227
Mapping he no he nmos ansna ional non-na i e popula ion o
Xenopus lae is using pooled eDNA sampling
Teun E e s1,2* , Loïc an Doo n3* , Tim Ad iaens4, Sab ina Ney inck1, Ke in Ke ckho s1,
Je oen Speyb oeck3, Rein B ys1
1 Resea ch Ins i u e o Na u e and Fo es (INBO), Gene ic Di e si y, Ge aa dsbe gen, Belgium
2 KU Leu en, Depa men o Biology, Plan Conse a ion and Popula ion Biology, He e lee, Belgium
3 Resea ch Ins i u e o Na u e and Fo es (INBO), Moni o ing and Res o a ion o Aqua ic Fauna, Linkebeek, Belgium
4 Resea ch Ins i u e o Na u e and Fo es (INBO), Wildli e Managemen and In asi e Species, B ussels, Belgium
Co esponding au ho : Teun E e s ([email p o ec ed])
Copy igh : © Teun E e s e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
Ea ly-de ec ion- apid esponse (EDRR) p og ams a e in aluable o managing incipien in asions,
bu equi e moni o ing s a egies ha maximize sea ch co e age and minimize cos s. While en i-
onmen al DNA (eDNA)-based moni o ing echniques a e now widely used o moni o ing aqua ic
in asions, he la ge moni o ing e o s in EDRR p og ams demand u he imp o emen s in sam-
pling s a egies. This need is exempli ied by he ecen ly disco e ed A ican clawed og Xenopus lae is
(Daudin, 1802) in asion along he F ench-Belgian bo de egion – i s no he nmos in oduced
popula ion – whe e i was i s de ec ed in F ance in 2018 nea he Belgian bo de . In esponse o
he isk o i s pe manen es ablishmen and c oss-bounda y sp ead, we employed quan i a i e eDNA
ba coding analyses in he con ex o an EDRR p og am. We (i) de eloped and alida ed a no el
cos -e icien sampling s a egy ha in ol es pooling wa e om mul iple si es allowing o b oade
a ea co e age, (ii) delinea ed he spa ial ex en o he in asion, and (iii) assessed whe he lo ic sys ems
se e as c oss-bounda y dispe sal co ido s. In 2020, 2022, and 2023, we de e mined he p esence
and eDNA concen a ions o X. lae is om a o al o 426 si es by pooling wa e om 366 si es in 83
samples, and sampling 59 addi ional si es indi idually. We ound ha his pooled app oach can ac-
cu a ely de e mine X lae is p esence and app oxima e i s a e age eDNA concen a ion ac oss pooled
si es. We de ec ed X. lae is in 26 samples, e ealing an in e connec ed popula ion spanning an a ea
o 103 km2, con adic ing p io assump ions o an ea ly in asion s age. We p o ide e idence ha he
i e Lys and he Dou ebeek acili a e c oss-bounda y mo emen . The pooled sampling echnique
p esen ed he e is a cos -e ec i e me hod o p o iding imely, ac ionable da a o in o m managemen
decisions; howe e , u he alida ion is needed o con i m i s eliabili y and b oade applicabili y.
The X. lae is in asion along he F ench-Belgian bo de unde sco es he complexi ies o managing
biological in asions ac oss na ional and adminis a i e bounda ies, p o iding aluable insigh s o
o he ansbounda y in asions.
Key wo ds: A ican clawed og, biological in asions, dispe sal co ido , d ople digi al PCR
(ddPCR), ea ly-de ec ion- apid- esponse (EDRR), en i onmen al DNA (eDNA), non-na i e in a-
si e species, apidly expanding dis ibu ion
Academic edi o : Filipe Ribei o
Recei ed:
14 Feb ua y 2025
Accep ed:
12 Augus 2025
Published:
7 Oc obe 2025
Ci a ion: E e s T, an Doo n L,
Ad iaens T, Ney inck S, Ke ckho s K,
Speyb oeck J, B ys R (2025) Mapping
he no he nmos ansna ional
non-na i e popula ion o Xenopus
lae is using pooled eDNA sampling.
In: Anas ácio P, Ribei o F, Chainho P
(Eds) In asions in Aqua ic Sys ems.
