Biodi e si y Da a Jou nal 12: e117169
doi: 10.3897/BDJ.12.e117169
Da a Pape
The InBIO Ba coding Ini ia i e Da abase: DNA
ba codes o Po uguese mo hs
Sónia Fe ei a , Ma in F. V. Co ley , João Nunes , Jo ge Rose e , Sasha Vasconcelos , Vanessa
A. Ma a , Joana Ve íssimo , Te esa L Sil a , Ped o Sousa , Rui And ade, José Manuel
G osso-Sil a , Ca a ina J. Pinho , Cá ia Cha es , Filipa MS Ma ins , Joana Pin o , Pamela
Puppo , An onio Muñoz-Mé ida , John A che , Joana Paupe io , Ped o Beja
‡ CIBIO, Cen o de In es igação em Biodi e sidade e Recu sos Gené icos, InBIO Labo a ó io Associado, Campus de Vai ao,
Uni e sidade do Po o, 4485-661 Vai ao, Vila do Conde, Po ugal
§ BIOPOLIS P og am in Genomics, Biodi e si y and Land Planning, CIBIO, Campus de Vai ao, 4485-661 Vai ao, Vila do
Conde, Po ugal
| EBM, Es ação Biológica de Mé ola, P aça Luís de Camões, Mé ola, Po ugal
¶ Pucke y Fa m Co age, Fa ingdon, Ox o dshi e SN7 8JP, U.K, Ox o dshi e, Uni ed Kingdom
# Rua Edua do Joaquim Reis Figuei a, 1104 RC AR, 4440-647, Valongo, Po ugal
¤ U banização Lou isol, Rua Manuel Ce quei a Nob ega, Lo e 16, 2. en e, P-3105-165 Lou içal, Pombal, Po ugal
« CIBIO, Cen o de In es igação em Biodi e sidade e Recu sos Gené icos, InBIO Labo a ó io Associado, Ins i u o Supe io
de Ag onomia, Uni e sidade de Lisboa, Lisboa, Po ugal
» Depa men o Ecology, Swedish Uni e si y o Ag icul u al Sciences, PO Box 7044, 750 07, Uppsala, Sweden
˄ Depa amen o de Biologia, Faculdade de Ciências, Uni e sidade do Po o, 4169-007, Po o, Po ugal
˅ In e na ional Union o Conse a ion o Na u e (IUCN), Species Su i al Commission (SSC), Spide and Sco pion Specialis
G oup, Gland, Swi ze land
¦ Rua Calous e Gulbenkian 237 4H3, 4050-145, Po o, Po ugal
ˀ Museu de His ó ia Na u al e da Ciência da Uni e sidade do Po o, Po o, Po ugal
ˁ Ma shall Uni e si y, Depa men o Biological Sciences, Hun ing on, Wes Vi ginia, Uni ed S a es o Ame ica
₵ CIBIO, Cen o de In es igação em Biodi e sidade e Recu sos Gené icos, InBIO Labo a ó io Associado, Campus de Vai ão,
Uni e sidade do Po o,, 4485–661 Vai ão, Vila do Conde, Po ugal
ℓ Eu opean Molecula Biology Labo a o y, Eu opean Bioin o ma ics Ins i u e, Hinx on, Camb idge, Uni ed Kingdom
Co esponding au ho : Sónia Fe ei a ([email p o ec ed])
Academic edi o : Richa d Mally
Recei ed: 11 Dec 2023 | Accep ed: 19 Feb 2024 | Published: 16 May 2024
Ci a ion: Fe ei a S, Co ley MFV, Nunes J, Rose e J, Vasconcelos S, Ma a VA, Ve íssimo J, Sil a T, Sousa P,
And ade R, G osso-Sil a JM, Pinho CJ, Cha es C, Ma ins FM, Pin o J, Puppo P, Muñoz-Mé ida A, A che J,
Paupe io J, Beja P (2024) The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs.
Biodi e si y Da a Jou nal 12: e117169. h ps://doi.o g/10.3897/BDJ.12.e117169
‡,§,| ¶ # ¤ «,»
‡,§ ‡,§,˄‡,§ ‡,˅¦
ˀ‡,§,˄‡,§ ‡,§ ‡,§
ˁ‡,§ ‡,§ ₵,ℓ‡,§,«
© Fe ei a S e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY
4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e
c edi ed.
Abs ac
Backg ound
The InBIO Ba coding Ini ia i e (IBI) Da ase - DS-IBILP08 con ains eco ds o 2350
specimens o mo hs (Lepidop e a species ha do no belong o he supe amily
Papilionoidea). All specimens ha e been mo phologically iden i ied o species o
subspecies le el and ep esen 1158 species in o al. The species o his da ase
co espond o abou 42% o mainland Po uguese Lepidop e a species. All specimens
we e collec ed in mainland Po ugal be ween 2001 and 2022. All DNA ex ac s and o e
96% o he specimens a e deposi ed in he IBI collec ion a CIBIO, Resea ch Cen e in
Biodi e si y and Gene ic Resou ces.
