al In oduc ion
Goi i, U., Aiha za, J., & Ga in, I. (2004). Die and p ey selec ion in he Medi e anean ho seshoe ba Rhinolophus
eu yale (Chi op e a, Rhinolophidae) du ing he p e-b eeding season. Mammalia, 68(4), 397-402.
h ps://doi.o g/10.1515/mamm.2004.039
Goi i, U., Aiha za, J. R., Ga in, I., & Zabala, J. (2003). In luence o habi a on he o aging beha iou o he
Medi e anean ho seshoe ba , Rhinolophus eu yale. Ac a Chi op e ologica, 5(1), 75-84.
Goi i, U., Aiha za, J. R., Almena , D., Salsamendi, E., & Ga in, I. (2006). Seasonal o aging by Rhinolophus eu yale
(Rhinolophidae) in an A lan ic u al landscape in no he n Ibe ian Peninsula. Ac a Chi op e ologica, 8(1),
141-155.
Goi i, U., Aiha za, J., Guiu, M., Salsamendi, E., Almena , D., Napal, M., & Ga in, I. (2011). Geo oy's ba , Myo is
ema gina us, p eys p e e en ially on spide s in mul is a i ied dense habi a s: a s udy o o aging ba s in he
Medi e anean. Folia Zoologica, 60(1), 17-24.
Goi i, U., Ga in, I., Almena , D., Salsamendi, E., & Aiha za, J. (2008). Fo aging by Medi e anean ho seshoe ba s
(Rhinolophus eu yale) in ela ion o p ey dis ibu ion and edge habi a . Jou nal o Mammalogy, 89(2), 493-502.
h ps://doi.o g/10.1644/07-MAMM-A-054R2.1
Goldsh ein, A., Handel, M., Ei an, O., Bons ein, A., Shale , T., Colle , S., ... & Yo el, Y. (2020). Rein o cemen
lea ning enables esou ce pa i ioning in o aging ba s. Cu en Biology, 30(20), 4096-4102.
Go don, R., I ens, S., Amme man, L. K., Fen on, M. B., Li le ai , J. E., Ra cli e, J. M., & Cla e, E. L. (2019).
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di e se echoloca ion and o aging s a egies. Ecology and E olu ion, 9(6), 3117-3129.
G uppo I aliano Rice ca Chi o e i (GIRC) (2004). The I alian ba oos p ojec : a p elimina y in en o y o si es and
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Hagen, E. M., & Sabo, J. L. (2011). A landscape pe spec i e on ba o aging ecology along i e s: does channel
con inemen and insec a ailabili y in luence he esponse o ba s o aqua ic esou ces in i e ine landscapes?.
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Hamil on, I. M., & Ba clay, R. M. (1998). Die s o ju enile, yea ling, and adul big b own ba s (Ep esicus uscus) in
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41

S a e o he A
III. STATE OF THE ART
Unde s anding he o aging ecology o ba s equi es a comp ehensi e app oach ha accoun s o
seasonal and egional a ia ions in die , and he species' in e ac ions wi h i s en i onmen , including
he su ounding habi a s and p ey a ailabili y. Addi ionally, in a- and in e speci ic compe i ion is
c ucial in shaping o aging s a egies and esou ce pa i ioning among coexis ing species.
High- h oughpu sequencing echniques, such as DNA me aba coding, p o ide high- esolu ion
axonomic iden i ica ion o p ey i ems, making hem in aluable ools o in es iga ing ophic
ecology. This disse a ion aims o enhance he unde s anding o he ophic ecology o he h ee
ho seshoe ba species p esen in he s udy a ea (Basque Coun y, No he n Ibe ian Peninsula), wi h a
pa icula emphasis on he lesse (Rhinolophus hipposide os) and g ea e (Rhinolophus
e umequinum) ho seshoe ba s. By in eg a ing DNA me aba coding wi h ecological ai analyses,
his esea ch seeks o p o ide no el insigh s in o die a y a ia ion, habi a dependences, and he
ecological ac o s in luencing p ey selec ion. The indings will con ibu e o a b oade unde s anding
o ho seshoe ba o aging s a egies and hei ole wi hin he ecosys em.
III.1. HYPOTHESIS
Recen ad ances in molecula echniques enable a comp ehensi e analysis o he ophic ecology o
bo h ho seshoe ba species (Rhinolophus hipposide os and R. e umequinum), including empo al and
spa ial a ia ion in die , habi a dependency, and hei unc ional ole as na u al pes supp esso s.
Fu he mo e, hese me hods will acili a e he iden i ica ion o die a y di e ences be ween ju enile
and adul indi iduals, p o iding insigh in o hei dis inc ophic equi emen s and ecological niches.
43
S a e o he A
III.2. OBJECTIVES
The speci ic objec i es ou lined in he ollowing chap e s unde sco e he signi icance o his esea ch;
he i s wo deal wi h me hodological imp o emen s, while he subsequen ones we e designed o
p o e he hypo hesis and con ibu e o a b oade unde s anding o ba o aging ecology:
OBJECTIVE 1: To iden i y and selec he mos sui able p ime s o analysing he die o ho seshoe
ba s, enabling accu a e and eliable insigh s in o hei eeding habi s and ecological in e ac ions.
This objec i e is de eloped in Chap e 1.
The apid ad ancemen s in DNA me aba coding du ing he de elopmen o he s udy
necessi a ed a e iew and upda e o he p e iously used p ime s, leading o an addi ional
analysis o ongoing esea ch. This sec ion is included in an addendum o Chap e 1.
OBJECTIVE 2: To de elop a eliable assay o de ec ing and semi-quan i ying mul iple pes species
in ba aeces using a mul iplex liga ion-dependen p obe ampli ica ion (MLPA) assay. This
objec i e is de eloped in Chap e 2.
OBJECTIVE 3: To analyse he undamen al ophic ecology o bo h ho seshoe ba s by examining
seasonal and geog aphical a ia ions in hei die h oughou hei ac i e season. The s udy also
aims o unde s and he habi a and landscape dependence o he species by examining he
ela ions o p e e ed p ey and hei habi a s. This objec i e is de eloped in Chap e 3 o R.
hipposide os and Chap e 4 o R. e umequinum.
OBJECTIVE 4: To s udy he p ima y pes species consumed by R. hipposide os and R.
e umequinum ac oss a ious ag o o es y landscapes h oughou he season and es ima e he
o al mass o pes s consumed, con ibu ing o unde s anding hei ole in pes con ol. This
objec i e is de eloped in Chap e 5
OBJECTIVE 5: To compa e he die s o ju eniles and adul s ac oss he h ee ho seshoe ba species
o he s udy a ea –R. hipposide os, R. e umequinum and R. eu yale– by analysing hei die a
he axonomic and p ey ai le els (size, lying speed, ha dness). This esea ch aims o es
age- ela ed die a y di e ences and he impac s o de elopmen and ophic specialisa ion on
hese di e ences. This objec i e is de eloped in Chap e 5.
44
IV. RESULTS
CHAPTER 1
1.2. MATERIALS AND METHODS
1.2.1. S udy A ea
The s udy was ca ied ou in Ka an za and Lea-A ibai Valleys (Basque Coun y, No he n Ibe ian
Peninsula). Ka an za is a hilly alley wi h ele a ions o 200–855 m a.s.l. (30T 46968E, 478950N)
whe e he p e ailing landscape consis s o a mosaic o small meadows and pas u es, dedica ed o
dai y ca le b eeding, su ounded by an impo an hedge ow ne wo k consis ing mainly o sh ubs and
deciduous ees. Lea-A ibai Valley is also a hilly and s eep alley wi h ele a ions anging ca 40–700
ma.s.l. (30T 53647E, 479442N), whe e p e ailing plan a ions o Pinus adia a–and less equen ly
Eucalyp us globulus–a e in e spe sed wi h small a ming pa ches and small deciduous and holm oak
woodland pa ches. Limes one massi s ha p o ide abundan na u al ca i ies su ound bo h alleys,
cha ac e ised by A lan ic empe a e oceanic clima e, whe e ain all occu s h oughou he yea (annual
mean 1400mm) (A izabalaga-Escude o e al., 2015)
1.2.2. Sample Collec ion
Sampling was ca ied ou du ing he b eeding season, in July 2012. Wi hin each sampling a ea
(Ka an za and Lea-A ibai), each ba species was sampled in a di e en cap u e si e. The e we e h ee
oos s in Ka an za–one o each species–and wo oos s in Aules i–one used by R. eu yale and R.
e umequinum, and ano he one by R. hipposide os. Ba s we e cap u ed wi h a 2 × 2 m ha p ap
(Tu le, 1974) loca ed in he en ance o he colony oos s om 00:30 a.m. onwa ds, as ba s e u ned
o he ca es a e o aging. Each cap u ed ba was held indi idually in a clean clo h bag un il i
de eca ed (a maximum o 40–90 min). Each bag was used only once o a oid c oss-con amina ion o
aecal samples. Faecal ma e ial collec ed om each indi idual ba was ozen wi hin 6 h since
collec ion ime. Ba s we e immedia ely eleased in o he ca e a e handling. Conside ing bo h cap u e
si es al oge he , 24 R. e umequinum, 31 R. hipposide os and 18 R. eu yale indi iduals we e sampled.
Indi idual ba s we e conside ed as sample uni s (Whi ake e al., 1996).
Cap u e and handling p o ocols ollowed published guidelines o ea men o animals in esea ch
and eaching (She win, 2006) and we e app o ed by he E hics Commi ee a he Uni e si y o he
Basque Coun y (Re . CEBA/219/2012/GARIN ATORRASAGASTI). Cap u es we e pe o med
unde license om he Depa men o he En i onmen o he Regional Council o Biscay (Pe mi
numbe s G13 1061; G13 1064 and G13 1066).
1.2.3. DNA Ex ac ion, PCR Amplifica ion, Lib a y P epa a ion and Sequencing
Indi idual aecal samples o 10–40 mg we e used o DNA ex ac ion wi h he DNeasy Powe -Soil
Ki (Qiagen, Valencia, CA), ollowing he manu ac u e s eps. Ex ac ed DNA was PCRampli ied
wice using o di e en p ime se s, a ge ing di e en mini-COI segmen s o he mi ochond ial DNA
cy och ome c oxidase subuni I ba code egion (COI): Zeale p ime s (ZBJ-A F1c and ZBJ-A R2c)
52

CHAPTER 1
(Zeale e al., 2011) we e used o ampli y a 157 bp sec ion, and Gille p ime s (modi ied LepF1 and
EPT-long-uni R, ollowing (Gille e al., 2015) o ampli y ano he 133bp sec ion. Bo h ampli ica ions
we e pe o med using QIAGEN Mul iplex PCR Ki (Qiagen Ibe ia, S.L. Mad id) in 25 μl PCR
eac ions. Each eac ion con ained 2.5 μl Bu e 10X, 1.5 μl MgCl2 50mM, 0.5 μl nNTPs 25mM and
0.125 μl o aq polyme ase. In he case o Zeale p ime s, 0.6 μl o each p ime (10μM) ( o wa d and
e e se), 17.175 μl deionised wa e and 2 μl sample DNA we e added. Wi h Gille p ime s, 0.75 μl o
each p ime (10μM), 14.875 μl deionised wa e and 4 μl sample DNA we e added. Each p ime se
had i s own PCR p og am, modi ied om he e e ence o he used eac i e. The mocycle condi ions
o Zeale p ime s we e: 95˚C– 15 min; 50 cycles o 94˚C– 30 sec, 52˚C– 30 sec, 72˚C– 30 sec; 72˚C–
6 min (modi ied om Razgou e al., 2011). Fo Gille p ime s we used: 95˚C– 15 min; 40 cycles o
94˚C– 30 sec, 45˚C– 45 sec, 72˚C– 30 sec; 72˚C– 10 min (Gille e al., 2015). Fo he lib a y
p epa a ion, each sample was agged wi h a unique combina ion o Mul iplex Iden i ie p ime s (MID)
(Binladen e al., 2007). PCR ou pu s we e sequenced by Ion To en sequencing pla o m, one un
making abo e one million eads.
1.2.4. Bioin o ma ic Analyses
Quali y con ol, sequence p e-p ocessing and collapsing o iden ical sequences in o a single sequence
we e pe o med using CUTADAPT (Ma in, 2011) and USEARCH (Edga , 2010). Clus e ing o
sequences in o Ope a ional Taxonomic Uni s (OTU) was ca ied ou wi h he VSEARCH (Rognes e
al., 2016), a a 97% simila i y h eshold. OTUs we e no malised in o de o a oid dispa i ies in sample
eads and he ones wi h less han 1% equency we e il e ed.
The axonomic assignmen o each OTU was pe o med by compa ing he ep esen a i e sequence o
each OTU agains e e ence sequences in he Ba code o Li e Da abase (BOLD;
www.boldsys ems.o g/) using BLAST (h p://blas .ncbi.nlm.nih.go /Blas .cgi) and GenBank
(h p://www.ncbi.nlm.nih.go ), ollowing he iden i ica ion c i e ia o Cla e e al. (2014). The
dis ibu ion ange o each species was checked in o de o e i y ha i encompasses ou s udy a ea.
Species le el assignmen s we e pe o med when que y sequences ma ched e e ence sequences abo e
98% simila i y and 75% o e lap (Cla e e al., 2014). When que y sequences ma ched mo e han one
species in he da abase, he hi wi h he longes alignmen leng h was selec ed. Besides, as a ule, only
hi s wi h e- alue below 1e-20 we e accep ed (Ves e inen e al., 2013) o make su e ha he ma ch did
no occu by chance. P ime ou pu s we e also es ed o see whe he any o he OTUs buil om hem
could also iden i y he p eda o s hemsel es (See Appendix A o sc ip de ails).
1.2.5. Da a Analysis
To s udy he e ec o p ime s on he species composi ion obse ed in he die , we pe o med a
pe mu a ional mul i a ia e analysis o a iance (PERMANOVA) using adonis wi h 999 andom
pe mu a ions in egan 2.5–1 package (Oksanen e al., 2018) o R e sion 3.3.2 (2016). Fi s , we
53
CHAPTER 1
measu ed he di e ence among colonies/sampling si es and, as i was no signi ican , i was no longe
conside ed. Then we used p ime se and ba species as p edic o a iables and he numbe o
occu ences o p ey as esponse a iable. Jacca d’s dis ance measu e was used o calcula e
dissimila i ies be ween samples. We pe o med NMDS in egan 2.5–1 package o R o isualise
dissimila i ies in species composi ion among samples. The pe cen age o occu ence (POO) o a gi en
p ey axon e e s o he pe cen age ob ained wi h he numbe o occu ences o each axon when
compa ed wi h he o al numbe o occu ences o all axa and he equency o occu ence (FOO) o
he numbe o ba indi iduals whe e each axon was ound compa ed wi h he sample size (Deagle e
al., 2019).
Pianka’s (Pianka, 1973) measu e o niche o e lap has been ca ied ou o compa e he in e speci ic
esou ce pa i ioning o he species. Fo he compa ison o he die o he h ee ba s, PERMANOVA
analyses we e pe o med using adonis and o he pai wise analysis we used pai wise.pe m.mano a
om RVAideMemoi e 0.9–72 package (He é, 2014). See Appendix C o R sc ip de ails.
1.3. RESULTS
1.3.1. Die o Ho seshoe Ba s
We success ully ex ac ed and ampli ied DNA om aeces o 24 Rhinolophus e umequinum
indi iduals, 18 R. eu yale and 31 R. hipposide os, ob aining abo e one million sequence eads (Table
1.1). 309 OTUs we e hen buil and 135 o hem we e assigned o po en ial p ey species consumed by
ba s.