NeoBio a 102: 227–248. h ps://doi.
o g/10.3897/neobio a.102.150311
NeoBio a 102: 227–248 (2025)
DOI: 10.3897/neobio a.102.150311
* Con ibu ed equally as he i s au ho .
Ad ancing esea ch on alien species and biological in asions
A pee - e iewed open-access jou nal
NeoBio a
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
In oduc ion
F eshwa e ecosys ems ha bou en pe cen o all species and one- hi d o all e e-
b a e species while co e ing less han one pe cen o he Ea h’s su ace (Dudgeon
e al. 2006). Howe e , eshwa e biodi e si y is declining a an ala ming a e (Say-
e e al. 2025), wi h he ongoing es ablishmen and sp ead o non-na i e in asi e
species (NIS) iden i ied as a signi ican con ibu o . These biological in asions can
esul in species ex inc ions, bio ic homogeniza ion, and dis up ions o ecosys em
unc ioning (Ka sane akis e al. 2014; Blackbu n e al. 2019; an Rees e al. 2021;
And és e al. 2023). Despi e ongoing managemen e o s, he in lux o NIS is ex-
pec ed o con inue (Seebens e al. 2017). To coun e newly incoming NIS, es ab-
lishing ea ly-de ec ion- apid- esponse (EDRR) sys ems is essen ial (Hulme 2006;
Vande Zanden and Olden 2008). To be e ec i e, EDRR sys ems mus employ
cos -e icien moni o ing me hods capable o de ec ing species a low densi ies and
ha ing he po en ial o ope a e e ec i ely ac oss ex ensi e spa ial scales (Mye s e
al. 2000; Meh a e al. 2007; G eenhalgh e al. 2022).
In ecen yea s, a g owing body o e idence has demons a ed he e ec i eness o
en i onmen al DNA (eDNA)-based analyses o he ea ly de ec ion o aqua ic NIS,
unde sco ing hei alue in EDRR sys ems (T ebi z e al. 2017; E e s e al. 2023).
Typically s emming om skin cells, mucus, game es, and o he biological ma e ials,
eDNA ep esen s he en i e y o DNA di ec ly ex ac able om en i onmen al sam-
ples, including wa e , soil, and ai , elimina ing he necessi y o obse e o cap u e
he o ganism i sel (Fice ola e al. 2008). eDNA-based de ec ion me hods a e gen-
e ally mo e cos -e icien and sensi i e compa ed o con en ional moni o ing me h-
ods – such as ne ing, acous ic moni o ing, o isual su eys – pa icula ly when
su eying inaccessible habi a s o a ge ing c yp ic species (Fediaje ai e e al. 2021;
Keck e al. 2022; Moss e al. 2022). Beyond species de ec ion, eDNA-based analyses
can, in ce ain ci cums ances, also p o ide app oxima e es ima es o species densi-
ies (Ya es e al. 2019; Rou ke e al. 2021; Ca alho e al. 2022; E e s e al. 2022).
Howe e , he accu acy o eDNA-based analyses can a y signi ican ly depending
on ac o s such as he speci ic ecosys em and a ge species (Fediaje ai e e al. 2021;
Keck e al. 2022). In e p e ing eDNA da a in open sys ems, such as ma ine o i e -
ine en i onmen s, can be mo e complex han in closed en i onmen s such as ponds
o small lakes due o in ica e e ec s o admix u e, di usion, and mixing on he
dis ibu ion o eDNA pa icles (Rou ke e al. 2021; Hinz e al. 2022). E en wi hin
a gi en sys em, he pe o mance o eDNA-based moni o ing can be a ec ed by
species-speci ic ac o s, including eDNA shedding a e, species densi y, phenology,
and mobili y (Adams e al. 2019; E e s e al. 2021; Thalinge e al. 2021). O e all,
eDNA-based me hods a e expec ed o p o ide he g ea es alue in de ec ing ea ly
in asion s ages o ish and amphibian species in ponds o small s eams (Secondi e
al. 2016; Fediaje ai e e al. 2021; E e s e al. 2022; Keck e al. 2022).
