249
In eg a i e axonomy e eals non-na i e Squalius hyb ids in he
Ibe ian Peninsula
Ch is os Gkenas1* , So ia Noguei a1* , Manuel Cu o2,3 , Diogo Dias1,4 , Diogo Ribei o1, Rui Ri aes1,
Joana Ga ido Noguei a2,3 , Manuel Lopes-Lima2,3 , Ma ia Judi e Al es4,5 , Filipe Ribei o1
1 MARE, Ma ine and En i onmen al Sciences Cen e /ARNET, Aqua ic Resea ch Ne wo k, Facul y o Sciences, Uni e si y o Lisbon, 1749-016 Lisbon, Po ugal
2 CIBIO, Resea ch Cen e in Biodi e si y and Gene ic Resou ces, InBIO Associa e Labo a o y, Vai ão Campus, 4485-661 Vai ão, Po ugal
3 BIOPOLIS, P og am in Genomics, Biodi e si y and Land Planning, Vai ão Campus, 4485-661 Vai ão, Po ugall
4 cE3c – Cen e o Ecology, E olu ion and En i onmen al Changes/Global Change and Sus ainabili y Ins i u e, Facul y o Sciences, Uni e si y o Lisbon, 1749-
016 Lisbon, Po ugal
5 MUHNAC – Na ional Museum o Na u al His o y and Science, Uni e si y o Lisbon, 1250-102 Lisbon, Po ugal
Co esponding au ho : Ch is os Gkenas ([email p o ec ed])
Copy igh : © Ch is os Gkenas 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
The in oduc ion o non-na i e species poses a c i ical h ea o biodi e si y in Medi e anean
eshwa e ecosys ems. This s udy documen s he i s eco d o non-na i e Squalius hyb ids in
he A e Ri e (Ibe ian Peninsula), combining mo phological and molecula da a o con i m hei
p esence and assess hei po en ial o igin. The specimens exhibi hyb id cha ac e is ics, wi h mos
mo phological and me is ic ai s o e lapping hose o bo h S. cephalus and S. squalus. Almos all
indi iduals analysed showed an admixed genomic backg ound o bo h species, sugges ing a hy-
b id o igin likely wi hin he species’ na i e ange be o e hei in oduc ion in o Po ugal h ough
human-media ed ac i i ies, likely ela ed o ec ea ional ishing. Fu he mo e, molecula da a also
suppo s hyb idiza ion wi h he na i e S. ca oli e ii, aising conse a ion conce ns gi en he nega-
i e impac s associa ed wi h hyb idiza ion wi h non-na i e species. By combining da a om social
media and ci izen science pla o ms, we we e able o map he apid expansion o his in asi e chub
in he A e Ri e om 2018 un il now. Ou indings emphasize he u gen need o popula ion
con ol measu es, while enhancing moni o ing, gene ic s udies, and inc eased public awa eness o
mi iga e he impac s o in asi e species on Ibe ian eshwa e biodi e si y.
Key wo ds: A e i e , chub, hyb idiza ion, in asi e species, mo phology, Po ugal, Squalius ceph-
alus, Squalius squalus
In oduc ion
The Medi e anean egion is conside ed a eshwa e biodi e si y ho spo , cha -
ac e ised by high le els o endemic species shaped by i s unique palaeogeog aphic
his o y (Za doya and Doad io 1999). Howe e , ish species ace se e e h ea s in
his egion, mainly caused by human p essu es including pollu ion, habi a de-
s uc ion, agmen a ion, and he in oduc ion o non-na i e species, wi h he ap-
id sp ead o hese species in ecen decades being pa icula ly conce ning (Cla e-
o e al. 2010; He moso e al. 2011; Mila di e al. 2022). Add essing his end
Academic edi o : Josie Sou h
Recei ed:
30 Janua y 2025
Accep ed:
15 July 2025
Published:
7 Oc obe 2025
Ci a ion: Gkenas C, Noguei a S, Cu o
M, Dias D, Ribei o D, Ri aes R, Ga ido
Noguei a J, Lopes-Lima M, Al es MJ,
Ribei o F (2025) In eg a i e axonomy
e eals non-na i e Squalius hyb ids in
he Ibe ian Peninsula. In: Anas ácio P,
Ribei o F, Chainho P (Eds) In asions
in Aqua ic Sys ems. NeoBio a 102:
249–268. h ps://doi.o g/10.3897/
neobio a.102.148337
NeoBio a 102: 249–268 (2025)
DOI: 10.3897/neobio a.102.148337
* These au ho s con ibu ed equally o his wo k.
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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NeoBio a 102: 249–268 (2025), DOI: 10.3897/neobio a.102.148337
Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
equi es he implemen a ion o e ec i e biosecu i y s a egies suppo ed by ac i e
moni o ing o new occu ences and esea ch in o hei dispe sal pa hways and
ec o s (Riccia di e al. 2021).