New in o ma ion
The au ho s enabled "The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o
Po uguese mo hs" in o de o elease he majo i y o da a o DNA ba codes o Po uguese
mo hs wi hin he InBIO Ba coding Ini ia i e. This da ase inc eases he knowledge on he
DNA ba codes o 1158 species om Po ugal belonging o 51 amilies. The e is an
inc ease in DNA ba codes o 205% in Po uguese specimens publicly a ailable. The
da ase includes 61 new Ba code Index Numbe s. All specimens ha e hei DNA ba codes
publicly accessible h ough BOLD online da abase and he dis ibu ion da a can be
accessed h ough he Global Biodi e si y In o ma ion Facili y (GBIF).
Keywo ds
Lepidop e a, occu ence eco ds, species dis ibu ions, con inen al Po ugal, DNA ba code,
cy och ome c oxidase subuni I (COI)
In oduc ion
The Po uguese auna o Lepidop e a is ela i ely ich wi h 2775 species eco ded so a
(Co ley e al. 2023). O hese, o e 85 species ha e been desc ibed only in his cen u y,
wi h an inc easing numbe o esea che s con ibu ing o he knowledge o he coun y's
Lepidop e an auna. Du ing his pe iod, 17 newly-endemic species ha e been desc ibed
(Phalac op e ix i schi Hä enschwile , 2003; Coleopho a lusi anica Baldizzone & Co ley,
2004; Sc obipalpa co leyi Hueme & Ka shol , 2010; In u ci inea co leyi Gaedike, 2011;
Dahlica es ela (A nscheid, 2012); Lobesia a zilae T ema e a, 2014; Willibaldiana cula ae
T ema e a, 2014; Denisia pi esi Co ley, 2014; Fila ima alga biella Co ley, 2014; A oga
ea oni Co ley & Goodey, 2014; Ekboa mia minia ia Skou, S üning & Sih onen, 2016;
Ch ysoclis a soniae Co ley, 2017; Megac aspedus occiden ellus Hueme & Ka shol , 2018;
A ibe ina salemae Skou & Sih onen, 2019; Mondeguina a lan icella Co ley & Rose e,
2Fe ei a S e al
2020; He e ogynis cyne is de F eina, Monas e io, Escobés, Hinojosa & Vila, 2020;
Epe menia lusi anica Gaedike, 2022) (Co ley 2015, Skou e al. 2017, Co ley 2017, Hueme
and Ka shol 2018, Mülle e al. 2019, Co ley e al. 2020, De F eina e al. 2021, Gaedike
2022) and o he s a e known o exis (Co ley, unpublished da a). Since 2006, addi ions
ha e been published on a yea ly basis in he se ies "New and in e es ing Po uguese
Lepidop e a eco ds" (e.g. Co ley e al. (2018), Co ley e al. (2019b)). In he las wo
decades, o e 500 species ha e been added and an anno a ed checklis was published in
2015 (Co ley 2015). In 2021, a dis ibu ion da ase wi h o e 50,000 eco ds e e ing o
specimens o 68 amilies, 925 gene a and 2311 species/subspecies has been eleased in
GBIF (Co ley and A onso 2021). Addi ionally, in 2021, a new ci izen-science p ojec c ea ed
he Po uguese Mo h Reco ding Scheme called "Rede de Es ações de Bo bole as
Noc u nas" (REBN). This p ojec p omo es public pa icipa ion in he p oduc ion o aunis ic
da a. I eleased a i s da ase ha consis s o a collec ion o pe sonal obse a ions by he
pa icipan s (Nunes e al. 2023) and he eco ds p oduced will con inue o be eleased a
egula in e als.
In pa allel wi h he e o s o p oduce and make a ailable eliable da a on he axonomy
and cho ology o Po uguese mo hs, he IBI was c ea ed - he InBIO Ba coding Ini ia i e, a
DNA ba coding ini ia i e by he Resea ch Ne wo k in Biodi e si y and E olu iona y Biology
- InBIO as a esul o he pauci y o gene ic da a on Po uguese biodi e si y. The InBIO
Ba coding Ini ia i e (IBI) makes use o High-Th oughpu Sequencing echnologies o
cons uc a e e ence collec ion o mo phologically iden i ied Po uguese specimens and
espec i e DNA ba codes (e.g. Rebelo e al. 2020, Fe ei a e al. 2021, Oli ei a e al. 2021,
Paupe io e al. 2023, Rosa e al. 2023). DNA ba coding o e s a apid, cos -e ec i e
al e na i e ool o bo h he iden i ica ion o desc ibed species and he disco e y o new
ones (Hebe e al. 2003, Sa olainen e al. 2005, Mi chell 2008, ou pape s). In ac , a ew
new species ha e seen hei dis inc ness con i med by DNA ba codes leading o hei
desc ip ion including Ypsolopha hinolophi Co ley, 2019 ha was ound o be pa o he
die o ho seshoe ba s in a s udy using DNA me aba coding (e.g. Ma in and Fe ei a
(2019), Co ley e al. (2019a), Co ley e al. (2020)). DNA ba coding p o ides a powe ul ool
by using a sho agmen o DNA o assign any o ganism o a species in a apid and
au oma ed way (Hebe , Cywinska, Ball and DeWaa d 2003). The numbe o DNA ba code
e e ence sequences a ailable in public da abases has inc eased ema kably and DNA
ba coding has been b oadly adop ed (e.g. Hebe e al. 2003,Mo água e al. 2019,
Aze edo e al. 2020, da Sil a e al. 2020, Fais e al. 2020, Ma a e al. 2020, Vela de‐
Ga céz e al. 2023). Howe e , o many g oups and geog aphical egions, hese da abases
a e s ill e y incomple e, which limi s he gene al applica ion o DNA ba coding in
biodi e si y esea ch (Elb ech e al. 2017). DNA ba codes o Lepidop e a o Ibe ian
Peninsula a e being p og essi elly documen ed in ecen yea s (e.g. O iz e al. 2017, O iz
e al. 2023) and he p esen wo k ep esen s he i s p ojec o gene a e DNA ba codes o
Po uguese mo hs a a aunal le el and, hus, ep esen s a majo s ep in documen ing he
gene ic di e si y o he Po uguese mo h auna.