The A ican clawed og, Xenopus lae is (Daudin, 1802), se es as a pe inen
example o a NIS o which an eDNA-based EDRR sys em may p o e highly
e ec i e (Measey e al. 2012; Secondi e al. 2016; Vime ca i e al. 2019). Na i e
o Sub-Saha an A ica, X. lae is has been used as a model o ganism in biological
esea ch and p egnancy es ing o decades (Gu don and Hopwood 2000; Wall-
ing o d e al. 2010). I has also become a luc a i e commodi y in he pe ade
(Measey 2016), leading o i s global expo a ion in he 20 h cen u y and o he
success ul es ablishmen o popula ions in a ious egions ac oss No h-Ame ica,
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
Sou h-Ame ica, Asia, and Eu ope. Gi en i s aqua ic li es yle, e ec i e camou lage,
and unde wa e ma ing calls (Tinsley and Kobel 1996), in oduc ions o X. lae-
is o en emain unno iced o ex ended pe iods (Measey e al. 2012). The spe-
cies p ima ily b eeds in ponds and dispe ses ia s eams and i e s (Mo ei a e al.
2017), wi h pe iodic o e land mo emen ha ypically occu s du ing we pe iods
and hea y ain all (Measey e al. 2012; Measey 2016). Xenopus lae is has a la gely
aqua ic ecology, can adap o a wide ange o en i onmen s, is highly e ile, and
can dispe se up o 5.4 kilome es pe yea (Pagano e al. 2024). I can exe a p o-
ound impac on na i e aqua ic auna due o p eda ion, compe i ion, and disease
ansmission (Weldon e al. 2004; Lillo e al. 2011; Cou an e al. 2018). The c yp-
ic and in asi e na u e o X. lae is makes i a highly sui able a ge o eDNA-based
moni o ing sys ems, which o e a sensi i e and cos -e ec i e app oach o gen-
e a ing c i ical in o ma ion o suppo he managemen o biological in asions
(Secondi e al. 2016; B ys e al. 2021; E e s e al. 2022, 2023, 2024).
In Eu ope, es ablished X. lae is popula ions can be ound in Po ugal, I aly, and
F ance (Measey e al. 2012). In F ance, X. lae is was i s eco ded in he wes e n
depa men Deux-Sè es in he 1980s (Fouque and Measey 2006). Since hen, he
species has sp ead a an es ima ed a e o 1.2 km pe yea , eaching an in aded a ea
o 8,400 km2 by 2024 (Pagano e al. 2024). Fu he no h, a new popula ion was
disco e ed in 2018 in he A men iè es can on (No d depa men , F ance), jus a ew
kilome es om he Belgian bo de ( an Doo n e al. 2022). By 2021, he p esence
o X. lae is had been con i med in wo adjacen adminis a i e egions o Belgium
– Flande s and Wallonia (Ad iaens e al. 2023; Pauwels e al. 2023). Howe e , he
spa ial ex en o his new in asion in Belgium, as well as he pa hways acili a ing
c oss-bounda y expansion, emained la gely unknown. To add ess his knowledge
gap, we sampled 426 wa e bodies in he F ench-Belgian bo de egion be ween
2020 and 2023 o quan i a i e eDNA-based ba coding analyses o de e mine he
p esence and associa ed eDNA concen a ions o X. lae is h oughou he a ea. We
de eloped and p elimina ily es ed he e ec i eness o a no el sampling s a egy
ha pools wa e om mul iple si es (Fig. 1), enabling ex ensi e a ea co e age in a
sho ime ame wi h limi ed esou ces, and discuss i s s eng hs and limi a ions.
We mapped he cu en dis ibu ion o X. lae is in he bo de egion as a pa o an
EDRR p og am, and assessed whe he wo wa e ways – he i e Lys and i s ib-
u a y, he Dou ebeek – se e as po en ial dispe sal co ido s (Pauwels e al. 2023).
Gi en ha X. lae is is cu en ly ega ded as being in an ea ly in asion s age whe e
comple e e adica ion migh s ill be easible (Renie s e al. 2023), he insigh s gained
om his s udy could suppo e ec i e ansbounda y managemen s a egies. We
u he add ess he challenges posed by he expansion o NIS ac oss na ional and
adminis a i e bo de s and p opose po en ial solu ions o mi iga e hese issues.