We iden i ied 62 p ey species o R. e umequinum: 34 lepidop e ans, 17 dip e ans, 7 coleop e ans, 2
neu op e ans and 1 ichop e an. Lepidop e a and Dip e a we e he mos equen ly consumed,
ollowed by Coleop e a (Tables A, B and C in S1.1 Suppo ing In o ma ion File). Among he mos
equen ly consumed species Pha macis usconebulosa (Hepialidae; FOO = 46%) p e ailed among
Lepidop e a and Rhipidia macula a (Limoniidae; FOO = 71%) and Tipula maxima (Tipulidae; FOO =
29%) among Dip e a. Wi hin Coleop e a, he mos consumed we e he ela e id S enagos us hombeus
(FOO = 46%) and he ce ambycids A hopalus us icus and P ionus co ia ius (FOO = 42% and 17%
espec i ely), comple ed wi h sca abeids Aphodius sp. and Se ica b unnea (FOO = 29% and 17%
espec i ely).
Ou o he 81 p ey species iden i ied in aeces o R. eu yale, 61 we e lepidop e ans, 10 dip e ans, 5
neu op e ans, 2 epheme op e ans, 1 ichop e an, 1 hemip e an and 1 hymenop e an. Lepidop e a was
he mos equen ly consumed o de ollowed by Dip e a (Tables A, B and C in S1.1 Suppo ing
In o ma ion File), al hough he FOO o mos o hem was less han 20%. The excep ions we e he
noc uids Capsula spa ganii, Cosmia apezina and Lycopho ia po phy ia (FOO > 28%), he
geome id Idaea bisela a (FOO = 56%) and he limonid Aus olimnophila och acea (FOO = 94%).
54
CHAPTER 1
Finally, 73 p ey species we e iden i ied o R. hipposide os, including 33 lepidop e ans, 28 dip e ans,
3 hemip e ans, 3 neu op e ans, 2 coleop e ans, 1 hymenop e an, 1 ichop e an, 1 spide and 1
psocop e an. Among hem, Dip e a we e he mos equen ly consumed, ollowed in descending o de
by Lepidop e a, Epheme op e a, T ichop e a and Neu op e a (Tables A, B and C in S1.1 Suppo ing
In o ma ion File). Among Dip e a, lesse ho seshoe ba s mos ly p eyed upon limonids
Aus olimnophila och acea (FOO = 100%), Rhipidia macula e (FOO = 58%), Neolimonia dume o um
(FOO = 52%), Limonia nubeculosa (FOO = 29%) and Dic anomyia modes a (FOO = 26%), and he
ipulid Tipula hel ola (FOO = 55%). They also consis en ly p eyed upon he neu op e ans
Heme obious humulinus and Wesmaelius ne osus (FOO > 19%). No ewo hy, he occu ence o mos
mo h species was below 3 wi h a maximum o 8 occu ences o he au os ichid Anania ho ula a.
Table 1.1. Resul s ob ained in he di e en s eps o bioin o ma ic analyses wi h each o he p ime se s. “Taxa”
a e he sum o OTUs iden i ied up o species and genus le el. (Sequence eads: To al o eads gene a ed om
he sequencing; P ima y OTUs: To al o buil OTUs; Iden i ied OTUs: Numbe o OTUs which ha e been
iden i ied in he da abases wi h he es ablished simila i y and o e lap le els; Po en ial p ey axa: To al numbe
o axa iden i ied up o genus o species le el; Po en ial p ey species: To al numbe o iden i ied species;
Occu ences o po en ial p ey: To al numbe o occu ences o he iden i ied OTUs.).
ZEALE
GILLET
TOTAL
Sequence eads
112191
1003689
1115880
P ima y OTUs
179
130
309
Iden i ied OTUs
122 (68%)
69 (53%)
191
Po en ial axa
112
58 (61)*
147 (150)*
Iden i ied species
101
54 (57)*
135(138)*
Occu ences o iden i ied sp.
350
278 (294)*
628 (644)*
*: The numbe in b acke s belongs o he o al species numbe iden i ied in Gille ’s samples, and he p e ious
one o he po en ial p ey species (i.e., excluding hose conside ed en i onmen al pollu ion).
As a whole, 12 p ey species ha e been iden i ied in he aeces o he h ee p eda o s: 6 we e
lepidop e ans (Ac onic a umicis, Cyclopho a punc a ia, Idaea degene a ia, Anaplec oides p asina,
Noc ua sp. and Udea e ugalis), 5 dip e ans (Rhipidia macula a, Aus olimnophila och acea,
Limonia nubeculosa, Neolimonia dume o um and Tipula hel ola) and one neu op e an (Heme obius
humulinus).
1.3.2. Pe o mance o P ime s
Gille p ime s yielded he highes numbe s o eads, whe eas Zeale ones go he highes numbe s o
ei he p ima y OTUs, posi i ely iden i ied OTUs, occu ences o p ey and p ey species iden i ied
(Table 1.1). Mo eo e , some o he OTUs buil om Gille p ime s we e iden i ied as belonging o
algae and mammal species (4.41% o he o al axa), and so hey mus be conside ed as en i onmen al
pollu ion ins ead o "po en ial p ey" consumed by ba s.
55
CHAPTER 1
Figu e 1.1. NMDS o dina ion o samples. S ess = 0.1997; k = 2; non-me ic i R2 = 0.96. Do s ep esen p ey
species and colou s di e en p ime se s (Red: Zeale; G een: Gille ). Mo e dis an do s indica e mo e di e en
p ey composi ion o samples. Indi idual ba samples a e ep esen ed as g ey iangles.
We i s es ed ha he e was no signi ican geog aphical e ec o he wo sampling si es in he die
(F(1,68) = 1.031; R2 = 0.132; p = 0.37). The e o e, he loca ion a iable was no conside ed in u he
analyses. The di e ence be ween species die s is signi ican o he whole da a se (F(2,135) = 8.277; R2
= 0.092; p = 0.001), bu also o he esul s ob ained wi h each o he p ime se s by hei own (Gille :
F(2,70) = 10.466; R2 = 0.230; p = 0.001; Zeale: F(2,65) = 4,772; R2 = 0.128; p = 0.001). The p ime choice
signi ican ly a ec ed he esul ing die composi ion (F(1,135) = 14.438; R2 = 0.082; p = 0.001; Figu e
1.1). Consequen ly, he sum o he pa ial esul s enla ged he en i e p ey species lis . O he o al
species iden i ied as po en ial p ey 40% ha e been iden i ied wi h Gille ’s se and 74.8% wi h Zeale’s,
i.e., only 21 ou o he 135 (15.5%) po en ial p ey species ha e been ampli ied by bo h p ime se s.
Anyway, we can see ha bo h p ime and ba species a ec he lis o consumed p ey, wi h a sligh ly
highe explana ion o he a ia ion in he case o he ba species. The in e ac ion o p ime and species
also shows a signi ican di e ence among he esul s, e en i i explains less a ia ion han p ime s
and species on hei own (F(2,135) = 4.841; R2 = 0.055; p = 0.001). The comple e lis s o po en ial p ey
species iden i ied wi h each p ime se is a e included in Tables A, B and C; sequences o all he
OTUs buil a e a ailable in Table D, all o hem in S1.1 Suppo ing In o ma ion File.
The e we e big quali a i e and quan i a i e di e ences among p ime s a a b oade axonomic le el as
well (F(1,135) = 61.157; R2 = 0.239; p = 0.001) (Figu e 1.2, Tables 1.2 and 1.3, and Table A in
Suppo ing In o ma ion File S1.1). Thus, Zeale p ime s we e able o iden i y i e o de s o po en ial
p ey–namely Lepidop e a, Dip e a, Neu op e a, Hemip e a and Psocop e a–whe eas OTUs yielded
56
CHAPTER 1
om Gille p ime s we e assigned o species o i een o de s: namely Lepidop e a, Dip e a,
Coleop e a, T ichop e a, Neu op e a, Epheme op e a, Hemip e a, Hymenop e a and A aneae o p ey
species, as well as Muco ales, A iodac yla, P ima es and Chi op e a o en i onmen al DNA.
Besides, Zeale p ime s yielded mo e occu ences han Gille ones (Table 1.1). The h ee p eda o
species we e iden i ied wi h Gille p ime s in all samples bu in wo R. e umequinum.
The in e speci ic o e lap o he die ob ained wi h Zeale p ime se s is no signi ican ly di e en han
he expec ed by chance (Ojk = 0.18, P = 0.076). R. e umequinum and R. hipposide os show he
highes o e lap (Ojk = 0.34), ollowed by R. eu yale and R. e umequinum (Ojk = 0.12), and R.
eu yale and R. hipposide os (Ojk = 0.08). On he con a y, he o e lap based on Gille p ime s was
signi ican ly highe han expec ed by chance (Ojk = 0.50, P = 0.002) wi h he leas o e lap be ween R.
eu yale and R. e umequinum (Ojk = 0.25) and he highes o e lap be ween he o he wo species
pai s (R. eu yale—R. hipposide os: Ojk = 0.74; R. e umequinum—R. hipposide os: Ojk = 0.52). In
any case, he e ec size is s ill gene ally la ge and he p- alue gene ally small. Simila ly, when esul s
o bo h p ime se s a e combined he o e lap was highe han expec ed by chance (Ojk = 0.34, P =
0.001). Again, he leas o e lap was showed by R. eu yale and R. e umequinum (Ojk = 0.15), while
he o he wo couples show a highe o e lap (R. eu yale—R. hipposide os: Ojk = 0.38; R.
e umequinum—R. hipposide os: Ojk = 0.50).
Table 1.2. Species iden i ied in aeces and hei occu ences wi h each p ime se (Zeale’s and Gille ’s), and
combining esul s, a anged by p ey o de s.
ORDER
ZEALE
GILLET
COMB
Occu ences
Species
Occu ences
Species
Occu ences
Species
A aneae
0
0
2
1
2
1
Coleop e a
0
0
44
10
44
10
Dip e a
68
19
165
37
269
55
Epheme op e a
0
0
3
2
3
2
Hemip e a
1
1
5
3
6
4
Hymenop e a
0
0
3
2
3
2
Lepidop e a
256
119
43
17
281
128
Neu op e a
26
6
10
5
33
10
Psocop e a
1
1
0
0
1
1
T ichop e a
0
0
5
2
6
3
57

CHAPTER 1
Figu e 1.2. Resul s o he h ee ba s’ die ob ained wi h each p ime and combining bo h p ime s. Resul s a e
ep esen ed as pe cen ages o occu ences (POO) 2a: R. e umequinum, 2b: R. eu yale, 2c: R. hipposide os).
“O he s” comp ise he o de s wi h lesse equencies: A aneae, hemip e a, hymenop e a, psocop e a and
ichop e a. GIL: Gille ; ZEA: Zeale; COMB: Combina ion o bo h p ime se s.
Table 1.3. Main o de s o p ey consumed iden i ied in aeces o he h ee species o ho seshoe ba s.
ORDER
R. e umequinum
R. eu yale
R. hipposide os
FOO
Species
FOO
Species
FOO
Species
A aneae
0.00
0
0.00
0
6.45
1
Coleop e a
70.83
8
0.00
0
9.68
2
Dip e a
95.83
17
94.44
10
100.00
28
Epheme op e a
0.00
0
16.67
2
0.00
0
Hemip e a
4.17
0
11.11
1
12.90
3
Hymenop e a
0.00
0
5.56
1
6.45
1
Lepidop e a
79.17
34
100.00
61
70.97
33
Neu op e a
20.83
2
22.22
5
48.39
3
Psocop e a
0.00
0
0.00
0
3.23
1
T ichop e a
4.17
1
5.56
1
12.90
1
58
CHAPTER 1
1.4. DISCUSSION
Ou esul s con i m he ele ance o combining complemen a y p ime s o desc ibe he die o
gene alis insec i o ous ba s wi h amplicon me aba coding echniques. In gene al, each pai o
p ime s e ealed a subse o he p ey composi ion, wi h a small ac ion o he species being de ec ed
by bo h o hem. As a esul , he in e play be ween he p ime axonomic a ini y and die a y
composi ion o he ba a ec ed he niche o e lap among he h ee ho seshoe ba s pic u ed by each
p ime .
Due o hei mo e gene alis cha ac e , Gille p ime s (Gille e al., 2015) ampli y and iden i y a highe
numbe o di e en o de s, showing a mo e di e se die composi ion. This gene alis cha ac e ,
hough, does no co e a ull ep esen a ion o impo an p ey o de s such as Lepidop e a. Mo eo e ,
hei high ampli ica ion success comes wi h he impossibili y o iden i ying a subs an ial ac ion o
he ampli ied DNA (Table 1.1). On he con a y, he highe selec i i y o he Zeale p ime se o
Lepidop e a and some Dip e a (Cla ke e al., 2014; Aizpu ua e al., 2018) migh elici he
unde es ima ion o o he g oups o consumed p ey, such as Coleop e a, Epheme op e a, Hymenop e a
o O hop e a (Aizpu ua e al., 2018). On he posi i e side, maybe due o hei lesse degene a ion
le el, a highe p opo ion o he OTUs go wi h Zeale p ime s we e assigned o know axa (68%,
Table 1.1), p o iding a deep co e age o lepidop e an p ey species.
As bo h p ime se s used in ou s udy ampli y egions o he same well- ep esen ed COI ma ke
egion, he inal p ey lis did no depend o he a ailabili y o model species’ sequences in he
da abases. Mo eo e , bo h p ime s a ge mini-COI segmen s o simila size, sho enough o be
p esen in aecal samples a e diges ion (Meusnie e al., 2008; Zeale e al., 2011; B andon-Mong,
2015; Gille e al., 2015). In ac , he sligh ly highe amoun o p ima y OTUs yielded by Gille
p ime s is consis en wi h he ac ha his p ime se ampli ies mode a ely sho e agmen s han
Zeale ones (133 s 157 bp, espec i ely). Ne e heless, he mo e degene a ed Gille p ime s could
ha e ampli ied mo e DNA agmen s, gene a ing mo e OTUs bu wi h a lowe assignmen o p ey
axa whe eas Zeale p ime s p oduced less OTUs bu wi h a highe assignmen o axa, consis en ly
wi h hei lowe deg ee o degene a ion.
The la es molecula s udy ca ied ou by Galan e al. (2018) desc ibed he die o Rhinolophus
e umequinum as mainly consis ing in Lepidop e a and Dip e a, whe eas mo phological s udies ha e
desc ibed Coleop e a and Lepidop e a as he mos impo an p ey o de s (Schobe & G immbe ge ,
1987; Du e gé & Jones, 1994; Beck, 1997; And eas e al., 2013). In ou s udy, Dip e a occu s in 96%
o he samples, closely ollowed by Lepidop e a (79%) and Coleop e a (71%). Ne e heless,
lepidop e ans we e he main p ey o de de ec ed wi h Zeale p ime s, ollowed by dip e ans, whe eas
wi h Gille ones coleop e ans and dip e ans p e ailed, lepidop e ans alling down o a modes hi d
place. In ac , coleop e ans we e only ampli ied by Gille p ime s and some o he mos impo an
lepidop e an amilies (namely Geome idae, Noc uidae and To icidae) we e disclosed by Zeale. We
59
CHAPTER 1
epo he amily Geome idae and equen ly occu ing species such as Rhipidia macula a (Dip e a,
Limoniidae), Se ica b unnea (Sca abaeidae) and Pha macis usconebulosa (Lepidop e a, Hepialidae),
o he i s ime among p ey o R. e umequinum.