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs 3
Gene al desc ip ion
Pu pose: This da ase aims o p o ide a con ibu ion o he knowledge on DNA ba codes o
Po uguese mo hs. Such a lib a y should acili a e DNA-based iden i ica ion o species o
bo h adi ional molecula s udies and DNA me aba coding s udies and cons i u e a
aluable esou ce o axonomic and ecological esea ch on Lepidop e a, wi h he ocus on
he Po uguese auna.
Addi ional in o ma ion: Figs 1, 2 illus a e examples o he di e si y o species ha a e
pa o he da ase o dis ibu ion da a and DNA ba codes o Po uguese mo hs. All
sequences o cy och ome c oxidase I (COI) DNA ba codes a e 658 base pai s (bp) long,
excep o one wi h 422 bp (Fig. 3, Suppl. ma e ial 1). Sequences a e dis ibu ed in 1199
Ba code Index Numbe s (BINs), 63 being unique o his da ase . A e age nucleo ide
composi ion o he sequences is 39.5% hymine (T), 15.5% cy osine (C), 30.5% adenine
(A) and 14.6% guanine (G), o a o al GC con en o 30.1% o he COI ba code agmen
analysed. Gene ic p-dis ances anged om 0.00% be ween he pai Pleu o a hono ella and
Pleu o a planella as p e iously ound wi h o he specimens in BIN BOLD:AEC9855; and
18.5% be ween Tinea ino ella and Tinea mu a iella. In aspeci ic gene ic p dis ances
anged om 0.00% o 11.1% in Pleu o a bicos ella g oup. BOLD Sys ems e ie ed BINs o
all specimens in he da ase (Ra nasingham and Hebe 2013). Only 4% o he species wi h
mo e han one specimen (n = 670) had sequences assigned o wo BINs (n = 31). Two
species in ou da ase p esen up o h ee BINs, namely Ephes ia welse iella
(BOLD:ABW1548, BOLD:AEC9178 and BOLD:AEC9179) and Cydia agiglandana (BOLD:
AAC5023, BOLD:ACS3074 and BOLD:AEB8145). Mul iple BINs a e ela i ely equen in
Lepidop e a species wi h highe pe cen ages ound in s udies wi h highe numbe o
specimens pe species and wi h la ge geog aphic scope. Fo example, Hueme e al.
(2020) and Dincă e al. (2021) ound ha 20% o species o Eu opean Gelechiidae and
12.2% species o Eu opean bu e lies exhibi deep spli s, while s udies o mo e limi ed
geog aphical scope ound 6% o species in Ba a ian Geome ids (Hausmann e al. 2011)
o e en no deep spli s in he case o Mal ese islands Vella e al. (2022). The da ase
includes 10 sha ed BINs be ween axa possibly in need o axonomical e ision:
BOLD:AAA7740 (Yponomeu a cagnagella and Yponomeu a e onymella); BOLD:AAA9515
(Chlo oclys a mia a and Chlo oclys a si e a a); BOLD:AAD0839 (Mac o hylacia dig amma
and Mac o hylacia ubi); BOLD:AAB4833 ( Oligia s igilis and Oligia e sicolo );
BOLD:AAD6780 (C yphia algae and C yphia pallida); BOLD:AAF0005 (Lozo aeniodes
cup essana and Lozo aeniodes o mosana); BOLD:ABV4113 (Cleonymia di luens and
Cleonymia y anii); BOLD:ACE8354 (Euxoa o ana ia and Euxoa i ici); BOLD:ACY5987
(Pleu o a andalusica and Pleu o a e icella); BOLD:AEC9855 ( Pleu o a hono ella and
Pleu o a planella).
F om he 2775 species belonging o 77 amilies eco ded om con inen al Po ugal (Co ley
e al. 2023), 235 species belonging o 40 amilies emain wi hou DNA ba codes and 26
amilies ha e no a single DNA ba coded species (Suppl. ma e ial 6). O hese, 103
4Fe ei a S e al
species ha e al eady specimens egis e ed in BOLD SYSTEMS lacking DNA ba codes,
while specimens o he emaining 132 species a e s ill needed.