Ma e ial and me hods
S udy a ea
The F ench-Belgian bo de egion is cha ac e ized by a empe a e oceanic clima e
wi h wa m summe s, bu is subs an ially colde compa ed o o he Eu opean a -
eas con aining es ablished non-na i e X. lae is popula ions. He e, as ag icul u al
ields a e in e spe sed wi h small owns (Fig. 2), simila o o he egions in Eu ope
ha a e in aded by X. lae is (Measey e al. 2012). The s udy a ea con ains nume -
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
Figu e 1. Schema ic o e iew o pooling eDNA samples ac oss loca ions unde h ee di e en scena ios. In his hypo he ical example,
h ee ponds we e sampled and il e ed h ough a single il e . a. When X. lae is is absen in all h ee ponds, no X. lae is eDNA is de ec ed
(i.e, no signal). This could also occu when X. lae is is p esen in e y low densi ies (i.e., alse-nega i e); b. When X. lae is is p esen in a
small p opo ion o he sampled ponds, a low amoun o X. lae is eDNA is expec ed o be ampli ied (i.e., weak signal), unless X. lae is
densi y and/o he a io a ge -posi i e o a ge -nega i e ponds a e high; c. When X. lae is is p esen in all h ee sampled ponds, in ei he
low o high densi ies, a la ge amoun o eDNA is expec ed (i.e., s ong signal).
ous ponds, ypically used o ca le o i iga ion, which a e known o be sui able
habi a s o X. lae is (Lobos and Jaksic 2005; Fa aone e al. 2008; Rebelo e al.
2010). Gi en he aqua ic na u e o X. lae is, he i e Lys and he Dou ebeek a e
assumed o se e as dispe sal co ido s be ween F ance and Belgium (Ad iaens e
al. 2023; Pauwels e al. 2023).
The Lys is a pa ially canalized i e , ex ending 202 kilome es in o al leng h.
Wi hin he s udy a ea, he i e anges om 30 o 70 me es in wid h and eaches
a maximum dep h o 4.5 me es (Fig. 2). I o igina es in Pas-de-Calais, F ance,
and lows no heas along he F ench-Belgian bo de be o e d aining in he i e
Scheld in Belgium. The Dou ebeek is a small ibu a y o he i e Lys ha is
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
Figu e 2. Map o he sampled a ea along he F ench-Belgian bo de . F ance is labelled as “FR”, while he wo Belgian Regions, Wallonia
and Flande s, a e labelled “BE(W)” and “BE(F)”, espec i ely. Colou ed ci cles indica e he sampled loca ions pe sampled yea . The i e
Lys and he Dou ebeek a e depic ed as hick blue lines, wi h o he wa e ways ep esen ed by hinne blue lines.
app oxima ely 21 kilome es long, o igina ing along he hillside o he Mon Noi
in F ance. This s eam con inues sou heas in o Flande s, whe e i lows along he
Flemish-Walloon bo de , d aining in o he i e Lys in he Walloon municipali y
o Comines-Wa ne on. The Dou ebeek can be cha ac e ized as a u bid c eek wi h
discha ge le els ha s ongly luc ua e in esponse o p ecipi a ion e en s. The
sou ce a ea is conside ed ecologically aluable wi h se e al adjacen na u al a -
eas ha bou ing high biodi e si y including la ge pondscapes wi h impo an elic
popula ions o na i e amphibians, such as ou local new species, including he
p o ec ed g ea c es ed new T i u us c is a us (Lau en i, 1768), which a e ul-
ne able o X. lae is in asion (Lillo e al. 2011; Cou an e al. 2018). The middle
and lowe eaches o he s eam, howe e , ha e poo wa e quali y due o e osion,
was ewa e discha ge, and nu ien in lows.
Sampling
Bo h len ic (i.e., ponds) and lo ic (i.e., di ches, s eams, canals, and i e s) sys ems
we e sampled o eDNA in Ap il 2020 (86 si es), Augus 2022 (211 si es), and
Augus 2023 (129 si es). These sampling pe iods coincide wi h he ep oduc i e
season and peak ac i i y o X. lae is, which spans om Ma ch o Oc obe , he eby
inc easing he likelihood o de ec ion (Cou an e al. 2018; E e s e al. 2021).