R. eu yale has been widely conside ed a mo h specialis (Schobe & G immbe ge , 1987) and,
acco ding o Koselj (2002) and Die z e al. (2009), lepidop e ans make up 90% o i s die . In ou
s udy, sepa a e molecula s udies pe o med wi h he wo p ime s showed a na ow specializa ion
le el o R. eu yale o lepidop e ans, bu seasonally complemen ed by epheme op e ans, hemip e ans,
hymenop e ans and ichop e ans (A izabalaga e al., 2015; Galan e al., 2018). In ac ,
epheme op e ans had been p e iously epo ed as p ey o R. eu yale in No h A ica (Ahmim &
Moali, 2013). No ewo hy, lepidop e ans a e almos he only p eyed o de i using Zeale, whe eas
Gille gi es simila impo ance o lepidop e ans and dip e ans. Fou ou o he i e mos equen ly
occu ing lepidop e ans—namely Capsula spa ganii (Noc uidae), Udea e ugalis C ambidae),
Lycopoho ia po phy ea (Noc uidae), Scopa ia sp. (C ambidae) whe e solely ampli ied by Zeale. The
wo mos p eyed species Capsula spa ganii and Aus olimnophila och acea ha e no been desc ibed
be o e in he die o R. eu yale.
R. hipposide os is known o p ey mos ly upon Dip e a Nema oce a, ollowed by Lepidop e a and
Neu op e a (McAney & Fai ley, 1989; Lino e al., 2014). In ou s udy, he combined use o bo h
p ime se s o e all con i ms he die composi ion depic ed in p e ious s udies (McAney & Fai ley,
1989; Beck, 1997; Feldman e al., 2000; Ahmim & Moali, 2013; Lino e al., 2014; Galan e al., 2018),
e en i he amily choices wi hin dip e ans and neu op e ans di e . When only Gille p ime s we e
used, hough, p e alence o dip e ans (mainly limonids) in la ed, while ha o lepidop e ans and
neu op e ans (heme obids) de la ed. Fo Zeale, ins ead, dip e ans and lepidop e ans appea ed almos
in he same equencies, closely ollowed by neu op e ans. This esul s ag ee wi h And eas e al.
(And eas e al., 2013) who epo ed a highly p e alence o Lepidop e a in he pelle s. Some o he
mos impo an amilies wi hin Dip e a epo ed by mo phological s udies (Vaughan, 1997), namely
Tipulidae, Empididae, Muscidae and Culicidae a e also ep esen ed wi hin he mos equen p ey
species in he cu en s udy, Empididae only ampli ied by Gille and Muscidae only by Zeale. Two o
he mos equen limonid p ey species—namely Neolimonia dume o um and Limonia
nubeculosa—had been p e iously epo ed by Galan e al. (2018). Con e sely, some o he equen
limonids such as Aus olimnophila och acea, Rhipidia macula a, Dic anomyia modes a o along wi h
o he equen p ey species—Heme obius humulinus (Neu op e a, Heme obiidae), Wesmaelius
ne osus (Neu op e a, Heme obiidae), o Pseuda emelia josephinae (Lepidop e a)—had no been
epo ed be o e.
No ewo hy, mos o he molecula die s udies ca ied ou on ba s exclusi ely wi h Zeale p ime s no
su p isingly ha e concluded ha mo hs o /and Dip e a we e hei main p ey: e. g. Ba bas ella
ba bas ellus, Pleco us mac obulla is, Chalinolobus gouldii, Vespadelus egulus, Nyc ophilus gouldi,
60
CHAPTER 1
Ep esicus nilssonii, Myo is b and ii, M. dauben onii, M. mys acinus and Pleco us au i us (Zeale e al.,
2011; Albe di e al., 2012; Bu ga e al., 2014; Ves e inen e al., 2016). In some o he s udies (Bu ga
e al., 2014) p e iously known p ey species–such as Coleop e a, Hymenop e a, Isop e a and
T ichop e a–we e lacking. No wi hs anding he wellse led impo ance o mo hs and dip e ans as p ey
o insec i o ous ba s (e.g., Beck, 1997; Whi ake e al., 2009), he eliabili y o ophic scena ios
depic ed so a only wi h Zeale p ime s is s ill o be asce ained. The e o e, new s udies using
combina ion o p ime s a e highly ad isable in o de o acqui e he ulles die a y iew whe he o
con i m he esul s ob ained wi h Zeale.
Fu he mo e, he s ong p ime bias epo ed he ein cas doub s on he esul s o p e ious s udies
compa ing he ophic niche o e lap be ween sibling ba species, ca ied ou exclusi ely wi h a single
p ime se (Zeale). Fo example, a s udy compa ing he niches o R. eu yale and R. mehelyi
(A izabalaga-Escude o e al., 218) showed a high deg ee o die o e lap. Razgou e al. (2011)
ob ained simila esul s o Pleco us aus iacus and P. au i us. Some o he s udies ha e also analysed
he die s o sympa ic ba species (Ves e inen e al., 2018) based on Zeale p ime s. E en hough he
die o e laps hese s udies epo ed canno be denied, o he p ime s may well un eil addi ional
consumed p ey and highe le els o esou ce pa i ioning among he species pai s.
Las bu no leas , p e ious s udies ha e shown ha Gille p ime s a e use ul o iden i y p eda o s’
DNA (Galan e al., 2018; Esnaola e al., 2018). In his s udy we iden i ied almos all he aecal
samples o hei p eda o , in excep om wo R. e umequinum samples. Galan e al. (2018) a gued
ha a misma ch (T/C) a he 30-end o he e e se p ime could be a he o igin o hei highe a es o
ampli ica ion ailu e o some ba species, including R. e umequinum. We also iden i ied DNA
emains indica ing unexpec ed in e ac ions, including seconda y p eda ion e en s. Thus, we ound one
R. eu yale aecal sample con aining Bos au us sequences, likely aces o bo ine animal exc emen s
coming om he common house ly (Musca domes ica). Lich heimia amose was iden i ied in R.
eu yale and R. hipposide os. This is a ungus li ing in soil and ege able was es ha in ec s bo h
animals and humans. These esul s mus be conside ed wi h cau ion, hough, because ield
con amina ion canno be ully disca ded.
61
CHAPTER 1
1.I.2. MATERIALS AND METHODS
We conduc ed sequencing and assessed he pe o mance and e iciency o selec ed p ime s (see
below) on 18 samples om each hinolophid species—Rhinolophus eu yale, R. hipposide os, and R.
e umequinum— o alling 54 samples. These samples we e p e iously ampli ied o Chap e 1.
1.I.2.1. Selec ion o Uni e sal P ime s
To add ess he biases o he p ime se s used in Chap e 1, we selec ed no el p ime se s a ge ing
mini-COI egions. Ou chosen p ime s ha e a highe deg ee o degene acy han hose o Zeale e al.
(2011) while ampli ying longe sequences han hose o Gille (2015). Speci ically, we employed he
ANML p ime pai (Jusino e al., 2019), which is de i ed om Zeale e al. (2010) bu exhibi s g ea e
degene acy and was designed o ampli y p ey DNA om ba aecal samples. Addi ionally, we used
FWH1 and FWH2 (Vamos e al., 2017), which a e based on mul iple a h opod- a ge ing p ime se s
(Folme e al., 1994; Zeale e al., 2011; Le ay e al., 2013; Gibson e al., 2015) and ha e an inc eased
le el o degene acy o enhance ampli ica ion success. These p ime s we e p e iously es ed on R.
e umequinum aecal samples, demons a ing good pe o mance (Tou nay e e al., 2020).
Table 1.I.1. P ime se s used in his analysis, including ID names o hei Fo wa d (Fo ) and Re e se (Re )
p ime s, hei leng h (bp), and hei e e ence.
ID
Fo wa d
Re e se
Leng h
Re e ence
ANML
LCOI1490
COI-CFMRa
180 bp
Jusino e al. 2019
FWH1
LCOI1490
ZBJ-A R2c
178 bp
Vamos e al. 2017
FWH2
mlCOIin F
A R5
205 bp
Vamos e al. 2017
1.I.2.2. PCR Amplifica ion, Lib a y P epa a ion And Sequencing
Th ee mini-COI segmen s o mi ochond ial DNA we e ampli ied using he FWH1, FWH2, and
ANML p ime s (178, 205, and 180 bp, espec i ely). PCR ampli ica ion was pe o med ollowing
Jusino e al. (2019) and Tou nay e e al. (2020), wi h modi ica ions. Lib a ies we e cons uc ed using
Illumina’s Nex e a XT ki , and samples we e sequenced on an Illumina MiSeq. PCR ampli ica ion,
DNA lib a y cons uc ion, and sequencing we e ca ied ou a he Genomics and P o eomics Gene al
Se ice (SGIke ) o he Uni e si y o he Basque Coun y (UPV/EHU).
1.I.2.3 Bioin o ma ic Analyses
Sepa a ion by p ime s, quali y con ol, sequence p e-p ocessing, collapsing o iden ical sequences,
and OTU clus e ing we e pe o med ollowing he s eps ou lined in Chap e 1. Simila ly, he
axonomic assignmen o each OTU was ca ied ou by compa ing he ep esen a i e sequence o each
OTU agains e e ence sequences in he Ba code o Li e Da abase (BOLD; www.boldsys ems.o g)
and GenBank (www.ncbi.nlm.nih.go ), accessed on 16 h July 2022, using he same c i e ia as in
Chap e 1 (S1.2 and S1.3 Suppo ing In o ma ion Files).
68

CHAPTER 1
1.I.2.4. Da a Analysis
We e alua ed he pe o mance o he h ee p ime se s based on ead coun s (p e iously no malised)
and es ima ed OTU numbe s ac oss p eda o , p ey, en i onmen al, con amina ion, and unassigned
ca ego ies. OTUs iden i ied a he genus (e.g., Rhinolophus sp.) o species le el in hei espec i e
samples (R. hipposide os, R. eu yale, o R. e umequinum) we e assigned as p eda o . A h opods
om he s udy a ea we e classi ied as po en ial p ey. Sequences om ungi, lichens, plan s, and he
mic obiome, along wi h hose unlikely o be na u ally ound in aeces—such as hose om humans,
o he ba s, o mammals—we e classi ied as en i onmen al con amina ion.
Addi ionally, we accoun ed o iden i ied p ey species ac oss di e en o de s. We analysed all h ee
ho seshoe ba species bo h collec i ely and indi idually. Finally, esul s we e combined by pe o ming
pai wise p ime combina ions o iden i y he op imal se .
1.I.3. RESULTS
The sequencing ou pu a ied signi ican ly among he di e en p ime se s, bo h in e ms o he
quali y o sequence eads and he numbe o ough ope a ional axonomic uni s (OTUs) iden i ied
(Figu e 1.1a). The FWH2 p ime s p oduced he highes numbe o ough OTUs, o alling 2,904,
ollowed by he ANML p ime s wi h 2,313 OTUs, and he FWH1 p ime s wi h 2,178 OTUs.
Howe e , wi h espec o he numbe o p ey eads, he ANML p ime s yielded he highes p opo ion,
accoun ing o 92.98% o he eads, ollowed by FWH2 (68.84%) and FWH1 (63.25%) (Figu e 1.1a).
In e ms o p ey OTUs iden i ied, he ANML p ime s led wi h 775 OTUs (33.51%), ollowed by
FWH1 wi h 659 OTUs (30.26%) and FWH2 wi h 579 OTUs (19.94%) (Figu e 1.1a). This pa e n was
consis en ac oss he pe o mance o each ba species (Figu e 1.1b).
FWH1 and FWH2 gene a ed he g ea es numbe o p eda o eads (6.98% and 6.30%, espec i ely),
wi h hei p eda o OTUs comp ising he la ges numbe s o sequences, eaching up o 250,418
sequences o FWH1 and 186,275 sequences o FWH2. In compa ison, he la ges p eda o OTU in
he ANML da ase consis ed o only 2,475 sequences (Figu e 1.1a). FWH1 also led o he highes
numbe o p eda o OTUs, wi h 11, while bo h ANML and FWH2 each iden i ied only 3 p eda o
OTUs (Figu e 1.1a). Addi ionally, R. eu yale had he mos OTUs (8 wi h FWH1, and 2 wi h FWH2
and ANML) and he highes numbe o eads (18.96% wi h FWH2, 18.17% wi h FWH1, and 0.52%
wi h ANML), while he ead coun s o R. hipposide os and R. e umequinum we e signi ican ly
lowe (2.% wi h FWH1, and below 0.01% wi h FWH2 and ANML; 0.94% wi h FWH1, 0.28% wi h
FWH2, and 0.02% wi h ANML, espec i ely) (Figu e 1.1b).
69
CHAPTER 1
Figu e 1.I.1. To al numbe o OTUs and Read Coun s ob ained by he h ee p ime se s (ANML, FWH1 and
FWH2) o he ca ego ies con amina ion ( ed), en i onmen al (blue), p eda o (g een), p ey (pu ple) and
unassigned (yellow). a) To al and b) di ided by ba species. REU: R. eu yale, RHI: R. hipposide os, RFE: R.
e umequinum.
Rega ding en i onmen al con amina ion, FWH1 showed he highes alues (13.49% o he eads;
6.89% o he OTUs, espec i ely), ollowed by FWH2 (6.63% o he eads; 6.06% o he OTUs,
espec i ely), while ANML showed he lowes (1.47% o he eads; 1.95% o he OTUs, espec i ely)
(Figu e 1.1a).
Al hough he pe cen ages o unassigned OTUs we e high o all h ee p ime s (ANML 64.42%;
FWH1 62.44%; FWH2 73.86%), he pe cen ages o unassigned eads we e much smalle (ANML
5.38%; FWH1 16.28%; FWH2 18.22%), showing ha he unassigned OTUs consis ed o small OTUs
composed o only a ew sequences, which may be a e ac s o sequencing e o s. Howe e , ANML
achie ed he bes esul s in his ega d (Figu e 1.I.1a).
FWH1 was he p ime se ha iden i ied he highes numbe o p ey species, wi h 534 species,
ollowed by ANML wi h 522 species, and FWH2 wi h 483 species. Howe e , ANML consis en ly
iden i ied mo e p ey species o each ba species (R. hipposide os: 379, R. eu yale: 334, R.
e umequinum: 239), while FWH2 iden i ied he ewes (R. hipposide os: 314, R. eu yale: 253, R.
e umequinum: 191). The highes o al numbe o p ey species was iden i ied in R. hipposide os
(529), ollowed by R. eu yale (454), and he ewes in R. e umequinum (368) (Table 1.I.2).
A he o de le el, all p ime s oge he iden i ied 13 p ey o de s. In all cases, he mos abundan p ey
o de s we e Lepidop e a and Dip e a, al hough he ela i e impo ance o he smalle g oups a ied
(Figu e 1.I.2). ANML led o a highe numbe o lepidop e an eads and occu ences, while FWH1
iden i ied mo e dip e ans and coleop e ans (Table 1.I.3). FWH1 and FWH2 iden i ied mo e
hemip e ans (Figu e 1.I.2).
70
CHAPTER 1
Table 1.I.2. Table showing he o al p ey species o di e en p ey o de s iden i ied in he die o h ee
Rhinolophus ba species (R. e umequinum, R. hipposide os, and R. eu yale) using di e en p ime
combina ions (ANML+FWH1; ANML+FWH2; FWH1+FWH2). The da a include absolu e coun s and he o al
pe cen ages o iden i ied species o each combina ion ac oss samples.
71
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Table 1.I.3. Pe cen age o occu ences and ead coun s o seconda y p ey o de s de ec ed by each p ime se
(ANML, FWH1, and FWH2).