P ojec desc ip ion
Ti le: The name "The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese
mo hs" e e s o he i s da a elease o DNA ba codes and dis ibu ion da a o Po uguese
mo hs wi hin he InBIO Ba coding Ini ia i e.
a
b
c
d
e
Figu e 1.
Examples o he di e si y o species ha a e pa o he da ase o dis ibu ion da a and DNA
ba codes o Po uguese mo hs. All pho os by João Nunes.
a: Ag iphila e sellus Lede e , 1855, C ambidae - BIN URI BOLD:AEC8773;
b: Hoyosia code i (Obe hü , 1883), Limacodidae - BIN URI BOLD:ADV3396;
c: Symmoca se a a Gozmany, 1985, Au os ichidae - BIN URI BOLD:AEO2252;
d: Paidia ica F eye , 1855, E ebidae - BIN URI BOLD:AEC7499;
e: Dyscia dis inc a ia (Bang-Haas, 1910), Geome idae - BIN URI BOLD:ACF1137;
: Idaea igu a ia (Bang-Haas, 1907), Geome idae - BIN URI BOLD:AEC6951.
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs 5
Pe sonnel: Ped o Beja (p ojec coo dina o ), Sónia Fe ei a ( axonomis and IBI manage ),
Ma in F.V. Co ley ( axonomis , lepidop e is ), Cá ia Cha es (p ojec echnician), Filipa M.S.
Ma ins (molecula biologis ), Vanessa A. Ma a (molecula biologis ), An onio Muñoz-
Mé ida (bioin o ma ician), John A che (bioin o ma ician), Joana Paupé io (molecula
biologis ), Ca a ina J. Pinho (p ojec echnician), Joana C. Pin o (p ojec echnician),
Pamela Puppo (molecula biologis ), Te esa L Sil a (molecula biologis ), Ped o Sousa
(p ojec echnician), Sasha Vasconcelos (con ibu o ), Joana Ve íssimo (molecula
biologis ), all a ilia ed o CIBIO-InBIO, Uni e si y o Po o, José Manuel G osso-Sil a
(en omologis ), a ilia ed o he MHNC-UP, Uni e si y o Po o and Rui And ade
a
b
c
d
e
Figu e 2.
Examples o he di e si y o species ha a e pa o he da ase o dis ibu ion da a and DNA
ba codes o Po uguese mo hs (con .). All pho os by João Nunes.
a: Pe igune con e ga a (Ville s, 1789), Geome idae - BIN URI BOLD:AEH2380;
b: Dichagy is cons an i Milliè e, 1860, Noc uidae - BIN URI BOLD:AED6734;
c: Dasyce a oli iella (Fab icius, 1794), Oecopho idae - BIN URI BOLD:ADT8549;
d: Lo yma eg egialis (He ich-Schä e , 1838), Py alidae - BIN URI BOLD:AEC8952;
e: C assico nella agenjoi (Pe e sen, 1957), Tineidae - BIN URI BOLD:ADT5404;
: Eana ne ana (de Joannis, 1908), To icidae - BIN URI BOLD:ADS8408.
6Fe ei a S e al
(en omologis ), João Nunes (lepidop e ologis ), Jo ge Rose e (lepidop e is ), independen
esea che s.
S udy a ea desc ip ion: Con inen al Po ugal (Fig. 3).
Design desc ip ion: Lepidop e a specimens we e collec ed in he ield, mo phologically
iden i ied and DNA ba coded.
Funding: The p esen wo k was unded by Na ional Funds h ough FCT-Fundação pa a a
Ciência e a Tecnologia in he scope o he p ojec LA/P/0048/2020. InBIO Ba coding
Ini ia i e was unded by he Eu opean Union’s Ho izon 2020 Resea ch and Inno a ion
P og amme unde g an ag eemen No 668981 and by he p ojec PORBIOTA –
Po uguese E-In as uc u e o In o ma ion and Resea ch on Biodi e si y (POCI-01-0145-
FEDER-022127), suppo ed by Ope a ional Thema ic P og am o Compe i i eness and
In e na ionaliza ion (POCI), unde he PORTUGAL 2020 Pa ne ship Ag eemen , h ough
he Eu opean Regional De elopmen Fund (FEDER). The wo k was pa ially Funded by
Ho izon Eu ope unde he Biodi e si y, Ci cula Economy and En i onmen call (REA.B.3);
co- unded by he Swiss S a e Sec e a ia o Educa ion, Resea ch and Inno a ion (SERI)
unde con ac numbe 22.00173; and by he UK Resea ch and Inno a ion unde he
Depa men o Business, Ene gy and Indus ial S a egy’s Ho izon Eu ope Gua an ee
Scheme. The ieldwo k bene i ed om EDP Biodi e si y Chai , he p ojec “P omoção dos
se iços deecossis emas no Pa que Na u al Regional do Vale do Tua: Con olo de P agas
Ag ícolas eFlo es ais po Mo cegos” unded by he Agência de Desen ol imen o Regional
Figu e 3.