Sampling loca ions co e ed he F ench-Belgian bo de egion (i.e., in Flande s,
Wallonia, and F ance), mainly ocusing on he i e Lys, he Dou ebeek, and he
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
su ounding a eas (Fig. 2). In 2020, sampling ocused on he wes e n pa o he
egion, based on he assump ion o o e land c oss-bo de dispe sal. Resul s om
his ini ial campaign in o med he 2022 sampling s a egy, which shi ed o he
eas e n pa o he s udy a ea and conside ed bo h o e land and lo ic dispe sal
pa hways. In 2023, he sampling design was u he e ined based on insigh s om
p e ious yea s, wi h a ocus on e alua ing he ole o he Dou ebeek and i e Lys
as po en ial co ido s o ansbounda y dispe sal (Fig. 2).
In o al, 303 len ic and 123 lo ic si es we e sampled. To minimize he numbe
o equi ed eDNA analyses while main aining spa ial co e age, we implemen ed a
no el sampling s a egy. This s a egy in ol ed pooling wa e om mul iple sam-
pling loca ions (mean: 4.4, ange: 2–8), and il e ing his pooled wa e h ough a
single eDNA il e (i.e., pooling ac oss loca ions). Adjacen ponds (mean dis ance
o cen oid: 309 m, ange 7–1,333 m), as well as segmen s and ibu a ies o he
Dou ebeek and i e Lys (mean dis ance o cen oid: 632 m, ange 52–2,692 m),
we e he e o e g ouped in o sampling clus e s. Wi hin each clus e , wa e samples
we e me ged in o one in eg a ed wa e sample (Fig. 1). This sampling s a egy was
used in a eas wi h unce ain p io knowledge o X. lae is p esence, and allowed us
o assess i s occu ence in 366 si es using only 83 eDNA il e s. An addi ional 59
si es, loca ed in egions whe e X. lae is p esence was conside ed mo e likely (e.g.,
he downs eam po ion o he Dou ebeek, p oximal o wa e ways) we e indi id-
ually sampled, yielding a g and o al o 142 eDNA il e s. To e alua e he pe o -
mance o he pooled sampling app oach, we selec ed wo clus e s, each comp ising
ou ponds, o which he ini ial pooled sample indica ed he p esence o X. lae is
DNA. These ponds we e hen sampled bo h indi idually and again ia pooled
sampling, gene a ing an addi ional en eDNA il e s o analysis.
Uppe wa e laye s we e sampled o eDNA om he sho e, using a s e ile sam-
pling pole wi h a 0.5 L s e ile bag a ached a he end. As eDNA pa icles a e he -
e ogeneously dis ibu ed in len ic sys ems, de ec ion sensi i i y was inc eased by
implemen ing a me ged sampling (i.e., pooling o wa e wi hin a loca ion), as de-
sc ibed by B ys e al. (2021). As such, wa e was collec ed om all si es a ibu ed o
a single sampling clus e , and immedia ely il e ed in he ield o e a single disc il e
con aining a 0.8 µM PES memb ane combined wi h a 5 µm glass ib e p e il e
(Na u eMe ics, Su ey, England). Wa e was il e ed un il clogging, and he o al
olume o il e ed wa e was eco ded. Fil e s we e hen sealed o a bo h ends and
immedia ely placed in a eeze box. A he end o he day, he il e s we e ans e ed
o s o age a -21 °C, whe e hey emained un il molecula analyses we e conduc ed.
All eusable sampling ma e ial (i.e., sampling pole and bucke ) was disin ec ed wi h
a 2% Vi kon S solu ion (An ec DuPon , Su olk, UK) o minimize he isk o DNA
c oss-con amina ion. To es o po en ial con amina ion du ing ield sampling, i e
nega i e con ols we e included by il e ing 2 L o mine al wa e in he ield (i.e.,
ield blanks): one a he end o he 2020 sampling campaign and one a he end o
each o he wo sampling ounds in bo h he 2022 and 2023 sampling campaigns.