Read Coun s
Pe cen age o Occu ences
O de
ANML
FWH1
FWH2
ANML
FWH1
FWH2
A aneae
0.21
0.48
0.00
0.47
1.00
0.12
Bla odea
0.00
0.39
0.06
0.04
0.43
0.49
Coleop e a
0.86
4.00
2.38
2.35
5.29
4.52
Dip e a
41.35
43.61
45.50
27.82
30.96
32.77
Epheme op e a
0.03
0.62
0.19
0.25
0.49
0.25
Hemip e a
0.21
0.89
2.36
1.38
5.10
7.47
Hymenop e a
0.36
0.31
0.20
0.91
1.73
2.38
Lepidop e a
53.87
46.73
46.80
62.37
47.58
44.35
Man odea
0.01
0.00
0.00
0.04
0.00
0.00
Neu op e a
2.14
2.38
1.61
2.95
6.20
6.28
Odona a
0.00
0.10
0.00
0.00
0.40
0.00
O hop e a
0.04
0.00
0.01
0.19
0.12
0.21
T ichop e a
0.92
0.49
0.87
1.22
0.70
1.15
Rega ding p ime combina ions, he pai ing ha iden i ied he mos p ey species was ANML+FWH1,
de ec ing 675 ou o 752 species, yielding an iden i ica ion a e o 89.76%. This was closely ollowed
by he ANML+FWH2 combina ion, which iden i ied 673 species, o 89.49%. The combina ion wi h
he lowes numbe o de e mined species was FWH1+FWH2, which iden i ied 636 species,
co esponding o 84.57% (Table 1.I.2). The co e age pa e n o each ba species was simila (Table
1.I.2).
Figu e 1.I.2. Pe cen age o occu ences and ead coun s o seconda y p ey o de s de ec ed by each p ime se
(ANML, FWH1, and FWH2), excluding Lepidop e a and Dip e a.
72
CHAPTER 1
When examining p ey spec um co e age, ANML+FWH1 exhibi ed he bes co e age ac oss he main
p ey o de s: Lepidop e a (94.12%), Dip e a (89.16%), Hemip e a (83.87%), and Coleop e a (88.46%),
bo h o e all and o each ba species (Table 1.I.2). In R. eu yale, a mo h specialis , 94.89% o he
species we e iden i ied by ANML+FWH1, ollowed by ANML+FWH2 (92.70%) and FWH1+FWH2
(76.64%). The pa e n o Lepidop e a emained simila ac oss he h ee ba species. In R.
e umequinum, he iden i ica ion o Dip e a and Coleop e a was also signi ican . Fo Dip e a, he bes
combina ion was s ill ANML+FWH1 (84.76%), ollowed by FWH1+FWH2 and ANML+FWH2
(81.90%). Fo Coleop e a, he op imal combina ion was FWH1+FWH2 (87.5%), ollowed by
ANML+FWH1 and ANML+FWH2 iden i ying he ewes (81.25%). Finally, o R. hipposide os, he
iden i ica ion o Dip e a and Hemip e a was also impo an . Fo Dip e a, he bes combina ion
emained ANML+FWH1 (89.61%), ollowed by ANML+FWH2 (88.96%), and FWH1+FWH2
(86.36%). Fo Hemip e a ANML+FWH2 achie ed he highes co e age (90.74%), ollowed by he
o he wo combina ions (87.04%).
1.I.4. DISCUSSION AND CONCLUSIONS
To o e come he biases obse ed in p e iously used p ime s—whe e he ZBJ p ime s (Zeale e al.,
2011) exhibi ed a na ow axonomic ange (Cla ke e al., 2014; B andon‐Mong e al., 2015; Mallo e
al., 2015), and he Gille p ime s (2015) ampli ied oo many uniden i iable sequences (Esnaola e al.,
2018)—we aimed o iden i y mo e e ec i e p ime s ha would b oaden axonomic co e age while
main aining speci ici y. The h ee selec ed p ime se s ou pe o med he p e ious ones, iden i ying a
g ea e numbe o p ey species and o de s. Among hese, ANML success ully ampli ied he highes
numbe o p ey species. No ably, e en he FWH2 p ime se , which de ec ed he ewes species among
he h ee, iden i ied 483 species, signi ican ly su passing he Zeale p ime s (101 species) and he
Gille p ime s (54 species) (Chap e 1; Aldaso o e al., 2019).
Addi ionally, when analysing bulk aecal samples collec ed om benea h he animals in he oos ,
iden i ying he p eda o is c ucial, as aeces om o he species may be mixed wi h hose o he a ge
species. Among he es ed p ime s, FWH1 yielded he bes esul s o p eda o iden i ica ion.
The ANML p ime s p o ided he highes co e age o p ey species, while he FWH1 p ime s we e he
mos e ec i e o de ec ing p eda o DNA. Al hough all h ee p ime s p ima ily ampli ied
lepidop e ans and dip e ans, hey a ied in hei abili y o iden i y smalle g oups, which could be
seasonally signi ican o each p eda o species. Fo ins ance, FWH1 iden i ied he mos coleop e an
species, which a e an impo an pa o Rhinolophus e umequinum’s die (e.g., Ransome, 1996;
Jones, 1990; Flande s & Jones, 2009; Tou nay e, 2021). Simila ly, la ge , ha de p ey such as
bla odeans o o hop e ans we e also ampli ied by FWH1 p ime se . In con as , small p ey a e
c ucial o R. hipposide os (e.g., McAney & Fai ley, 1989; Bon adina e al., 2008; Lino e al., 2014).
In his case, he iden i ica ion o hemip e ans and neu op e ans is impo an , and FWH1 and FWH2
we e he p ime s ha ampli ied he highes numbe o species om his o de .
73

CHAPTER 1
We con i med ha using mul iple p ime s inc eased he numbe o iden i ied p ey axa and p o ided
mo e comp ehensi e di e si y co e age, consis en wi h p e ious s udies (Esnaola e al., 2018;
Aldaso o e al., 2019; Jusino e al., 2019). The combina ion o ANML and FWH1 iden i ied 89.8% o
he o al species, ollowed by ANML and FWH2 wi h 89.5%. Fo he mos consumed p ey o de s,
Lepidop e a and Dip e a, he ANML and FWH1 combina ion demons a ed he highes species
co e age, while allowing he iden i ica ion o he p eda o i sel .
While iden i ying a highe numbe o p ey species can be bene icial, mo e is no always be e , and
u he analysis is equi ed o assess he ecological ele ance o hese esul s. Fo ins ance, i emains
unce ain whe he R. hipposide os could ealis ically consume 19 species o Coleop e a, highligh ing
he need o species-le el in es iga ions in o p ey size and ecological plausibili y. Some de ec ions
may esul om c oss-con amina ion be ween samples, po en ially om R. e umequinum.
Addi ionally, many iden i ied species belong o low- ead, low-occu ence OTUs, which could
ep esen a e species o seconda y p ey (e.g., Tou nay e e al., 2021; Aiha za e al., 2023). The e o e,
when in es iga ing o aging ecology beyond simply lis ing p ey, i is c ucial o de e mine whe he he
ecological signi icance lies in he mos equen ly consumed species o whe he mo e de ailed
axonomic analyses a e necessa y. None heless, selec ing app op ia e p ime s emains undamen al o
ob aining a eliable species lis , which se es as he ounda ion o any subsequen ecological
in e p e a ion.
In his con ex , we concluded ha he combina ion o ANML and FWH1 was he mos e ec i e o
he h ee ho seshoe ba species s udied, maximising p ey di e si y co e age, p ey quan i y, and
p eda o iden i ica ion. We will he e o e choose his combina ion o u u e s udies.
74
CHAPTER 1
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Taylo , P. J., Ma amba, E., S eyn, J. N. K., Nangammbi, T., Zepeda-Mendoza, M. L., & Bohmann, K. (2017). Die
de e mined by nex gene a ion sequencing e eals pes consump ion and oppo unis ic o aging by ba s in
macadamia o cha ds in Sou h A ica. Ac a Chi op e ologica, 19(2), 239-254.
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CHAPTER 1
1.I.6. SUPPORTING INFORMATION
S1.2 Suppo ing In o ma ion File. Table A. Iden i ica ions o OTUs. Table C. OTU
Table. (xlsx) h ps:// inyu l.com/aldaso oS12
S1.3 Suppo ing In o ma ion File. Table B. Sequences o all he OTUs buil wi h he
h ee p ime se s. ( x ) h ps:// inyu l.com/aldaso oS13
77
CHAPTER 2
Figu e 2.1. Schema ic ep esen a ion o he Mul iplex Liga ion-dependen P obe Ampli ica ion (MLPA) p ocess
(adap ed om Schou en e al., 2002). (1) Dena u a ion and hyb idisa ion: Ta ge DNA is dena u ed, and MLPA
p obes hyb idise o complemen a y sequences. (2) P obe liga ion: I bo h p obe hal es hyb idise co ec ly, a
DNA ligase joins hem in o a single s and. (3) Ampli ica ion: Liga ed p obes a e ampli ied using a uni e sal
p ime pai wi h luo escen labelling o de ec ion. (4) F agmen sepa a ion and da a analysis: PCR p oduc s a e
sepa a ed ia capilla y elec opho esis, p oducing an elec ophe og am whe e peak heigh s co espond o he
ela i e quan i y o a ge DNA.
Howe e , some COI p ime s may exhibi species-speci ic misma ches, leading o biased ampli ica ion
and educed de ec ion e iciency (Deagle e al., 2014), leading o he explo a ion o al e na i e DNA
me aba coding ma ke s (Cla ke e al., 2014; Deagle e al., 2014). The in e nal ansc ibed space (ITS)
egion has been ecognised as a iable al e na i e, demons a ing success ul applica ion in a ious
axa (C uickshank, 2002; Wieme s e al., 2009).
In mos euka yo es, ibosomal DNA ( DNA) is o ganised as andemly epea ed mul icopy gene
clus e s (A nheim e al., 1980; Coen e al., 1982), unde going conce ed e olu ion in animals o
84

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main ain sequence homogenei y (Hillis e al., 1991; Schlö e e & Tau z, 1994). Copy numbe s o he
animal RNA genes ange om 39 o 19,300 (P okopowich e al., 2003), making hese genes aluable
o molecula species iden i ica ion. Each ansc ibed uni con ains 18S, 5.8S, and 28S RNA genes
sepa a ed by ITS1 and ITS2 (He nandez e al., 1993). Rema kably, he ITS egion o DNA is
wo hwhile due o i s conse ed lanking egions and species-speci ic size a iabili y. ITS1, a nuclea
noncoding agmen wi h high in e speci ic a iabili y, has been widely used o de elop
species-speci ic p ime s o single-s ep PCR in pes iden i ica ion (Liu, 2004; Asokan e al., 2007;
Yeh e al., 2015; Fa is e al., 2020). I has acili a ed species iden i ica ion in mosqui oes (Collins &
Paskewi z, 1996; Pe e a e al., 1998) and mo hs (Pe e a e al., 2015; Tsai e al., 2020; Pa k e al.,
2022), showing consis ency ac oss mo h popula ions (Lew e e al., 2006). Howe e , no a emp s ha e
been made o ampli y a h opod ITS om aecal samples.
Conside ing all he aspec s men ioned, his wo k aims o de elop an MLPA assay o be used in ba
aeces o he simul aneous sc eening and quan i ica ion o DNA om a ious ag icul u al pes s using
he ITS1 egion as species-speci ic.
2.2. MATERIALS AND METHODS
2.2.1. Sample Selec ion
Ta ge species we e chosen based on 1) hei p e alences in p e ious samplings and me aba coding
analysis and hei consump ion by di e se ba species (Aizpu ua e al., 2018; Ba oja e al., 2019;
Ga in e al., 2019; Aiha za e al., 2024); 2) Ou specimen a ailabili y in he collec ion o he
possibili y o ob aining specimens o DNA ex ac ion; 3) whe he genome in o ma ion was a ailable
in he NCBI da abase; and 4) hei impac and in e es as ag icul u al pes s (Gil e al., 2014; Eu opean
and Medi e anean Plan P o ec ion O ganiza ion (EPPO) Global Da abase (h ps://gd.eppo.in /)).
Conside ing all his, we selec ed ou species o design he MLPA p obes: he pine p ocessiona y
(Thaume opoea pi yocampa, TPI), he codling mo h (Cydia pomonella, CPO), he sil e Y
(Au og apha gamma, AGA) and he Eu opean ine mo h (Lobesia bo ana, LBO).
2.2.2. Design o MLPA Oligonucleo ide P obes o 18S DNA -ITS1 Region
We ex ac ed i e specimens o each species (single specimen in AGA due o a ailabili y) using he
DNeasy Blood & Tissue DNA ex ac ion ki (Qiagen) and ollowing he manu ac u e ´s ins uc ions.
DNA concen a ion was measu ed wi h a NanoD op ND-1000 spec opho ome e (NanoD op
Technologies Inc., Mon chanin, DE).
To sequence he ITS1 egion o he a ge ed species, we compa ed he genome da abase o di e en
mo h species in NCBI (Na ional Cen e o Bio echnology In o ma ion; h p://www.ncbi.nlm.nih.go ;
accessed on 9 June 2021) o iden i y he p ese ed sequences a he ibosomal DNA agmen s 18S o
5.8S. Then, we designed a degene a ed p ime se o Lepidop e a In e nal T ansc ibed Space 1
85
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(ITS1) which pa ially ampli ied 18S and 5.8S DNA, and he whole ITS1 sequence, which will a y
om species o species: Fw: 5´-AGTCGTAACAAGGTTTCCGTAGG-3´; R :
5´-CKATGACGCRCAGTTTRCTGC-3´.
B ie ly, we pe o med a PCR on he ex ac ed samples. Fi s , he DNA was dena u ed a 95ºC o 15
minu es, ollowed by 40 cycles o ampli ica ion (30 seconds a 94ºC o dena u a ion, 30 seconds a
57ºC o annealing, and 30 seconds a 72ºC o ex ension), and a inal ex ension a 72ºC o 6 minu es.
The PCR was pe o med wi h he Ho S a Taq DNA Polyme ase (Qiagen) in a Mas e cycle p o S
The mocycle (Eppendo ). Amplicons we e obse ed in 2% aga ose gel s ained wi h E hidium
B omide. Then, he posi i e amplicons we e Sange sequenced by he eam o he Genomics Se ice
o SGIke (UPV/EHU/ERDF, EU).
Once we had ou a ge sequences, hey we e aligned (Clus alW) and analysed (Figu e 2.2) o ind
sui able egions o he design o he MLPA species-speci ic p obe se (Table 2.1). The speci ici y o
he p obes was e alua ed in silico: 1) by BLAST sea ch wi hin NCBI GenBank (accessed on 5
Oc obe 2021), and 2) looking a he po en ial seconda y s uc u e wi h OligoAnalyze Tool (IDT,
h ps://www.id dna.com/pages/ ools/oligoanalyze ; accessed on 5 Oc obe 2021), in his way we
a oided in e e ing c oss- eac i i ies wi h he a ge mo hs and o he ela ed species. As a as
possible, he oligonucleo ide p obe a ibu es we e designed ollowing he gene al sugges ions o
MRC-Holland (Ams e dam, The Ne he lands). The 5´ end has o be phospho yla ed since i allows he
ligase o join p obe hal es, ensu ing selec i e ampli ica ion o co ec ly hyb idised a ge s, which
imp o es assay speci ici y and eliabili y (Schou en e al., 2002). P obes we e pu chased om IDT
(In eg a ed DNA Technologies, Iowa, USA) (h ps://eu.id dna.com/si e).
86
CHAPTER 2
Figu e 2.2. Alignmen o he consensus sequences belonging o he ou a ge species (AGA, TPI, CPO, and
LBO) along wi h he p obe binding si es. The Le P obe Oligo binding si e is highligh ed in blue, while he
Righ P obe Oligo binding si e is shown in yellow. A eas belonging o 18S DNA and 5.8S DNA a e inside
black squa es.
87
CHAPTER 2
Table 2.1. P obes and p ime s o he 18S DNA-ITS1-5.8S DNA MLPA sys ems. The p ime binding si es a e
highligh ed in bold capi als, and he 5' end o RPOs we e phospho yla ed (*).