Map o he locali ies whe e DNA ba coded Lepidop e a samples we e collec ed in con inen al
Po ugal.
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs 7
do Vale doTua and includes esea ch conduc ed a he Long Te m Resea ch Si e o Baixo
Sabo (LTER_EU_PT_002). SF and VM we e unded by he FCT h ough he p og amme
‘S imulus o Scien i ic Employmen , Indi idual Suppo —3 d Edi ion’ (h ps://doi.o g/
10.54499/2020.03526.CEECIND/CP1601/CT0010; h ps://doi.o g/10.54499/2020.02547.
CEECIND/CP1601/CT0006). CJP, JV and FMSM by PhD g an s (SFRH/BD/145851/2019;
SFRH/BD/133159/2017; SFRH/BD/104703/2014) unded by FCT.
Sampling me hods
Desc ip ion: Con inen al Po ugal (Fig. 3).
Sampling desc ip ion: Specimens we e collec ed du ing ield expedi ions h oughou
con inen al Po ugal, om 2001 o 2020. They we e cap u ed a nigh using ligh aps, he
la e wi h UV LEDs, mixed ligh o me cu y apou lamps o du ing he day by di ec
sea ch. All specimens we e obse ed in he ield and, in mos cases, hey could be eadily
iden i ied o species le el by an expe ienced axonomis (Ma in Co ley). Such specimens
we e p ese ed in 96% e hanol and s o ed a he InBIO Ba coding Ini ia i e e e ence
collec ion (Vai ão, Po ugal), whe e hey can be e-examined and geni alia dissec ed, i
needed. Specimens ha could no be iden i ied in he ield (n = 84) we e pinned and d ied
o subsequen examina ion in he labo a o y. They we e hen s o ed a he Resea ch
Collec ion o Ma in Co ley o he P i a e Collec ions o Jo ge Rose e o J.M. G osso-Sil a.
DNA ex ac ion was pe o med using ei he he 96-Well Pla e Animal Genomic DNA Mini-
P eps Ki (Bio Basic, On a io, Canada) o he QIAamp DNA Mic o Ki (Qiagen, Ge many)
which is designed o ex ac highe concen a ions o gene ic ma e ial om samples wi h
small amoun s o DNA. Ampli ica ion was pe o med using wo di e en p ime pai s ha
ampli y pa ially o e lapping agmen s (LC + BH) o he 658 bp ba coding egion o he
COI mi ochond ial gene. We used he p ime s FwhF1 (Vamos e al. 2017) + C_R (
Shok alla e al. 2015) o LC and BF3 (Elb ech e al. 2019) + BR2 (Elb ech and Leese
2017) o BH ampli ica ion, all modi ied wi h 5’ adap o s sequences o be compa ible wi h a
wo-s ep p o ocol. PCRs we e pe o med in 10 μl eac ions, con aining 5 μl o Mul iplex
PCR Mas e Mix (Qiagen, Ge many), 0.3 μl o each 10 mM p ime and 1-2 μl o DNA, wi h
he emaining olume in wa e . PCR cycling condi ions consis ed o an ini ial dena u a ion
a 95ºC o 15 min, ollowed by 45 cycles o dena u a ion a 95ºC o 30 sec, annealing a
45ºC and 50ºC, espec i ely, o 45 sec and ex ension a 72ºC o 45 sec and a inal
elonga ion s ep a 60ºC o 10 min.
Success ul ampli ica ion was alida ed h ough 2% aga ose gel elec opho esis s ained
wi h GelRed (Bio ium, USA) and samples selec ed o sequencing p oceeded o a second-
ound PCR whe e Illumina P5 and P7 adap e s wi h cus om 7 bp long ba codes we e
a ached o each i s PCR p oduc . The second PCR was pe o med in a olume o 10 μl,
including 5 μl o KAPA HiFi PCR Ki (KAPA Biosys ems, Cape Town, Sou h A ica), 0.5 μl o
each 10 mM indexing p ime and 2 μl o dilu ed i s PCR p oduc (usually 1:4). PCR
cycling condi ions we e as ollows: ini ial dena u a ion a 95ºC o 3 min, wi h 8-10 cycles
(adjus ed o sample quali y) o dena u a ion a 95ºC o 30 sec, annealing a 50ºC o 60
8Fe ei a S e al
sec and ex ension a 72ºC o 45 sec and a inal elonga ion s ep a 60ºC o 10 min. The
amplicons we e pu i ied using AMPu e XP beads (Beckman Coul e , U.S.A.) and quan i ied
using NanoD op 1000 (The mo Scien i ic, U.S.A.). Clean PCR p oduc s we e hen pooled
equimola ly pe agmen . Each pool was quan i ied wi h KAPA Lib a y Quan i ica ion Ki
Illumina Pla o ms (KAPA Biosys ems, Cape Town, Sou h A ica) and he 2200 Tapes a ion
Sys em (Agilen Technologies, Cali o nia, USA) was used o agmen leng h analysis p io
o sequencing (Paupé io e al. 2018). DNA sequencing was done a CIBIO acili ies on an
Illumina MiSeq bench op sys em, using V2 MiSeq sequencing ki s (2 x 250 bp) (Illumina,
Cali o nia, U.S.A.).