Molecula analyses
All eDNA samples we e s o ed and p ocessed in a PCR- ee building dedica ed o
low copy numbe empla e ex ac ions. This acili y hos s DNA- ee high-e icien-
cy pa icula e ai (HEPA)- il e ed compa men s wi h posi i e p essu e o p e en
eDNA sample con amina ion. To es o PCR inhibi ion and o e alua e he ex-
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
ac ion e iciency o each il e , an in e nal posi i e con ol (IPC) was added in he
i s s ep o he ex ac ion oge he wi h he lysis bu e ( o de ails on he IPC see
E e s e al. 2021). DNA ex ac ion was pe o med using Qiagen’s DNeasy Blood
& Tissue Ki and pu i ied wi h he DNeasy Powe Clean Cleanup Ki (Qiagen)
acco ding o he guidelines p o ided by he manu ac u e . To de e mine X. lae is
p esence and es ima e i s abundance, an 83-bp ba code loca ed on he 12S egion
o he ob ained DNA ex ac s was analysed and quan i ied ia d ople digi al PCR
(ddPCR) using he p ime /p obe assay de eloped by Secondi e al. (2016). E en
hough his assay canno disc imina e be ween 12 (sub)species o Xenopus, i is ex-
pec ed ha only X. lae is cu en ly occu s in he sampled a ea (Pauwels e al. 2023),
and hus a posi i e de ec ion indica es he p esence o X. lae is. Fo each eDNA
sample, an a e age o 3 ( ange: 1–6) eplica e ddPCR eac ions we e conduc ed.
The unequal numbe o eplica es ac oss samples e lec s he adap i e na u e o he
EDRR amewo k in which his s udy was conduc ed, whe e esou ce alloca ion
was adjus ed in subsequen yea s based on ield condi ions and p elimina y ind-
ings. Speci ically, a highe numbe o eplica es was gene ally analysed o samples
collec ed in a eas wi h limi ed p io knowledge o species p esence (e.g., loca ions
inc easingly dis an om he known F ench popula ion in A men iè es), in mo e
inland si es gi en he expec a ion ha X. lae is p ima ily dispe ses ia aqua ic sys-
ems, and in la ge wa e bodies whe e de ec ing a ew indi iduals would equi e
inc eased analy ical e o (e.g., he i e Lys compa ed o small ponds). To es o
po en ial PCR ailu e o con amina ion, each ddPCR pla e included wo posi i e
con ols con aining 10 pg/µL o DNA ex ac ed om X. lae is issue, along wi h
no- empla e con ols (NTCs) ha se ed as PCR blanks by omi ing DNA om
he eac ion. The ad an ages o ddPCR compa ed o con en ional quan i a i e
PCR (qPCR) a e ou old: (i) i elimina es he need o calib a ion cu es h ough
absolu e quan i ica ion, (ii) i s high le el o sample pa i ioning enables highly p e-
cise measu emen s, (iii) i eliably de ec s low concen a ions o a ge DNA e en
in he p esence o abundan non- a ge DNA, and (i ) i exhibi s g ea e esilience
o PCR inhibi o s, making i pa icula ly sui able o complex en i onmen al sam-
ples (Gu i e al. 2024).
Da a analyses
Xenopus lae is was conside ed p esen i success ul PCR ampli ica ion occu ed in
a leas one ddPCR eplica e. Ta ge eDNA concen a ions (in copies/µL pe L
il e ed wa e ), used as a p oxy o species densi y, we e calcula ed ollowing E e s
e al. (2022), and a e aged ac oss eplica es. The esul s o pooled samples (i.e.,
X. lae is p esence/absence and a ge eDNA concen a ion) we e plo ed as he
cen oid o all he si es a ibu ed o a single sampling clus e , ep esen ing a con-
se a i e app oach o isualizing pooled eDNA da a. We es ed he e ec i eness o
pooled eDNA sampling o wo clus e s each con aining ou ponds. Speci ically,
we compa ed (i) he p esence/absence o X. lae is in each indi idually sampled
wa e body wi h i s de ec ion in he pooled sample, and (ii) whe he he a e age
a ge eDNA concen a ions om indi idually sampled ponds co esponded o
he a ge eDNA concen a ion in he pooled sample, unc ioning as measu es o
simila i y ac oss sampling me hods.
To quan i y he spa ial dis ibu ion o X. lae is in he sampled a ea, we cal-
cula ed a bu e -based app oxima ion o he po en ially occupied ange. This
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
app oach inco po a es he loca ions o posi i e de ec ions and he known dis-
pe sal capaci y o he species o delinea e a eas whe e X. lae is may plausibly be
p esen . Al hough he dispe sal capaci y o X. lae is a ies widely (Pagano e al.