88
CHAPTER 2
2.2.3. MLPA Assay Valida ion Wo kflow
We conduc ed he MLPA assay ollowing he s eps below (Figu e 2.3):
1. Tes ing he MLPA design in pu e samples
We i s conduc ed an in i o analysis o de ec a speci ic a ge DNA, ensu ing he p ope
unc ionali y o he p obes wi h indi idual DNA ex ac ions. DNA empla es ex ac ed om he a ge
mo h species we e indi idually analysed in sepa a e eac ions using hei co esponding MLPA p obe
se s. This analysis was pe o med using TPI, CPO, AGA, and LBO. We s udied h ee specimens pe
species, excep o AGA, o which only a single specimen was a ailable. Each sample was es ed in
duplica e, and a no- empla e con ol (NTC) was included o each p obe combina ion. The DNA
concen a ion anges (ng/µL) o he mo h ex ac ions o each species we e as ollows: TPI
(11.6–19.7), CPO (6.2–20.6), LBO (15.4–19.7), and AGA (6.0).
2. De e mining MLPA de ec ion limi in pu e mo h DNA samples
A e e i ica ion, we pe o med se ial dilu ions o he DNA ex ac ions o de e mine he de ec ion
limi o TPI, CPO, and AGA a 1:2.5, 1:5, and 1:10. Since we ailed o de ec LBO in he i s s ep,
we se i aside o he subsequen s eps. Due o he low ini ial DNA concen a ion, sample
concen a ion was no easible, and u he dilu ion o he ini ial samples would be nonsense;
he e o e, he samples we e no equimola ised. We analysed h ee TPI and CPO samples and one
AGA sample, wi h wo eplica es pe dilu ion and an NTC pe p obe combina ion.
3. Mul iplexing he p obes in pu e mo h DNA samples
A e con i ming he e ec i eness o indi idual p obes, we conduc ed a mul iplex assay using a single
a ge DNA and mul iple MLPA p obes simul aneously. This assay included a iplex combina ion
(CPO + TPI + AGA) and h ee duplex combina ions (CPO + TPI, CPO + AGA, and TPI + AGA). We
included an NTC sample o each combina ion. Analyses we e pe o med in duplica es.
4. Mo h MLPA assay in ba aeces
Finally, we conduc ed a p elimina y es o iden i y pes species in ba aeces. Samples we e selec ed
based on p e ious s udies whe e he a ge species had been de ec ed in DNA ex ac ions h ough
me aba coding (posi i e samples om Chap e 6 and Aiha za e al., 2023) (Table 2.2). Analyses we e
pe o med in iplica es, and an NTC sample was included. The samples in which he a ge DNA was
de ec ed we e dilu ed o hei o iginal 1:10 concen a ions h ough se ial dilu ions o es ablish he
de ec ion limi .
2.2.4. MLPA Assay and Analysis
All s eps o he MLPA assay we e pe o med using a SimpliAmp The mal Cycle (The mo Fishe
Scien i ic, USA) and he SALSA MLPA EK1 eagen ki (MRC-Holland) acco ding o he
manu ac u e 's ins uc ions. A nega i e sample (NTC) was included in each es o de ec possible
con amina ion o o he po en ial p oblems. The eac ion componen s a e included in he SALSA
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CHAPTER 2
MLPA EK1 (FAM-6) eagen ki , excluding he DNA sample, p obe mix, wa e , and TE bu e .
B ie ly, 4 µL o sample DNA we e dena u ed wi h 1 µL o TE a 95ºC o 5 minu es using 0.2 µL
ubes. A non- empla e con ol con aining 5 µL o TE was included in each eac ion o de ec any
po en ial eagen con amina ion. A e dena u a ion, he samples we e cooled o 25ºC and emo ed
om he he mocycle o add he hyb idisa ion mix, which consis ed o 1.5 µL o SALSA MLPA
bu e and 1.5 µL o he syn hesised p obe mix u e in TE. The samples we e hen hea ed o 95ºC o 1
minu e and incuba ed a 70ºC o 16 hou s o acili a e hyb idisa ion. Fo he liga ion eac ion o he
hyb idised p obes, 32 µL o he Ligase mas e mix (comp ising 3 µL o ligase bu e A, 3 µL o ligase
bu e B, 25 µL o H2O, and 1 µL o Ligase-65) we e added and incuba ed o 15 minu es a 54ºC.
A e wa ds, he samples we e hea ed o 98ºC o 5 minu es o inac i a e he enzyme. Simul aneous
ampli ica ion o he liga ion p oduc s was pe o med using uni e sal SALSA PCR p ime s. To ini ia e
he ampli ica ion, we added 10 µL o he polyme ase mas e mix (con aining 7.5 µL o H2O, 2 µL o
PCR P ime Mix, and 0.5 µL o SALSA polyme ase). We execu ed he ollowing p og am: 35 cycles
o ampli ica ion (30 seconds a 95ºC o dena u a ion, 30 seconds a 60ºC o annealing, and 1 minu e
a 72ºC o ex ension), ollowed by a inal ex ension a 72ºC o 20 minu es and a cool-down s ep o
15ºC. The PCR p oduc s we e s o ed in he da k un il u he analysis. In each expe imen , DNA
samples we e analysed in duplica e o e alua e he ep oducibili y o he de eloped assay. Finally, he
amplicons we e analysed by elec opho esis using he ABI PRISM 3130XL (The mo Fishe
Scien i ic, USA) by he Genomics Se ice o he SGIke eam (UPV/EHU/ERDF, EU).
Table 2.2. The aecal samples used o he MLPA es in ba aeces, including hei o iginal DNA concen a ion
(ng/µL), ela i e ead abundance (RRA), and weigh ed pe cen age o occu ence (wPOO) om he
me aba coding analysis. Ampli ica ion indica es whe he he MLPA assay success ully de ec ed he expec ed
peak. Fo FaTPI1 and FaTPI2, DNA concen a ions o he se ial dilu ions a e also p o ided.
SAMPLE
[DNA]
(ng/µL)
[1:2.5]
(ng/µL)
[1:5]
(ng/µL)
[1:10]
(ng/µL)
RRA (%)
wPOO (%)
FaCPO1
37.97
63.4
12.60
FaTPI1
5.1
2.04
1.02
0.51
62.1
9.09
FaTPI2
7.6
3.04
1.52
0.76
69.8
9.09
FaTPI3
3.9
94.2
33.33
FaTPI4
8.0
66.1
14.28
FaTPI5
7.7
61.4
10
90
CHAPTER 2
Figu e 2.3. Schema ic ep esen a ion o he MLPA assay wo k low o de ec ing mo h pes species in ba aeces.
The op sec ion illus a es he a ge mo h species and hei co esponding p obes, in i o mo h DNA
ex ac ions, and DNA ex ac ions om ba aeces. The bo om sec ion p esen s he expe imen al s eps: (1)
es ing he MLPA design wi h pu e samples, (2) de e mining he de ec ion limi o MLPA, (3) mul iplexing he
p obes, and (4) applying MLPA o ba aeces samples o assess de ec ion e iciency. G een checkma ks indica e
success ul de ec ion, while ed c osses signi y ailed de ec ion o de ec ion limi s. TPI: Thaume opoea
pi yocampa; CPO: Cydia pomonella; AGA: Au og apha gamma; LBO: Lobesia bo ana.
2.3. RESULTS
2.3.1. E alua ion o he ITS1 MLPA P obe Specifici y
The designed p ime s success ully ampli ied he 18S-ITS1-5.8S egion. A e analysing he o wa d
and e e se Sange sequencing esul s, we ob ained he consensus sequence o each species by
compa ing ou sequenced agmen s wi h sequences a ailable in GenBank (The consensus sequences
ha e been deposi ed in GenBank unde he ollowing accession numbe s: PQ605054, PQ605056,
PQ605057, and PV156760). We obse ed homology in he 3´ egion o he 18S DNA, while he
5´ egion o he 5.8S egion exhibi ed nucleo ide a ia ions be ween species (Figu e 2.2). In leng h, he
91
CHAPTER 2
ITS1 egion a ied signi ican ly be ween species, anging om he sho es in LBO (146 bp) o he
longes in CPO (468 bp), wi h AGA measu ing 331 bp and TPI 432 bp.
The ITS1 MLPA design was i s in silico es ed o possible c oss-homologies be ween DNA om
he a ge species (TPI, CPO, AGA, LBO) and non- a ge a h opod species. To do so, we aligned he
comple e hyb idising sequences o each p obe (le and igh hyb idisa ion sequences) agains
sequences a ailable wi hin NCBI GenBank using BLASTn. Ou analysis showed 100% iden i y o
TPI, 100% o CPO, 99.01% o LBO, and 95.92% o AGA compa ed o he expec ed species in a
blas sea ch agains he en i e nucleo ide da abase. The analysis o po en ial he e odime o ma ion o
he p obes indica ed a gene ally low isk, wi h only one high- isk he e odime (ΔG < -9 kcal/mol)
obse ed o each species p obe combina ion. In any case, hese did no align wi h he expec ed
leng h.
2.3.2. Tes ing he MLPA Design in Pu e Samples
DNA empla es ex ac ed om he a ge mo h species we e indi idually analysed in sepa a e
eac ions using hei co esponding MLPA p obe se s. Th ee ou o he ou species (TPI, CPO, and
AGA) we e success ully de ec ed, p oducing ep oducible signals o he expec ed sizes (TPI: 155 n ,
CPO: 180 n , AGA: 168 n ), while LBO was no de ec ed (Figu e 2.4). Due o he absence o
de ec able peaks in LBO, we excluded i om u he analyses.
Figu e 2.4. Elec ophe og ams o he MLPA assay o independen de ec ion in pu e samples. (a) Cydia
pomonella (CPO): 180 n ; (b) Thaume opoea pi yocampa (TPI): 155 n ; (c) Au og apha gamma (AGA): 168 n ;
and (d) Lobesia bo ana (LBO), which was no de ec ed. The ed peak a 50 bp ep esen s he lowe ma ke o
he elec ophe og am. The X-axis ep esen s sequence leng h (n ), while he Y-axis shows luo escence uni s.
A sligh 1–2 n shi om he expec ed agmen leng hs was consis en ly obse ed (Table 2.1). This
shi was likely due o a ia ions in DNA agmen mobili y and/o di e ences in labelling be ween
92
CHAPTER 2
he s anda d and ampli ied agmen s, as epo ed in p e ious s udies (Ehle e al., 2009; Un e be ge
e al., 2014; Ga cia-Ga cia e al., 2017).
2.3.3. De e mining MLPA De ec ion Limi
E en hough we could de ec speci ic peaks in he dilu ions ( anging om 1:2.5 o 1:10) o TPI and
AGA, he peak heigh s emained nea ly cons an despi e he a ia ions in DNA concen a ions (Figu e
2.5). Only one o he TPI samples (da k blue in Figu e 2.5b) showed a decline in luo escence wi h
dec easing DNA concen a ions. Fo CPO, howe e , he MLPA assay did no wo k as expec ed, and
we iden i ied nonspeci ic peaks (a 113 bp, 126 bp and 146 bp, which likely co esponded o di e en
dime s) in addi ion o he expec ed 180 bp peak.
Figu e 2.5. DNA concen a ion (ng/µL) and speci ic peak heigh s om elec opho esis esul s o se ial dilu ions
(1:1, 1:2.5, 1:5, and 1:10) in in i o samples o (a) Au og apha gamma (AGA), showing dilu ions om a single
sample and i s duplica es, and (b) Thaume opoea pi yocampa (TPI), showing dilu ions om h ee di e en
samples and hei duplica es. Di e en shades o blue in (b) ep esen he h ee o iginal TPI samples. The ed
do ed line shows he mean end line, and he blue lines show he end line o each sample.
2.3.4. Mul iplexing he P obes
No speci ic peaks co esponding o he a ge species we e de ec ed when we conduc ed he in i o
mul iplex assay using a single a ge DNA and mul iple MLPA p obes simul aneously (including all
h ee p obes). Ins ead, a peak o app oxima ely 115 bp was obse ed in all samples (Figu e 2.6a).
When he assay was pe o med using p obe pai s (TPI+CPO, TPI+AGA, and CPO+AGA), he 115 bp
93
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Seasonal and geog aphic a ia ion in he
ophic ecology and habi a dependence o
Rhinolophus hipposide os
ARTICLE
Aldaso o, M., de Ce io, O. D., Russo, D., Vallejo, N., Olasagas i, L.,
Goi i, U., & Aiha za, J. (2025). Molecula Die a y Analysis Re eals
Plas ici y in Habi a Requi emen s o a Clu e Specialis Ba . Basic
and Applied Ecology, 84, 101-109.
CONGRESS
XVII h Eu opean Ba Resea ch Symposium (EBRS). Pos e
communica ion. “The Lesse Ho seshoe ba , a lexible clu e o aging
specialis ”. Gi ona, Ca alonia.
CHAPTER 3
LABURPENA
Azken hama kade an au e apen handiak izan di a saguza en bazka-habi a en az e ke an. Hala e e,
ike ke a hauek be e beha en ule men muga ua bes e ik ez du e eskain zen. Me aba coding-ak auke a
ema en du kon sumi u ako ha apakinak espezie mailan iden i ika zeko, haien ja o izko habi a ak
zehaz eko auke a emanez. Az e ke a hone an, Rhinolophus hipposide os-en hi u kolonie ako go o z
laginak ha u zi en, e emu klima iko ezbe dine an, udabe i ik uda bukae a a e. Me aba coding-a
balia uz, e a-saguza xikia en die a denbo an zeha eko die a aldako asuna az e u zi en, e a haien
ha apakin nagusiak sasoi e a e emu ezbe dinen a abe a alda zen di en. Emai zek e akus en du enez,
saguza en die ak alda u egi en di a u a oa en a abe a, e a kolonien a eko die a ezbe dina da, asko an
ha apakinen age aldiei e an zunez. Saguza en jandako ha apakine a ik habi a eskakizun
desbe dinak di uz ela ondo ioz a u genuen. Basoak e a zuhaixkak habi a nagusiak di a, nahiz e a
bes e ingu une ba zuekiko e e menpeko asuna e akus en du en. Habi a i ekiak be eziki us e baino
maizago us ia zen di ela ondo ioz a u zen, bazka ze habi a en beha e an plas iko asun-maila handia
e aku siz. Ho az, saguza en kon se bazio ako asko a iko ha apakinak eskain zen di uz en e a elka
konek a u a dauden paisai ani zak babes ea un sezkoa da.
ABSTRACT
In ecen decades, he e has been signi ican p og ess in s udying he o aging habi a s o ba s.
Howe e , hese s udies p o ide only a limi ed unde s anding o hei equi emen s. Me aba coding
allows species-le el iden i ica ion o consumed p ey, allowing us o de e mine hei sou ce habi a s. In
his s udy, we sampled aeces om h ee Rhinolophus hipposide os colonies in di e en clima ic
zones om sp ing o la e summe . Using me aba coding, we examined how he lesse ho seshoe ba
die changes o e ime and whe he hei mos -consumed p ey a ies seasonally ac oss landscapes.
Ou esul s show ha ba die s change seasonally and di e be ween colonies, o en p esumably in
esponse o new p ey ou b eaks. We deduced om he p ey ea en by ba s ha hey ha e a ied habi a
equi emen s. While woodland and sh ubs a e p ima y p ey sou ce habi a s, ba s also ely on o he
en i onmen s. We in e ed ha , in pa icula , open habi a s a e exploi ed mo e equen ly han
expec ed, indica ing a high deg ee o plas ici y in hei ophic habi a needs. The e o e, p o ec ing
di e se, in e connec ed landscapes wi h a ied p ey is c ucial o hei conse a ion.