Illumina sequencing eads we e p ocessed using OBITools (Boye e al. 2015) and
VSEARCH (Rognes e al. 2016). B ie ly, pai ed-end eads we e aligned, collapsed in o
exac sequence a ian s, il e ed by leng h, denoised and checked o chime as. The
esul ing sequences om bo h LC and BH agmen s o each sample we e u he
assembled using CAP3 (Huang and Madan 1999) o p oduce a single 658 bp con ig pe
sample.
Quali y con ol: All DNA ba codes sequences we e compa ed agains he BOLD
da abase and he 99 op hi s we e inspec ed in o de o de ec possible issues due o
con amina ions o misiden i ica ions.
S ep desc ip ion: 1. Specimens we e collec ed in 234 di e en locali ies. Fieldwo k was
ca ied ou be ween 2001 and 2022.
2. Selec ed specimens we e pinned and d ied and a e p ese ed in h ee p i a e
collec ions. O he wise, specimens collec ed as issue samples we e s o ed in 96% e hanol
in he IBI collec ion a CIBIO, Resea ch Cen e in Biodi e si y and Gene ic Resou ces
(Vai ão, Po ugal).
3. All specimens we e mo phologically iden i ied and DNA ba coded. To sequence he 658
bp COI DNA ba code agmen , one leg was emo ed om each indi idual, DNA was
ex ac ed and hen ampli ied. All DNA ex ac s we e deposi ed in he IBI collec ion.
4. All sequences in he da ase we e submi ed o BOLD and GenBank da abases and, o
each sequenced specimen, he mo phological iden i ica ion was con as ed wi h he esul s
o he BLAST o he newly-gene a ed DNA ba codes in he BOLD Iden i ica ion Engine.
Geog aphic co e age
Desc ip ion: Con inen al Po ugal
Coo dina es: 36.960 and 42.124 La i ude; -9.467 and -6.229 Longi ude.
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs 9
• Hueme P, Ka shol O (2018) Re ision o he genus Megac aspedus Zelle , 1839, a
challenging axonomic igh ope o species delimi a ion (Lepidop e a, Gelechiidae).
ZooKeys 800: 1‑278. h ps://doi.o g/10.3897/zookeys.800.26292
• Hueme P, Ka shol O, Aa ik L, Be gg en K, Bidzilya O, Junnilainen J, Land y J,
Mu anen M, Nupponen K, Sege e A, Šumpich J, Wiese C, Wiesmai B, Hebe PN
(2020) DNA ba code lib a y o Eu opean Gelechiidae (Lepidop e a) sugges s g ea ly
unde es ima ed species di e si y. ZooKeys 921: 141‑157. h ps://doi.o g/10.3897/
zookeys.921.49199
• Ma in CFV, Fe ei a S (2019) A axonomic e ision o he Wes e n Palaea c ic genus
Cacoch oa Heinemann, 1870 (Lepidop e a, Dep essa iidae, C yp olechiinae) wi h
desc ip ion o a new genus and a new species. Zoo axa 4683 (2). h ps://doi.o g/
10.11646/zoo axa.4683.2.2
• Ma a V, Fe ei a S, Campos R, da Sil a L, Ve íssimo J, Co ley MV, Beja P (2020)
E icien assessmen o noc u nal lying insec communi ies by combining au oma ic
ligh aps and DNA me aba coding. En i onmen al DNA 3 (2): 398‑408. h ps://doi.o g/
10.1002/edn3.125
• Mi chell A (2008) DNA ba coding demys i ied. Aus alian Jou nal o En omology 47 (3):
169‑173. h ps://doi.o g/10.1111/j.1440-6055.2008.00645.x
• Mo água A, Vasselon V, Oli ei a R, Elias C, Cha don C, Bouchez A, Rime F, João Feio
M, F.P. Almeida S (2019) Applicabili y o DNA me aba coding app oach in he
bioassessmen o Po uguese i e s using dia oms. Ecological Indica o s 106 h ps://
doi.o g/10.1016/j.ecolind.2019.105470
• Mülle B, E lache S, Hausmann A, Rajaei H, Sih onen P, Skou P (2019) Ennominae II.
In: Hausmann A, Sih onen P, Rajaei H, Skou P (Eds) The geome id mo hs o Eu ope.
Vol. 6. B ill, Leiden, 906 pp.
• Nunes J, Ca doso H, Valada es A, Banza P, Tomás J, Valkenbu g T (2023) Po uguese
Mo h Reco ding Scheme. 1.3. GBIF Po ugal. URL: h ps://doi.o g/10.15468/mbpxnd
• Oli ei a D, Cha es C, Pin o J, Paupé io J, Fonseca N, Beja P, Fe ei a S (2021) DNA
ba coding o Po uguese lacewings (Neu op e a) and snake lies (Raphidiop e a)
(Insec a, Neu op e ida). ZooKeys 1054: 67‑84. h ps://doi.o g/10.3897/zookeys.