2024), we adop ed a easonable es ima e o 2 km pe yea . This is conside ably
lowe han he maximum p edic ed dispe sal speed o 5.4 km in Chile, ye
sligh ly highe han he 1.2 km pe yea epo ed in Sou he n F ance (Pagano
e al. 2024), ensu ing ha he po en ial in aded a ea is no unde es ima ed.
Bu e s wi h a 2 km adius we e cons uc ed a ound each a ge -posi i e eDNA
de ec ion (i.e., ei he he cen oid o pooled samples o he exac loca ions o
indi idual samples). O e lapping bu e s we e me ged and he o al su ace a ea
was calcula ed. The numbe o esul ing polygons can be in o ma i e on he
his o ical ajec o y o he in asion, including he occu ence and numbe o
in oduc ions (E e s e al. 2023).
In o de o acili a e he isualiza ion o he me ged eDNA sampling, we gen-
e a ed hea maps using he ke nel densi y es ima ion ool in QGIS 3.34.7. The
ob ained ddPCR da a om each o he ponds and s eams we e analysed collec-
i ely o ob ain an o e all unde s anding o he in asion in he s udied a ea. Gi en
he highly localized na u e o eDNA signals in small s eams (Van D iessche e al.
2022), we expec he esul s om lo ic sys ems o p ima ily e lec local condi ions,
a he han an accumula ion o ups eam signals (c . Deine e al. 2016). Weigh
was gi en o ei he he p esence (0 when X. lae is was no de ec ed o 1 when
X. lae is was de ec ed) o he log- ans o med a ge eDNA concen a ions in
wo sepa a e hea maps. Bo h hea maps we e gene a ed using a 2 km linea decay,
meaning ha he in luence o any gi en poin on he hea map alue diminishes
linea ly o ze o a a dis ance o 2 km. O he se ings we e le a de aul . Whe eas
his app oach does no accoun o une en sampling densi y, weigh ing by species
p esence o a ge eDNA concen a ion allows he gene a ed hea maps o p o ide
an app oxima e quali a i e (in e pola ion o X. lae is p esence) and quan i a i e
(i.e., in e pola ion o X. lae is eDNA concen a ions) o e iew o he in aded a ea.
Howe e , in cases whe e sampling si es a e excessi ely spa ially clus e ed, we ec-
ommend employing mo e ad anced in e pola ion me hods ha accoun o he
une en densi y o sampling loca ions, such as k iging o spa ially explici gene al-
ized linea mixed models (GLMMs).
Resul s
The gene a ed ddPCR da a exhibi ed high quali y, wi h no DNA de ec ed in ield
o PCR blanks, consis en eco e y o expec ed a ge DNA concen a ions om
posi i e con ols, and success ul IPC ampli ica ion con i ming he absence o PCR
inhibi ion. Consequen ly, he likelihood o alse posi i e de ec ions due o labo-
a o y o ield con amina ion, as well as alse nega i e de ec ions esul ing om
ex ac ion and PCR ailu e was conside ed unlikely.
Indi idual sampling o each o he ou ponds wi hin he wo pond clus e s
ha we e selec ed o he pooled sampling alida ion and es ed posi i e on X.
lae is p esence, e ealed ha X. lae is was p esen in only one pond pe clus e
(Fig. 3). Despi e a subs an ially lowe a ge eDNA concen a ion o ha pond in
one clus e compa ed o he o he (0.18 s 4.20 copies/µL pe L il e ed wa e ), X.
lae is eDNA could clea ly be de ec ed in he pooled sample o bo h clus e s. Ta -
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
Figu e 3. The X. lae is in asion along he F ench-Belgian bo de . Samples o s eams and ponds a e ep esen ed by iangles and squa es,
espec i ely. Fo pooled samples, he cen oid o he sampled loca ions is mapped. Xenopus lae is de ec ion is indica ed in ed, while i s
non-de ec ion is shown in g ey. The g ey shading ep esen s he bu e -based occupied a ea and was cons uc ed wi h a bu e o 2 km
a ound he a ge -posi i e loca ions. Inse s display wo clus e s whe e he pooled sampling me hodology was empi ically es ed. Whi e
and ed do s ep esen ponds whe e he indi idual sample was nega i e and posi i e, espec i ely. The ed-colou ed il e ep esen s a
a ge -posi i e pooled sample. The able shows X. lae is eDNA concen a ions (exp essed in copies/µL), he a e age concen a ion ac oss
indi idually sampled ponds, he concen a ion o he pooled sample, and he simila i y be ween he la e wo.