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3.1. INTRODUCTION
Unde s anding habi a p e e ences o elusi e, a e, o small o aging animals unde 20g is challenging,
e en wi h GPS ad ancemen s. VHF adio ags a e he p ima y acking me hod o small passe ines
(Baue e al., 2005) and ba s (Die z e al., 2016). Howe e , analysing hei die can indi ec ly e eal
he impo ance o some habi a s by iden i ying ood sou ces. This app oach p o ides c ucial habi a
in o ma ion o species conse a ion (e.g., Collins e al., 2005; Mi schunas & Wagne , 2015).
Mo phological die s udies we e labou -in ensi e and o en inaccu a e, o e ing limi ed ecological and
beha iou al insigh s and cap u ing only s a ic snapsho s o ba die s (Hope e al., 2014; Albe di e al.,
2020). These echnical limi a ions cons ained accu a e habi a iden i ica ion. Howe e , DNA
me aba coding now allows de ailed species-le el iden i ica ion o p ey emains (K üge e al., 2014;
Galan e al., 2018; Ba oja e al., 2019), pa ing he way o mo e accu a e o aging habi a
iden i ica ion (Cla e e al., 2011; S ockdale, 2018).
Such s udies ha e p o ided insigh s in o he habi a s associa ed wi h ba p ey (Albe di e al., 2012).
While hey canno pinpoin whe e p ey was cap u ed (sink habi a ), hey indica e p ey o igins (sou ce
habi a ) (A izabalaga-Escude o e al., 2015). Molecula s udies show ba s consume hund eds o
species ha a y o e ime (Razgou e al., 2011; A izabalaga‐Escude o e al., 2015; Aiha za e al.,
2023), space (Cla e e al., 2014), and among indi iduals (Ma a e al., 2016), adjus ing hei die o
p ey a ailabili y (e.g., Almena e al., 2013; Napal e al., 2013; Aiha za e al., 2023; Vallejo e al.,
2023). Consequen ly, molecula die analysis has become pa amoun o de e mining ba s' o aging
dependencies by iden i ying p ey-o igin habi a s h oughou he yea .
Di e en ba guilds, de ined by echoloca ion and ligh abili ies, ha e a ied o aging s a egies
(Fen on, 1990; Schni zle & Kalko, 2001). Clu e specialis use sho b oadband calls o na iga e
dense ege a ion, while ae ial hawke s use high- equency calls o ca ch insec s in open spaces.
Su p isingly, molecula die s udies show open-space ba s also o age in di e se habi a s, including
o es s, elying on p ey a ailabili y peaks (Ga in e al., 2019; Aiha za e al., 2023). Simila ly, clu e
specialis ba s likely exploi p ey ou b eaks in di e se habi a s, using hei echoloca ion and ligh
skills a he mic ohabi a le el by lying close o ege a ion.
The lesse ho seshoe ba , Rhinolophus hipposide os (Bechs ein, 1800), is he smalles Eu opean
hinolophid and uses high CF calls (abou 112 kHz) o hun nea ege a ion, making i a specialized
clu e hun e (Jones & Rayne , 1989; Scho ield, 1996). I is widely dis ibu ed in he wes e n and
cen al Palaea c ic, ound in almos all Eu opean coun ies (Taylo , 2016), pa ly due o i s
adap abili y o an h opic a eas. Despi e his, i is classi ied as o communi y impo ance in he EU,
equi ing Special A eas o Conse a ion (Council Di ec i e 92/43/EEC). The species is a isk due o
he des uc ion o na i e woodlands, (Bon adina e al., 2002; Mo e & Libois, 2002; Rei e , 2004;
106
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Zahn e al., 2008), oos loss o dis u bance, a i icial illumina ion and use o pes icides (Scho ield e
al., 2023).
The lesse ho seshoe ba p e e s b oadlea woodland habi a s, bu also uses o he habi a s o o aging
and commu ing, such as g azed g asslands and a eas wi h high habi a di e si y (Bon adina, 2002). In
ag o o es y a eas, i a ou s woodland pa ches and hedge ows nea cul i a ed ields (Mo e & Libois,
2002). The p esence o ma e ni y oos s, c ucial o he species' pe sis ence, depends on he
a ailabili y o wooded elemen s nea small buil a eas and he in eg a ion o oos s in o a connec ed
ne wo k (Mo e & Libois, 2002; Tou nan , 2013).
R. hipposide os has a o aging a ea wi h an a e age adius o 600 m om home, wi h a
maximum- eco ded dis ance o 4.2 km om he oos (Bon adina e al., 2002). The e o e, i s
conse a ion elies on he a ailabili y o sui able oos ing si es and p oduc i e o aging a eas nea by.
As a esul , unde s anding hei habi a dependence is essen ial. Thei simila die ac oss p is ine and
modi ied habi a s in Eu ope (e.g., McAney & Fai ley, 1989; Russo & Jones, 2003; Lino e al., 2014;
Ba oja e al., 2019), sugges s ha main aining high insec di e si y is c ucial o hei o aging.
S udying his species' die a y pa e ns in human-modi ied habi a s may help enhance hese a eas o
o aging (Lino e al., 2014).
In his s udy, we analysed he die changes o h ee ma e ni y colonies o R. hipposide os in he
Basque Coun y (No he n Ibe ian Peninsula) occu ing in a eas a ying in landscape, u banisa ion
le el, and clima e. We examined he species’ die ac oss hei ac i e season, paying a en ion o he
seasonal and la i udinal a ia ions. We also analysed he habi a s in which he mos consumed p ey
depends. Ou hypo heses we e:
The lesse ho seshoe ba die will change (1) wi h ime, showing a seasonally shi ing o
mos -consumed p ey, and (2) wi h he si e, depending on he p ey a ailabili y o each colony. (3) Mos
consumed p ey will depend on di e en landscapes o habi a s, causing seasonal changes in he
equi ed en i onmen al esou ces o he ba s, and hus, (4) such p ey-habi a dependence may also
a y among colonies.
3.2. MATERIALS AND METHODS
3.2.1. S udy A ea
We sampled h ee R. hipposide os ma e ni y oos s wi h a ying land use and clima e (Figu e 3.1).
Using QGIS 3.16 and Spanish Go e nmen (SIGPAC) maps, we mapped habi a and land co e uni s
wi hin 1, 2, and 3 km bu e s a ound he oos s. These bu e s co e all po en ial o aging a eas, as
ba s ypically mo e up o 4.2 km om hei oos s (Bon adina e al., 2002).
Ma xinben a (MA): O e 100 R. hipposide os oos ed in a u al chu ch dome in a illage su ounded
by deciduous and coni e o es s, meadows, and pas u e. The a ea has an A lan ic empe a e oceanic
clima e (293 MASL) wi h yea - ound ain all (annual mean 1400 mm).
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Za au z (ZA): Abou 100 R. hipposide os oos ed in a chu ch dome in an u ban a ea on he
Can ab ian coas . The su oundings include ineya ds, ui ees, pas u es, and deciduous and
coni e ous o es s. The a ea has an A lan ic empe a e oceanic clima e (4 MASL).
Caseda (CA): Up o 30 R. hipposide os oos ed in an emp y building ha was once pa o a
hyd oelec ic powe plan . Loca ed nea a la ge i e , he a ea is domina ed by d y ce eal a mland o
he no h and Medi e anean bush and o es o he sou h, wi h some ineya ds and ui o cha ds
nea by. The wea he is Medi e anean empe a e, wi h ho , d y summe s (367 MASL).
Figu e 3.1. S udy A ea: P ima y habi a and land co e uni s wi hin 3 km a ound Rhinolophus hipposide os
oos s whe e die a y analysis was conduc ed. ZA: Za au z colony; MA: Ma xinben a colony; CA: Caseda
colony. Cha s show habi a a ailabili y a 1, 2, and 3 km bu e s. Sea in ZA is excluded as i ’s no a p ey sou ce.
Map c ea ed wi h QGIS 3.16, based on Spanish Go e nmen (SIGPAC) public maps.
3.2.2. Sampling Ba D oppings
We sampled he h ee colonies in 2022, h oughou he ma e ni y season (mid-sp ing and summe ).
Samplings began in Julian Week 17 (la e Ap il) when ba s a i ed. MA ba s we e he las o lea e
(Sep embe 30 h, Week 39), while ba s om ZA and CA le wo weeks ea lie (Week 37). Sampling
occu ed e e y o nigh in e en weeks using non-in asi e me hods o a oid dis u bing he animals.
We placed pape collec o s unde he animals o keep he d oppings as d y as possible, and we
examined he aeces isually o only collec he mos ecen ba aeces o a oid deg ada ion o he
sample's DNA. Pape was changed a e each sampling. We collec ed up o 24 small “communi y
samples” o ou o six pelle s each o each sampling e en (adap ed om And iollo e al. 2019,
2021). These samples aim o e lec he die o he en i e ma e ni y colony h oughou he sampling
108
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pe iod, a he han he con ibu ions om indi idual animals. In o al, 413 aecal samples o
sequencing p ocedu es and u he analysis.
3.2.3. DNA Ex ac ion, PCR Amplifica ion, Lib a y P epa a ion and Sequencing
We ex ac ed DNA om 413 ozen aecal samples weighing up o 40 mg (median o 19.4 mg) wi h
he King ishe ex ac ion ool (The mo Fishe Scien i ic Inc.), p e ious homogenisa ion wi h P ecellys
24 Touch Homogenize (Be in Technologies SAS, F ance). F om he ex ac s, wo 178 and 180 bp
di e en mini-COI segmen s o he mi ochond ial DNA cy och ome c oxidase subuni I (COI)
ba code egion we e ampli ied wi h FWH1 ( whF1/ whR1) (Vamos e al., 2017) and ANML
(CO1490/CO1‐CFMRa) (Jusino e al., 2019) p ime s, espec i ely. Ampli ica ions we e pe o med
using he QIAGEN Mul iplex PCR Ki (Qiagen Ibe ia, S.L. Mad id) in 25 μl PCR eac ions,
ollowing he o iginal p o ocols. PCR p oduc s we e mig a ed in aga ose gel elec opho esis o es he
success o he ampli ica ion p ocess. Subsequen ly, a second PCR eac ion was pe o med o a ach a
unique combina ion o ags and Illumina sequencing adap e s o each amplicon using he Nex e a XT
Index Ki (Illumina, 2013). Finally, 413 samples we e pooled and sequenced using Illumina No aSeq
echnology (SP Flow Cell ki 2 PE: 2 x 250bp (500 cycles) / 800 M eads). DNA lib a y cons uc ion
and sequencing p ocesses we e ca ied ou a he Genomics and P o eomics Gene al Se ice (SGIke )
o he Uni e si y o he Basque Coun y.
3.2.4. Bioin o ma ic analyses
We pe o med sepa a ion by p ime s, quali y con ol, sequence p e-p ocessing, and collapsing
iden ical sequences in o single ones using CUTADAPT (Ma in, 2011) and VSEARCH (Rognes e al.,
2016). We clus e ed sequences in o Ope a ional Taxonomic Uni s (OTU) by VSEARCH a a 97%
simila i y h eshold (Hebe e al., 2003) using he “–clus e _size” command. Subsequen ly, we
cleaned up chimae a OTUs wi h VSEARCH’s “-uchime3_deno o” command. Only OTUs wi h an
abundance highe han 0.5% in any sample uni we e selec ed o he analysis (See Appendix B o
sc ip de ails).
The axonomic assignmen o each OTU was pe o med by compa ing he ep esen a i e sequence
agains e e ence sequences in GenBank (www.ncbi.nlm.nih.go ; accesed on 2023-08-28) using he
“-blas n” command (Chen e al., 2015). Only hi s wi h pai wise iden i y abo e 98% and e- alues
below 1e-20 we e accep ed (Ves e inen e al., 2013; Cla e e al., 2014) o ensu e ha he ma ch did no
occu by chance. The species da abases o a h opods o Spain, F ance and Po ugal we e
downloaded om GBIF (www.gbi .o g; accessed on 2022-11-03) o e i y ha he iden i ied species
encompassed ou s udy a ea. The Ibe ian Fauna Da abase (Ibe auna) was also checked o assu ing
he species dis ibu ion (ibe auna.mncn.csic.es; accessed on 2022-11-03). OTUs showing mul iple
po en ial assignmen s we e ca e ully e iewed and e ined manually o assign he mos accu a e axa
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Figu e 3.5. (a-b) RLQ analysis esul s o Rhinolophus hipposide os die : (a) Eigen alues and weekly colony
sample sco es (MA G een: Ma xinben a; ZA Blue: Za au z; CA O ange: Caseda). (b) P ey ai coe icien s.
Panels display he i s wo axes wi h d- alues indica ing g id size. Mon e-Ca lo es : obse ed s a is ic = 0.419,
p < 0.001. (c) 4 h co ne analysis esul s. Red indica es posi i e co ela ion, and blue indica es nega i e
co ela ion (p < 0.001).
Fo es s and sh ub pas u es we e c ucial o all colonies yea - ound. In empe a e colonies (ZA, MA),
hey p o ided o e 50% wPOO o die s, while, in he Medi e anean colony (CA), hese habi a s we e
less signi ican bu s ill impo an (Figu e 3.6).
In MA, he die consis ed o 32.1% o es , 23.5% sh ub pas u es, and 13.5% g assland. Despi e being
he main p ey-sou ce habi a , o es s we e used less han a ailable (Figu e 3.6a). Key woodland
species included dip e ans Dic anomyia sp., Neph o oma la ipalpis, Helina impuc a, and Neolimonia
dume o us, along wi h lepidop e ans Teleiodes wagae and S a hmopoda pedella. Sh ub pas u e
species, such as he mo h A gy es hia goeda ella and hemip e an Piezodo us li u a us, we e
posi i ely selec ed. G asslands, wi h species like Limonia nubeculosa, Lasius sp., and Nabis
pseudo e us, we e also a ou ed, especially in summe . Ripa ian o es s, wi h ichop e ans
116

CHAPTER 3
Limnephilus hombus and S enophylax ibex, and ui ees, including Cydia agiglandana and
Zeuze a py ina, we e impo an o he colony’s die , especially la e in he season (Figu e 3.6a).
In he ZA colony, o es and sh ub pas u es we e he main p ey-sou ce habi a s (32.9% and 21.6%,
espec i ely), wi h "o cha ds" in hi d place a 12.9%. Acco ding o he o es a ailabili y, we can
in e ha hey lew a he han 1km o hun in hese habi a s (Figu e 3.7b), he same as in sh ub
pas u es. O cha ds became signi ican om week 27, peaking in Augus (weeks 31-33) wi h in ense
p eda ion o Opogona saccha i (Figu e 3.6b). Al hough o cha ds we e posi i ely selec ed, u ban
species we e a ely consumed, indica ing his habi a was unde used (Figu e 3.7b).
Figu e 3.6. Pe cen age habi a con ibu ions as p ey sou ces (in wPOO in die ) o weekly die s o lesse
ho seshoe ba s: colonies in CA: Caseda, MA: Ma xinben a and ZA: Za au z. Numbe s on he x-axis e e o
Julian Weeks.
Figu e 3.7. Mosaic plo s o a ailable e sus impo an habi a s o co e p ey in Rhinolophus hipposide os die s:
(a) MA: Ma xinben a; (b) ZA: Za au z; (c) CA: Caseda. Squa e sizes show ca ego y pe cen ages; colou s
indica e s anda d esiduals—blue o posi i e selec ion, ed o nega i e. As e isks (*) ma k habi a s wi h
signi ican posi i e selec ion (p < 0.05).