1054.64608
• O iz A, Rubio R, Gue e o J, Ga e M, Se ano J, Hebe P, Hausmann A (2017) Close
cong uence be ween Ba code Index Numbe s (bins) and species bounda ies in he
E ebidae (Lepidop e a: Noc uoidea) o he Ibe ian Peninsula. Biodi e si y Da a Jou nal
5 h ps://doi.o g/10.3897/bdj.5.e19840
• O iz A, Rubio R, de F eina J, Gue e o J, Ga e M, Yela JL (2023) DNA ba coding and
mo phology e eal Eu opean and wes e n Asian A c ia illica (Linnaeus, 1758) as a
complex o species (Lepidop e a, E ebidae, A c iinae). ZooKeys 1159: 69‑86. h ps://
doi.o g/10.3897/zookeys.1159.95225
• Paupe io J, Gonzalez LM, Ma inez J, González M, Ma ins FM, Ve íssimo J, Puppo P,
Pin o J, Cha es C, Pinho C, G osso-Sil a JM, Quaglie a L, Sil a TL, Sousa P, Al es P,
Fonseca N, Beja P, Fe ei a S (2023) The InBIO ba coding ini ia i e da abase: DNA
ba codes o Ibe ian T ichop e a, documen ing biodi e si y o eshwa e biomoni o ing
in a Medi e anean ho spo . Biodi e si y Da a Jou nal 11 h ps://doi.o g/10.3897/bdj.
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• Ra nasingham S, Hebe PN (2013) A DNA-Based Regis y o All Animal Species: The
Ba code Index Numbe (BIN) Sys em. PLoS ONE 8 (7). h ps://doi.o g/10.1371/
jou nal.pone.0066213
• Rebelo H, Fe ei a S, Amo im F, Ho a P, Raposei a H, San os H, Beja P, Ma a V (2020)
Hidden in ou pocke s: building o a DNA ba code lib a y un eils he i s eco d o
Myo is alca hoe o Po ugal. Biodi e si y Da a Jou nal 8 h ps://doi.o g/10.3897/bdj.
8.e54479
• Rognes T, Flou i T, Nichols B, Quince C, Mahé F (2016) VSEARCH: a e sa ile open
sou ce ool o me agenomics. Pee J 4 h ps://doi.o g/10.7717/pee j.2584
• Rosa P, Wood T, Sil a TL, Ve íssimo J, Ma a V, Michez D, Beja P, Fe ei a S (2023) The
InBIO Ba coding Ini ia i e Da abase: con ibu ion o he knowledge on DNA ba codes o
cuckoo wasps, wi h he desc ip ion o new species om he Ibe ian Peninsula
(Hymenop e a, Ch ysididae). Biodi e si y Da a Jou nal 11 h ps://doi.o g/10.3897/bdj.
11.e98743
• Sa olainen V, Cowan RS, Vogle AP, Rode ick GK, Lane R (2005) Towa ds w i ing he
encyclopaedia o li e: an in oduc ion o DNA ba coding. Philosophical T ansac ions o
he Royal Socie y B: Biological Sciences 360 (1462): 1805‑1811. h ps://doi.o g/
10.1098/ s b.2005.1730
• Shok alla S, Po e T, Gibson J, Dobosz R, Janzen D, Hallwachs W, Golding GB,
Hajibabaei M (2015) Massi ely pa allel mul iplex DNA sequencing o specimen
iden i ica ion using an Illumina MiSeq pla o m. Scien i ic Repo s 5 (1). h ps://doi.o g/
10.1038/s ep09687
• Skou P, S üning D, Sih onen P (2017) Re ision o he Wes -Medi e anean geome id
genus Ekboa mia, wi h desc ip ion o a new species om Po ugal (Lepidop e a,
Geome idae, Ennominae). No a Lepidop e ologica 40 (1): 39‑63. h ps://doi.o g/
10.3897/nl.40.10440
• Vamos E, Elb ech V, Leese F (2017) Sho COI ma ke s o eshwa e
mac oin e eb a e me aba coding. Me aba coding and Me agenomics 1 h ps://doi.o g/
10.3897/mbmg.1.14625
• Vela de‐Ga céz D, Ma a V, Beja P, da Sil a L (2023) DNA me aba coding, di e si y
pa i ioning and null models e eal mechanisms o seasonal ophic specializa ion in a
Medi e anean wa ble . Molecula Ecology h ps://doi.o g/10.1111/mec.17245
• Vella A, Mi sud CM, Mag o D, Vella N (2022) DNA Ba coding o Lepidop e a Species
om he Mal ese Islands: New and Addi ional Reco ds, wi h an Insigh in o Endemic
Di e si y. Di e si y 14 (12). h ps://doi.o g/10.3390/d14121090
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs 17
Supplemen a y ma e ials
Suppl. ma e ial 1: Lis o species ha we e collec ed and DNA ba coded wi hin
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs
Au ho s: Sónia Fe ei a, Ma in F. V. Co ley, João Nunes, Jo ge Rose e, Sasha Vasconcelos,
Vanessa A. Ma a, Te esa L Sil a, Ped o Sousa, Rui And ade, José Manuel G osso Sil a, Ca a ina
J. Pinho, Cá ia Cha es, Filipa MS Ma ins, Joana Pin o, Pamela Puppo, An onio Muñoz-Mé ida,
John A che , Joana Paupe io, Ped o Beja
Da a ype: occu ences
B ie desc ip ion: Lis o species ha we e collec ed and DNA ba coded wi hin his p ojec
including he sample code (IBI code), he P ocess ID (BOLDcode), he BIN URI (BOLD BIN) and
GenBank acession numbe (GenBank). * Indica e species wi h new BINs.