ge eDNA concen a ion o he pooled sample in he highe -concen a ion clus e
(1.02 copies/µL) closely ma ched he a e age concen a ion o he indi idual sam-
ples (1.05 copies/µL; 97.5% simila i y). This simila i y was less p onounced in he
lowe -concen a ion clus e (pooled sample: 0.07 copies/µL; a e age o indi idual
samples: 0.045 copies/µL; 64.3% simila i y).
Xenopus lae is DNA was de ec ed in 26 o he 142 collec ed samples (12 lo ic
and 14 len ic; 11 indi idual and 15 pooled samples; 0 in 2020, 3 in 2022, and 23
in 2023), esul ing in a o al occupied a ea o 103 km2 wi hin he s udy a ea (Fig. 3;
Suppl. ma e ial 1). The spa ial p oximi y o posi i e X. lae is de ec ions esul ed in
a single, con inuous polygon, sugges ing he p esence o one in e connec ed popu-
la ion. All posi i e de ec ions occu ed wi hin one kilome e o ei he he i e Lys
o he Dou ebeek. The dis ibu ion o X. lae is ex ends om he headwa e s o he
Dou ebeek o i s con luence wi h he i e Lys, and ups eam along he i e Lys
and a small ibu a y (Cou an du Pon Be in) nea A men iè es, encompassing
bo h F ance and he Walloon and Flemish Regions o Belgium. The equency o
posi i e de ec ions and X. lae is eDNA concen a ions we e highe in he Dou e-
beek han in he i e Lys (Fig. 4). Posi i e de ec ions we e ela i ely e enly dis-
ibu ed wi hin he Dou ebeek, whe eas he majo i y o samples exhibi ing high
a ge eDNA concen a ions we e loca ed mo e downs eam nea i s con luence
wi h he i e Lys. Nine si es (6 len ic, 3 lo ic; 3 sampled in 2022, 6 sampled in
2023) exhibi ed a ge eDNA concen a ions simila o o exceeding hose a he
h ee known b eeding si es epo ed in Ad iaens e al. (2023); eigh we e nea he
Lys-Dou ebeek con luence, and one len ic si e was nea he sou ce o he Dou e-
beek (Suppl. ma e ial 1).
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Teun E e s e al.: Pooled eDNA sampling o map he no henmos Xenopus lae is in asion
Use o AI
No use o AI was epo ed.
Funding
Publica ion o his s udy was pa ly suppo ed by Resea ch Founda ion Flande s (1S01822N) and he
Eu opean Union’s Ho izon Eu ope HORIZON-CL6-2024-BIODIV-01 p ojec “Gua dIAS – Gua d-
ing Eu opean Wa e s om IAS”, unde g an ag eemen no. 101181413 (Ka sane akis e al. 2024).
Au ho con ibu ions
Concep ualiza ion: RB, TE. Da a cu a ion: KK, TE. Fo mal analysis: KK, TE. Funding acquisi ion:
TE, LD, RB, TA. In es iga ion: LD, TE. Me hodology: TE, RB, SN. P ojec adminis a ion: RB,
TA. Supe ision: RB. Valida ion: SN, RB, KK, LD, TE. Visualiza ion: TE, KK. W i ing – o iginal
d a : TA, TE, LD. W i ing – e iew and edi ing: SN, RB, JS.
Au ho ORCIDs
Teun E e s h ps://o cid.o g/0000-0001-7862-4209
Loïc an Doo n h ps://o cid.o g/0000-0003-3497-9168
Tim Ad iaens h ps://o cid.o g/0000-0001-7268-4200
Sab ina Ney inck h ps://o cid.o g/0000-0002-7379-9269
Je oen Speyb oeck h ps://o cid.o g/0000-0002-7241-7804
Rein B ys h ps://o cid.o g/0000-0002-0688-3268
Da a a ailabili y
All o he da a ha suppo he indings o his s udy a e a ailable in he main ex o Supplemen a y
In o ma ion.
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