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CHAPTER 3
In CA, o es s emained a key p ey-sou ce-habi a bu dec eased o 21.9%, ollowed closely by
ipa ian o es a 21.7% and g asslands a 20.8%. Sh ub pas u es con ibu ed 17.8%, bu habi a
impo ance a ied by da e (Figu e 3.6c). Woodland species' high consump ion was linked o o es
a ailabili y wi hin 3 km, peaking in May-June (weeks 19-25) and mid- Sep embe (week 37) (Figu e
3.7c). Howe e , hei die con ibu ion declined in summe as o he habi a s, especially ipa ian o es ,
became mo e signi ican . The la e was hea ily used om Augus , pa icula ly in week 35,
con ibu ing 40% o he die due o dip e ans like Gonomyia enella and Rheopelopia o na a, and he
epheme op e an Caenis luc uosa. In con as , g asslands we e a majo p ey sou ce in sp ing,
con ibu ing up o 49% o he die , while sh ub pas u es gained impo ance la e . C oplands we e
unde used excep a he end o Ap il (week 17).
3.4. DISCUSSION
While ba die s a e widely s udied, his esea ch, alongside Aiha za e al. (2023), is among he i s o
use de ailed molecula da a o in e habi a dependencies. The lesse ho seshoe ba s' die has been
add essed in a ious egions o Eu ope (McAney & Fai lay, 1989; Lino e al., 2014; Mi schunas &
Wagne , 2015; Ba oja e al., 2019), bu ou s udy is he mos comp ehensi e o da e, iden i ying p ey
a he species le el using molecula me hodologies, h ough ex ensi e aecal sample analysis. Using
No aSeq "ul a-high- h oughpu sequencing" p o ided deepe sequencing han p e ious s udies, wi h
a consis en species-le el p ey iden i ica ion. Addi ionally, by examining colonies in a clima ic
ansi ion zone, we compa ed empe a e and Medi e anean en i onmen s. Las ly, conduc ing he
s udy h oughou he ma e ni y season o e ed a ho ough unde s anding o lesse ho seshoe ba s' die
and ecology.
The indings on he lesse ho seshoe ba die align wi h p e ious mo phological s udies (e.g., McAney
& Fai ley, 1989; Lino e al., 2014; Mi schunas & Wagne , 2015). Lepidop e a was he mos consumed
o de , ollowed by Dip e a and Hemip e a. No ably, caddis lies play a signi ican ole in he
Medi e anean colony (Caseda: CA), sugges ing ba s ely mo e on i e s and s eams in
Medi e anean egions, consis en wi h Lino e al. (2014).
P e ious molecula die s udies on his species a e limi ed. The 197 axa iden i ied by Ba oja e al.
(2019) om May o Sep embe we e all ound in ou esea ch, wi h 63 pa o ou co e die s. Aldaso o
e al. (2019) (Chap e 1) assessed he die in July in a simila a ea o he ZA and MA colonies,
iden i ying key dip e an species such as Rhipidia macula a, Neolimonia dume o um, Limonia
nubeculosa, Dic anomyia modes a and Tipula hel ola. Limonia nubeculosa and Neolimonia
dume o um we e no ably he mos consumed species in he MA colony in la e July.
The species-le el die analysis suppo s ou hypo hesis o die a y di e ences be ween colonies. Ou
indings align wi h p e ious R. hipposide os (Mi schunas & Wagne , 2015) and o he ba s udies
(Aizpu ua e al., 2018; Vallejo e al., 2019), indica ing ha ba die s a y by loca ion. Ou esul s ha e
118
CHAPTER 3
also alida ed a seasonal die shi wi hin each colony, as ba s ake ad an age o he al e na ion o
occasional occu ence o many di e en p ey peaks. This oppo unis ic beha iou aligns wi h pa e ns
in insec i o ous ba s, which p o i om ho spo s and insec ou b eaks (Aizpu ua e al., 2013; Lino e
al., 2014; Aiha za e al., 2023; Vallejo e al., 2023). In his con ex , we would like o emphasise he
subs an ial consump ion o he c op pes Opogona saccha i by he ZA colony. This obse a ion
unde sco es he species' oppo unis ic na u e and i s po en ial ole as a consume and con olle o
ag icul u al pes s (Ba oja e al., 2019; Chap e 5).
Insec i o ous ba s use su ounding habi a s in di e se ways. Fen on (1990) ca ego ised hem as
"open-space o age s," "edge and gap o age s," and "na ow-space o age s." Schni zle and Kalko
(2001) ca ego ised habi a s as "unclu e ed," "backg ound-clu e ed," and "highly clu e ed," wi h
subca ego ies based on hun ing echniques: ae ial and gleaning ba s. R. hipposide os i s he
"na ow-space o age " o "ae ial in highly clu e ed space" guilds. Radio- acking s udies ha e
con i med so, showing hese ba s mainly o age in woodlands and hedge ows (e.g., Mo e & Libois,
2002; Russo & Jones, 2003; Rei e e al., 2013). Though R. hipposide os is hough o be es ic ed o
speci ic habi a s, i also uses g asslands, o cha ds (Zahn e al., 2008; Mi schunas & Wagne , 2015),
and u ban a eas nea building walls o hun ing (Jones & Rayne , 1989). Die z ound ha sou he n
egions show a di e gence om he p e alen use o o es s in no he n Eu ope, wi h ga dens,
o cha ds, hedge ows, and g azed pas u es being mos used in Bulga ia.
This s udy con i ms ha he lesse ho seshoe ba elies on di e se p ey om a ious habi a s. Ou da a
sugges s ha al hough classi ied as "clu e - eede s", hey o age o species o igina ing in se e al
ege a ion ypes besides woodlands. Like o he "open-space o age " Miniop e us sch eibe sii
(Aiha za e al., 2023), clu e specialis ba s also shi hei habi a eliance seasonally and ac oss
loca ions, acking eme ging p ey ou b eak peaks (Ba oja e al., 2021; Aiha za e al., 2023; Vallejo e
al., 2023). This adap abili y unde lines hei gene alis and oppo unis ic eeding beha iou , enabling
hem o exploi ho spo and abundan esou ces h oughou he yea .
As p e iously poin ed ou (e.g., Lino e al., 2014; A izabalaga-Escude o e al., 2015; Ancillo o e al.,
2023), we con i m he impo ance o p o ec ing well-connec ed he e ogeneous habi a s, mosaic
landscapes and eco ones wi h high p ey di e si y a ound hei oos s o ensu e he conse a ion o he
lesse ho seshoe ba .
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3.6. SUPPORTING INFORMATION
Suppo ing In o ma ion 3.1. Table A. Species o he co e die s used o he
Landscape analysis and hei habi a desc ip ions. Table B. Bibliog aphy o assessing
hei sou ce habi a s (xlsx) h ps:// inyu l.com/aldoso oS31
Suppo ing In o ma ion 3.2. Table A. Iden i ied p ey axa and hei wPOO
(weigh ed pe cen age o occu ence) and FOO ( equency o occu ence) o each
colony; Table B. P esence/absence alues o indi idual ba s. Table C. wPOO alues
o indi idual ba s. (xlsx) h ps:// inyu l.com/aldaso oS32
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Seasonal and geog aphic a ia ion in he
ophic ecology and habi a dependence o
Rhinolophus e umequinum
CHAPTER 4
a e ac ion cu es wi h iNEXT o de e mine whe he he colonies had eached a species di e si y
asymp o e, indica ing ha he sample sizes we e su icien o meaning ul compa isons. We compa ed
he di e ences in he die composi ion be ween colonies using Pianka’s (1973) measu e o niche
o e lap wi h he pianka unc ion in he package EcoSimR (Go elli e al. 2015) a he o de and species
le el. The ollowing s eps we e ca ied ou using he R package egan (Oksanen e al. 2020). We
calcula ed B ay-Cu is dissimila i ies be ween all samples using he unc ion egdis (Oksanen e al.,
2020). Using he adonis unc ion, we explo ed he ela ionship o die composi ion wi h Mon h,
Colony and hei in e ac ion h ough PERMANOVA (Ande son, 2001) a he o de and species le el.
We pe o med an NMDS (Non-me ic Mul i-Dimensional Scaling) wi h he me aMDS unc ion o
isualise di e ences in species composi ion be ween samples. See Appendix C o R sc ip de ails.
4.2.5. Cha ac e iza ion o P ey’s Sou ce Habi a s
We collec ed in o ma ion on he habi a p e e ences o p ey om a ious bibliog aphic sou ces
(Suppo ing In o ma ion S4.1B). Nex , we ca ego ised hese habi a s based on land use, aligning hem
as closely as possible wi h he SIGPAC habi a s. This p ocess enabled us o c ea e g oupings o
habi a s ha ha e unc ional alue o ba s, conside ing he le el o clu e and he po en ial
p oduc i i y o p ey (Table 4.1). This classi ica ion will be e e ed o as “p ey-sou ce-habi a ” om
now on.
Table 4.1. Habi a ca ego ies o he second RLQ analysis and hei desc ip ion.
Habi a ca ego y
Desc ip ion
C opland
Ag icul u al ields, c opland
Fo es
Coni e o b oadlea ed woodland
Ripa ian o es
Ri e s, s eams, and ipa ian ege a ion
O he wa e bodies
Ponds, small s eams wi hou ipa ian ege a ion
G assland
G assland, meadows pas u es, g azing pas u es (ca le), we lands
Sh ub g assland
Sc ubs, sh ubland, hea hland, woodland edges, hedge ows
Vineya ds
Vineya ds
F ui ees
F ui - ees, nu - ees, oli e g oo es
O cha ds
O cha ds
G eenhouse
G eenhouses, c ops unde plas ic
U ban
U ban, a i icial habi a s…
4.2.6. Landscape in e ence analysis
We pe o med RLQ and ou h-co ne analyses using he ade4 package o R (Thioulouse e al., 2018)
o explo e how ela ionships wi h p ey sou ce habi a s a y ac oss di e en loca ions and seasons. We
aimed o emphasize he signi icance o he sou ce-habi a s o p ey and o de e mine whe he pa icula
axa we e consumed in speci ic habi a ypes, he eby iden i ying habi a -speci ic p ey g oups. In his
way, we analysed how he consump ion o habi a -speci ic p ey g oups a ies mon hly in he h ee
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colonies. This app oach is based on cons uc ing h ee ma ices acco ding o he me hod ou lined by
A izabalaga-Escude o e al., (2019).
In ou da ase , he sampling e en (colony and sampling mon h) ep esen s he “en i onmen al
a iables”, he habi a -speci ic p ey g oup (e.g., Fo es Lepidop e a, G assland Coleop e a…)
ep esen s he “ ai ” ma ix, and he in e ac ion ma ix consis s o he wPOO abundance o each p ey
species wi hin each ba ’s die . We es ic ed he analysis o he co e die s o each ba colony
(Suppo ing In o ma ion S4.1A) o ob ain a be e pic u e o he sou ce habi a s o he mos consumed
p ey, which in u n we e possibly posi i ely selec ed. Be o e he RLQ analysis, mul i a ia e
o dina ion is needed on all h ee ma ices, namely a Co espondence Analysis on he L ma ix since
he la e includes quan i a i e da a, and Mul iple Co espondence Analysis (MCA) on ma ices R and
Q, which include ca ego ical a iables. We pe o med a andomiza ion es using he unc ion and es
o es he ela ionship be ween he en i onmen al ma ix (R) and he ai s ma ix (Q) h ough he
species' occu ence o abundance (L) (Model 2). The numbe o pe mu a ions was ele a ed o 9,999.
This model examines how ai s espond o en i onmen al ai s indi ec ly ia species dis ibu ions. In
ou case, we in es iga edhow habi a -speci ic p ey g oups espond o seasonali y (sampling mon h)
and space (colony). A e inding ha sampling e en s we e g ouped by colony (see esul s, Figu e
4.4), we pe o med an RLQ analysis o each colony o see he seasonali y o habi a -speci ic p ey
g oups wi hou he in e e ence o he si e.
Fo he h ee bu e s in each colony —see abo e— we compa ed habi a a ailabili y wi h hei ophic
con ibu ion (used habi a om now on), calcula ed by mul iplying each p ey-sou ce-habi a
a ailabili y by hei wPOO alues in he die . This way, we ob ained he “use” alues o each o he
p ey-sou ce-habi a s. A e ha , we pe o med a Pea son's Chi-Squa e Tes o ind co ela ions
be ween habi a a ailabili y and use, and we added he Mon e Ca lo es op ion o ob ain a s a is ical
signi icance p- alue (simula e.p. alue = T). See Appendix C o R sc ip de ails.
4.3. RESULTS
4.3.1. Die composi ion
A e sequencing, we ob ained aluable sequences om 377 ou o 379 samples. The No aSeq
sequencing led o 66,726,490 eads, which gene a ed up o 150,000 OTUs, o which only 5,349 had an
abundance highe han 0.5% in any o he samples. These esul s p o ided a comp ehensi e iew o
he ba 's die composi ion. We iden i ied 2320 OTUs (43.37% o he o al): i e OTUs belonged o he
p eda o , Rhinolophus e umequinum (1.85% o he eads), 200 (4.43% o eads) we e classi ied as
en i onmen al con amina ion, and 2,115 (51.22% o eads) we e iden i ied as po en ial p ey i ems. The
emaining 3,029 (42.5% o eads) did no ma ch wi h any sequence in he da abases, a leas a he
98% iden i y le el.
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CA and UX colonies we e mixed along wi h Myo is ema gina us, and a e sequencing, we de ec ed
DNA con amina ion in some o he samples. The e o e, we decided o wi hd aw he con amina ed
samples, me ge he clean samples, and analyse hem based on he sampling mon h ins ead o he week.
This decision was made o ensu e a s a is ically aluable sample size and o accoun o po en ial
a ia ions in he ba 's die o e longe pe iods. A possible co ela ion be ween Myo is ema gina us
OTUs and p ey species was analysed a e il e ing samples. No clea co ela ions we e ound, leading
us o assume ha he species examined in he die we e po en ial p ey o Rhinolophus e umequinum.
A e disca ding he con amina ed samples, we wo ked wi h 231 small communi y samples. We
iden i ied up o 924 p ey species belonging o 624 gene a, and ano he 44 p ey axa we e ecognised a
he genus le el. In addi ion, 34 gene a had OTUs iden i ied a bo h he genus and species le els. In
hese cases, a genus-le el occu ence was added o he die a y da a. O e all, we analysed 1,002 p ey
axa, iden i ied a he genus o species le els, belonging o 14 o de s (Suppo ing In o ma ion S4.2).
Ou da a analysis was ho ough, ensu ing a comp ehensi e unde s anding o he ba species' die .
The mos consumed o de was Lepidop e a (wPOO 46.99%), ollowed by Dip e a (26.67%) and
Coleop e a (6.79%) (Figu e 4.2). The mos equen p ey species was he mo h Coleopho a sp. —Fam.
Coleopho idae—, iden i ied in 66 samples (F equency o occu ence (FOO) 27,85%), accoun ed o
2.07% wPOO. The nex wo mos equen p ey i ems we e he caddis ly Hyd opsyche exocella a
—Fam. Hyd opsychidae—, and he mo h Opogona saccha i —Fam. Tineiidae—, iden i ied in 63 and
52 samples, wi h 1.58% and 0.85% wPOO, espec i ely. Mos p ey i ems appea ed occasionally in
aeces: 449 ou o 1,002 p ey axa we e only iden i ied in a single sample each, 166 in wo samples,
302 in a ange be ween h ee and en samples, and 87 species occu ed in mo e han en samples.
Finally, we added he species wi h a equency o occu ence (FOO) g ea e han 5% (Tou nay e e al.,
2021) o each colony o ob ain he co e die s. These co e die s, a signi ican pa o ou indings, ga e
a o al o 173 axa (species and gene a) o he analysis.