Download ile (212.71 kb)
Suppl. ma e ial 2: IBI - Lepidop e a 08 lib a y - Specimen de ails
Au ho s: Sónia Fe ei a, Ma in F. V. Co ley, João Nunes, Jo ge Rose e, Sasha Vasconcelos,
Vanessa A. Ma a, Te esa L Sil a, Ped o Sousa, Rui And ade, José Manuel G osso Sil a, Joana
Paupe io, Ped o Beja
Da a ype: Specimen da a eco ds
B ie desc ip ion: The ile includes in o ma ion abou all eco ds in BOLD o he IBI -
Lepidop e a 08 lib a y. I con ains collec ing and iden i ica ion da a. The da a a e as downloaded
om BOLD in he s o ma , wi hou u he p ocessing.
Download ile (924.30 kb)
Suppl. ma e ial 3: IBI - Lepidop e a 08 - Specimen de ails - Da win Co e S anda d
Au ho s: Sónia Fe ei a, Ma in F. V. Co ley, João Nunes, Jo ge Rose e, Sasha Vasconcelos,
Vanessa A. Ma a, Te esa L Sil a, Ped o Sousa, Rui And ade, José Manuel G osso Sil a, Joana
Paupe io, Ped o Beja
Da a ype: Specimen da a eco ds in he Da win Co e S anda d o ma
B ie desc ip ion: The ile includes in o ma ion abou all eco ds in BOLD o he IBI -
Lepidop e a 08 lib a y. I con ains collec ing and iden i ica ion da a. The da a a e as downloaded
om BOLD in he Da win Co e S anda d o ma , wi hou u he p ocessing.
Download ile (1.08 MB)
18 Fe ei a S e al
Suppl. ma e ial 4: IBI- Lepidop e a 08 lib a y - DNA sequences
Au ho s: Sónia Fe ei a, Ma in F. V. Co ley, João Nunes, Jo ge Rose e, Sasha Vasconcelos,
Vanessa A. Ma a, Te esa L Sil a, Ped o Sousa, Rui And ade, José Manuel G ossoSil a, Ca a ina
J. Pinho, Cá ia Cha es, Filipa MS Ma ins, Joana Pin o, Pamela Puppo, An onio Muñoz-Mé ida,
John A che , Joana Paupe io, Ped o Beja
Da a ype: Specimen genomic da a, DNA sequences
B ie desc ip ion: COI sequences in as a o ma . Each sequence is iden i ied by he
BOLDP ocessID, species name, gene ic ma ke name and GenBank accession numbe , all
sepa a ed by a e ical ba . The da a a e as downloaded om BOLD.
Download ile (1.59 MB)
Suppl. ma e ial 5: Phylogene ic ee (NJ) o all DNA ba codes used in he s udy
gene a ed in BOLD Sys ems
Au ho s: Sónia Fe ei a, Ma in F. V. Co ley, João Nunes, Jo ge Rose e, Sasha Vasconcelos,
Vanessa A. Ma a, Te esa L Sil a, Ped o Sousa, Rui And ade, José Manuel G osso Sil a, Ca a ina
J. Pinho, Cá ia Cha es, Filipa MS Ma ins, Joana Pin o, Pamela Puppo, An onio Muñoz-Mé ida,
John A che , Joana Paupe io, Ped o Beja
Da a ype: Phylogene ic ee
B ie desc ip ion: Phylogene ic ee (NJ) o all he specimens DNA ba codes wi hin DS-IBILP08:
he IBI-Lepidop e a 08 da ase collec ed in con inen al Po ugal, all o which ha e been
mo phologically iden i ied o species le el.
Download ile (100.77 kb)
Suppl. ma e ial 6: Specimens wi h occu ence in con inen al Po ugal missing DNA
ba codes
Au ho s: Sónia Fe ei a, Ma in F. V. Co ley, João Nunes, Jo ge Rose e, Sasha Vasconcelos,
Vanessa A. Ma a, Te esa L Sil a, Ped o Sousa, Rui And ade, José Manuel G osso Sil a, Ca a ina
J. Pinho, Cá ia Cha es, Filipa MS Ma ins, Joana Pin o, Pamela Puppo, An onio Muñoz-Mé ida,
John A che , Joana Paupe io, Ped o Beja
Da a ype: Gap Checklis
B ie desc ip ion: Specimens wi h occu ence in con inen al Po ugal missing DNA ba codes. To
each species is disc imina ed i he species has specimens in BOLD, bu s ill has no DNA ba code
o specimens a e s ill needed.
Download ile (14.92 kb)
The InBIO Ba coding Ini ia i e Da abase: DNA ba codes o Po uguese mo hs 19