We iden i ied 695 p ey axa o he die o he Balmaseda (BA) colony. No p ey ype p edomina ed
wi h ele ance in he die , as he banana mo h Opogona saccha i was he mos consumed species in
he whole season (accoun ing o 1.96% o he wPOO) and om May o July. The second mos
consumed species was he ichop e an Hyd opsyche exocella a (1.34%), peaking in July and Augus ,
and hi dly, we ound he limonid Dic anomyia sp. (1.27%) wi h a consump ion main ained o e ime.
In he die o he Caseda (CA) colony, we iden i ied 528 p ey axa. The mos consumed species was
he mo h Eu odach ha canigella (2.83%), ollowed by he caddis ly Hyd opsyche exocella a (2.25%),
bo h especially consumed in sp ing, and he mo h Coleopho a sp. (1.95%) showing a sus ained
consump ion un il July.
Finally, we assigned 289 p ey axa o he Uxue (UX) colony's die . The mos consumed species we e
mo hs. Coleopho a sp. (4.09%) was widely consumed du ing he season, Phalonidia con ac ana
134
CHAPTER 4
(3.98%) was especially consumed in Ma ch, while Rhodome a sac a ia (2.91%) was mainly
consumed in Ma ch and June.
Figu e 4.2. (a) To al die in he h ee colonies o Rhinolophus e umequinum, showing he consumed p ey a he
o de le el, measu ed as he weigh ed pe cen age o o al occu ences (wPOO) (Deagle e al., 2019) and mon hly
die o (b) BA, Balmaseda (c) CA, Caseda and (d) UX, Uxue. Axis y ep esen s he wPOO alues, while Axis x
indica es he colony in plo (a) and he mon h in plo s (b), (c), and (d).
4.3.2. Mul i a ia e analysis o die a iabili y and homogenei y
The colonies eached sample co e age o o e 75% (81% BA, 80% CA and 77% UX). Ou Pianka's
niche o e lap analysis a he p ey o de le el e ealed ha all h ee colonies sha e a simila gene al
die . Howe e , hei die s a ied signi ican ly a he p ey species le el, wi h a simila i y index o
46.52%. No ably, he geog aphic dis ance be ween he colonies played a signi ican ole in hese
die a y di e ences. Fo ins ance, BA and UX, being he mos dis an , exhibi ed he mos dis inc die s,
wi h a simila i y index o only 33.90%, while UX and CA, being close and ha ing a mo e simila
clima e, had he mos simila die s a 59.43%. BA and CA, alling in be ween, showing a 46.25%
135
CHAPTER 4
simila i y. Rega ding he co e die , he di e ences be ween colonies inc eased, wi h a o al simila i y
index o 20.55%.
Figu e 4.3. NMDS mul idimensional o dina ion o he samples based on hei die s measu ed as wPOO,
conside ing he whole die . BA: Balmaseda, CA: Caseda, UX: Uxue. Poin s belong o ba aecal samples, and
colou s con ey he di e en sampling da es. G ey do s show samples om he o he colonies.
The PERMANOVA analysis con i med wha he NMDS sugges ed (Figu e 4.3), indica ing ha die
composi ion a he species and o de le el di e ed signi ican ly based on he a iables "colony" and
"sampling mon h", as well as hei in e ac ion a he species le el (p < 0.001; R2 = 0.044; R2 = 0.054;
R2 = 0.055, espec i ely), and a he o de le el (p < 0.001: R2 = 0.046; R2 = 0.075; and p = 0.002; R2
= 0.055, espec i ely). Addi ionally, signi ican di e ences (p < 0.001) we e obse ed be ween he
sampling mon hs when analysing he die s o each colony sepa a ely a he species le el (R2BA =
0.122; R2CA = 0.097; R2UX = 0.112, espec i ely).
4.3.3. Landscape in e ence analysis
The RLQ andomiza ion es s we e s a is ically signi ican o he whole analysis and o he h ee
colonies (Model 2: p < 0.0001), es ing he ela ionship be ween sampling e en /seasons (R) and he
habi a -p ey g oup associa ion (Q) h ough he die ma ix (L). In he comple e RLQ analysis, he i s
wo axes accoun ed o 24.97% and 39.24% o he o al a iance. The o dina ion showed a clea
g ouping o sampling e en s by colonies, sugges ing ha he si e plays a c ucial ole in he di e ing
consump ion o habi a -speci ic p ey g oups (Figu e 4.4a). BA sampling e en s we e g ouped on he
igh side o he plo and we e associa ed wi h a ying g oups, such as o cha d- and
136
CHAPTER 4
u ban-lepidop e ans o di e se o es g oups. Besides, CA e en s we e mainly g ouped on he
down-le , associa ed wi h ipa ian species and some c opland g oups. Finally, UX e en s we e
g ouped on he op le and linked o g assland and sh ubland g oups. None heless, no clea
associa ion can be made om his analysis. A e pe o ming he RLQ o each colony, we con i med
ha he consump ion o some habi a -speci ic p ey g oups was associa ed wi h speci ic mon hs
(Figu e 4.3a,b,c). In con as , he consump ion o o he g oups showed a mo e homogeneous
dis ibu ion o e ime. Howe e , hese associa ions di e be ween colonies.
In he RLQ analysis o BA, he i s wo axes accoun ed o 44.8% and 22.6% o he o al a iance,
espec i ely (see Figu e 4.4b). We obse ed ha du ing sp ing (Ap il-May), o es dip e ans we e he
mos commonly consumed p ey g oup in BA and emained signi ican h oughou he en i e season.
Dip e a associa ed wi h di e en habi a s we e consumed homogeneously, al hough especially
p e alen du ing sp ing. In Ap il, g assland coleop e ans, dip e ans, and hymenop e ans we e highly
consumed. S a ing in May, he o cha d lepidop e an Opogona saccha i became one o he mos
consumed species and con inued o be signi ican un il he end o he season. Fo es coleop e ans we e
p ima ily consumed om May o July, wi h S enagos us hombeus being he dominan species in May
and June, while A hopalus us icus became mo e p e alen in July and Augus . Du ing summe ,
ichop e ans, linked o ipa ian habi a s, eme ged as a key p ey wi h a signi ican in ake o
Hyd opsyche exocella a. Fo es lepidop e ans we e mainly associa ed wi h July, highligh ing he
consump ion o Thaume opoea pi yocampa o Dend olimus pini, species linked o pine plan a ions. In
con as , g assland and sh ubland lepidop e ans showed a mo e gene al in ake and we e no explici ly
linked o any pa icula mon h (see Figu e 4.4b).
Fo CA, he i s wo axes o he RLQ accoun ed o 43.4% and 24.9% o he o al a iance,
espec i ely (see Figu e 4.4c). Ripa ian p ey g oups we e p ima ily associa ed wi h he ea ly season.
In Ap il, he mos consumed species was he ichop e an Hyd opsyche exocella a. Fo es and
g assland coleop e ans we e linked o he summe mon hs (June o Augus ), including he dung
geo upids Typhaeus yphoeus and Geo upes sp. and o es T icho e us ascicula us. P ey g oups
ela ed o c oplands we e also p e alen in summe (July and Augus ), wi h no able consump ion o
species like Tipula epanda (Dip e a), and Ag o is sege um (Lepidop e a). In Sep embe , he p ima y
p ey species shi ed back o lepidop e ans mainly associa ed wi h g asslands and sh ublands.
Lepidop e a associa ed wi h ui ees (Cydia agiglandana) we e s ongly linked o Sep embe .
Howe e , hese g oups did no show a clea associa ion because he consump ion o lepidop e ans
om sh ublands, g asslands, and o es s was dis ibu ed h oughou he season. The e o e, hey we e
posi ioned in he middle o he plo (see Figu e 4.4c).
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CHAPTER 4
Figu e 4.4. Resul s o RLQ analyses o he Rhinolophus e umequinum die , all oge he (a) and by colony: (b)
B: Balmaseda; (c) C: Caseda; (d) U: Uxue. The plo s on he le show he eigen alues and sco es o colony
mon hly samples. The plo s on he igh side show coe icien s o p ey ai s (habi a -speci ic p ey g oups).
Panels display he i s wo axes only, wi h d‐ alues e e ing o g id size.
138
CHAPTER 4
Figu e 4.5. Pe cen age con ibu ion o habi a s as co e p ey sou ce (in wPOO in die ) o he mon hly die s o he
g ea e ho seshoe ba s colonies in CA: Caseda, BA: Balmaseda, and UX: Uxue.
Finally, in he RLQ analysis o UX, he i s wo axes explained 46.7% and 32.7% o he a ia ion,
espec i ely (see Figu e 4.4d). The consump ion o dung coleop e ans (Geo upes sp.) dis inguished
May om he o he mon hs. In con as , o es coleop e ans (S enagos us hombeus) we e consumed
du ing Ma ch, Ap il, and June. G assland dip e ans like Symplec a s ic ica we e p edominan ly
consumed in Ma ch. Fo es and sh ubland lepidop e ans, and sh ubland dip e ans and hemip e ans,
we e especially associa ed wi h Ap il and July. Epheme op e ans (Hab ophlebia lau a) we e linked o
bo h Ap il and May. Fo es species gained p ominence in June, signi ican ly consuming lepidop e ans
(Coleopho a sp.) and dip e ans (Zai a cine ea). Finally, in July, he p ima y p ey g oup consis ed o
mo hs om sh ubland (Gymnoscelis u i ascia a), al hough mo hs om bo h o es and g assland we e
also consumed in la ge quan i ies. G assland lepidop e ans we e loca ed in he igh pa o he plo
because hey we e consumed h oughou he season, excep in May, when Geo upes we e he mos
consumed p ey (see Figu e 4.4d).
Fo es s we e an impo an p ey-sou ce-habi a h oughou he yea , bu did no ha e he same
signi icance in he di e en colonies and h oughou he en i e season (Figu e 4.5). They we e he
mos used p ey-sou ce-habi a in he empe a e clima e colony (BA), wi h a mean alue o 45% o he
die . In he Medi e anean colonies, (CA and UX) o es s we e ela i ely less signi ican , and
g asslands and sh ub g asslands gained impo ance.
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Figu e 4.6. Mosaic plo s o a ailable habi a ypes e sus hei impo ance as p ey sou ce habi a s o he mos
consumed p ey species in Rhinolophus e umequinum die : colonies in (a) BA, Balmaseda (b) CA, Caseda (c)
UX, Uxue. The habi a ca ego ies a e a anged in descending o de acco ding o hei use. The size o he
squa es ep esen s he combined pe cen age o ha ca ego y in he da ase , and colou s show he alues o he
s anda d esiduals. Blueish colou s ep esen a posi i e selec ion and eddish nega i e selec ion. The as e isks
(*) show he habi a s ha ha e been posi i ely selec ed wi h signi ican Pea son’s co ela ion coe icien (p >
0.05). Boxes wi h an “X” ep esen he absence o such habi a .
In BA, 45.2% o he co e die came om woodlands, ollowed by g asslands (18.5%) and sh ub
g asslands (16.4%) (Figu es 4.5a, 4.6a). As p ey-sou ce-habi a , woodlands we e unde used ela i e o
a ailabili y, while g asslands and sh ub g asslands we e used p opo ionally (Figu e 4.6a). Ripa ian
o es s, ins ead, we e impo an in July and Augus .
In he CA colony, sh ub g asslands we e he mos used co e p ey-sou ce-habi a (26.4%), ollowed by
o es s (23.7%) and g asslands (22.1%) (Figu es 4.5b, 4.6b). Fo es s o e 1 km away we e a ou ed,
and g asslands we e also posi i ely selec ed (Figu e 4.6b). Ripa ian o es s we e posi i ely selec ed
and we e c ucial in Ap il-May (Figu e 4.5b). Finally, c oplands gained impo ance in July-Augus . In
con as , ui ees we e mo e used in Sep embe , e en i hey we e o e all unde used (Figu e 4.5b).
Finally, in UX, g asslands (27.8.5%) and o es s (25.9%) we e he main p ey sou ce habi a s, wi h
hei impo ance a ying by mon h. Bo h sh ub g asslands and ipa ian o es s accoun ed o 19.9% o
usage (Figu es 4.5c and 4.6c). Sh ub g asslands peaked in July wi h 43.3% o consump ion, while
ipa ian o es s we e c ucial in sp ing. O cha ds we e used acco ding o a ailabili y, pa icula ly in
Ma ch, May, and June (Figu es 4.5c, 4.6c). G asslands and o es s we e posi i ely selec ed, while
ipa ian o es s we e a ou ed wi hin 1-3 km (Figu e 4.6c). P ey species linked o sh ub g asslands
we e used less han a ailable, and i is no ewo hy o men ion ha axa om c oplands we e s ongly
unde used.
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4.4. DISCUSSION
Ou s udy, which del es in o wo o he h ee c i ical aspec s o ba conse a ion iden i ied by Fen on
(1997) and Pie son (1998) — he p ey base and he in e ence o hei o aging equi emen s— has
signi ican ly ad anced ou unde s anding o he g ea e ho seshoe ba s' undamen al ophic niche. On
he i s subjec , we ha e un eiled subs an ial di e ences in he species’ die and main
p ey-sou ce-habi a s in empe a e and Medi e anean egions and seasonally, in oducing a weal h o
new knowledge o he ield o ba ' ecology.
Ou comp ehensi e s udy, in conjunc ion wi h Tou nay e’s esea ch (2021), indica es ha
Rhinolophus e umequinum has a ema kably di e se die . Ou sampling pe iod is he mos ex ensi e
o da e, encompassing h ee dis inc colonies in as ly di e en clima es and wi h a ied landscapes.
Fu he mo e, he No aSeq sequencing me hod we employed has a highe sequencing dep h han any
p e iously used, enabling us o iden i y app oxima ely one housand p ey axa down o he species o
genus le els.
Ou ini ial hypo hesis was suppo ed by he esul s, indica ing ha p ey a ailabili y is a c ucial ac o
in luencing he composi ion and spa io- empo al a ia ions o he R. e umequinum die (Tou nay e
e al., 2021), as well as he ela i e impo ance o di e en p ey g oups. O e all, he consump ion o
Lepidop e a, he p ima y o de o p ey, emained s able. Howe e , i exhibi ed a ia ions linked o he
consump ion o o he p ey g oups, such as Dip e a, Coleop e a, and Hemip e a. Ins ead, he sou ce
habi a o hese p ey g oups was ound o a y h oughou he season.
E en i he co e die s p ima ily consis ed o Lepidop e a, Dip e a and Coleop e a (Jones, 1990;
Flande s & Jones, 2009; Tou nay e e al., 2021), o he o de s, such as T ichop e a, we e signi ican
seasonally, pa icula ly in sp ing. The g ea e ho seshoe ba is known o hun ing bee les (Jones, 1990;
Ransome, 1996, 2002), bu hei consump ion a ies by colony in species, iming, and habi a . In UX,
Geo upes sp. was he mos consumed species in May, wi h bee les being less ele an in o he
mon hs. In CA, while bee les we e consumed h oughou he season, hey we e no he main p ey. In
BA, bee les cons i u ed a signi ican pa o he die om May o Augus , wi h p ey species changing
seasonally and being linked o o es habi a s a he han g azed pas u es.
Ou esul s sugges ha he species' abili y o selec p ey, a signi ican ai in ba ecology (Koselj e
al., 2011), is lexible, depending on he habi a and season. These indings a e consis en wi h a
gene al end obse ed in insec i o ous ba s, highligh ing hei adap able beha iou in exploi ing
ho spo s and insec ou b eaks (Aizpu ua e al., 2013; Lino e al., 2014; O’Rou ke e al., 2022; Aiha za
e al., 2023; And eas e al., 2023; Vallejo e al., 2023).
On he second ma e , ega ding he o aging habi a equi emen s o he g ea e ho seshoe ba s, i is
wo h men ioning ha ou landscape in e ence analysis unde lines he ela i e impo ance o hei
main p ey-sou ce-habi a s. This e alua ion, while no de e mining whe e such main p ey ha e been
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