Wi hin he Medi e anean egion, he Ibe ian basins ha e some o he highes
p opo ions o na i e species a isk o ex inc ion (Cos a e al. 2021), a si ua ion
ha has been signi ican ly exace ba ed by he p oli e a ion o non-na i e species
(Cla e o e al. 2013; Anas ácio e al. 2019; Vale io e al. 2022). Mos o he ecen ly
es ablished species in he Ibe ian Peninsula o igina e om o he pa s o Eu ope
(Anas ácio e al. 2019). The no heas e n Ibe ian basins a e ega ded as a key ga e-
way o species in oduc ions om F ance, acili a ing a no heas - o-wes sp ead
ac oss bo h Spanish and Po uguese basins (Ga cía-Be hou e al. 2005; Cla e o
and Ga cía-Be hou 2006), al hough some e idence sugges s al e na i e in oduc-
ion pa hways (Ribei o and Ve íssimo 2014; Ga cia-Ra en ós e al. 2020). The e
is, howe e , a consensual unde s anding ha non-na i e ishes a e p ima ily in o-
duced o ec ea ional ishing pu poses, ei he as bai o as a ge ed species (Banha e
al. 2017a; Anas ácio e al. 2019), a end ha con inues o g ow (Banha e al. 2024).
In ecen yea s, echnological ad ancemen s and inc eased awa eness o non-na-
i e species impac s ha e imp o ed ou capaci y o moni o new species a i als.
Ne e heless, moni o ing aqua ic habi a s emains challenging due o logis ical
cons ain s, including limi ed accessibili y o emo e loca ions, insu icien und-
ing and equipmen a ailabili y, and a lack o ained pe sonnel. Addi ionally, he
in oduc ion o species ha a e mo phologically simila o indis inguishable om
o he al eady es ablished non-na i e species, such as Ca assius au a us (Linnaeus,
1758) and Ca assius gibelio (Block, 1782) (Ribei o e al. 2015) o hose esembling
na i e species, p esen s signi ican challenges o accu a e iden i ica ion, moni o -
ing, and managemen (e.g., Apa icio e al. 2013; Pé ez-Bielsa e al. 2025). These
mo phological simila i ies can delay he iden i ica ion o newly occu ing species,
allowing hem o sp ead unde ec ed o ex ended pe iods. In his con ex , species
om highly di e se gene a including minnows (A ae e al. 2024), chubs (Buj e
al. 2020) and ba bels (Rossi e al. 2021), which sha e ambiguous mo phological
ai s and ha e axonomic classi ica ions ha emain ac i ely deba ed, a e pa icu-
la ly suscep ible o hese iden i ica ion and moni o ing challenges.
This is he case o he genus Squalius Bonapa e, 1837, which is p esen ac oss Eu-
opean wa e sheds and comp ises mo e han 50 species, many o which a e endemic
o speci ic d ainages in he Me idional peninsulas (Ko ella and F eyho 2007; Pe ea
e al. 2020). Mos o hese species sha e mo phological ea u es and exhibi o e -
lapping me is ic ai s. In he Ibe ian Peninsula, hi een species o Squalius ha e
been iden i ied (Mendes e al. 2024), many o which ha e been desc ibed mainly
based on molecula da a, wi h sub le mo phological di e ences (e.g., Doad io and
Ca mona 2006; Doad io e al. 2007). Un il now, no non-na i e species om his
genus has been eco ded in Po ugal. Howe e , ecen gene ic analyses ha e iden-
i ied a chub popula ion o Squalius cephalus (Linnaeus, 1758) and e idence o i s
hyb idiza ion wi h he endemic Squalius laie anus Doad io, Ko ela and de Sos oa,
2007 in Spain (Pé ez-Bielsa e al. 2025). Fu he mo e, eDNA da a om he A e
Ri e de ec ed he p esence o Squalius squalus (Bonapa e, 1837), which needs o
be con i med (Cu o e al. 2025). This s udy examines he occu ence o non-na-
i e Squalius hyb ids in he A e Ri e using mo phological and molecula analyses.
Addi ionally, we used da a mined om social media o um g oups o map ecen
occu ences and o cons uc hei cu en po en ial dis ibu ion in Po ugal.
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Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
Ma e ials and me hods
S udy a ea
This s udy was conduc ed in he A e Ri e , no he n Po ugal, which hos s a single
na i e Squalius species: he No he n Ibe ian chub, Squalius ca oli e ii (Doad io,
1988). This species a ely exceeds 25 cm, has a mode a ely comp essed body, ounded
snou wi h sligh ly sub e minal mou h, and sil e y-oli e scales wi hou da k ma gins.
I s pai ed, anal, and caudal ins a e hyaline o pale ambe wi h no i id pigmen a-
ion (Colla es-Pe ei a e al. 2021). In con as , non-na i e chubs in Po uguese ba-
sins, documen ed h ough angle pho os and iNa u alis eco ds, exceed 40 cm, ha e
bulkie heads wi h e minal mou hs, display o ange- ed o black en al and anal ins,
and show scales wi h da k edges o ming a e icula e pa e n. (Ko ela and F eyho
2007). These di e ences in size, in colo a ion, and scale pa e n p o ide clea c i e ia
o angle s and ci izens o dis inguish na i e S. ca oli e ii om non-na i e chubs.
Be ween 2017 and 2025, we collec ed 37 uniden i ied chubs, using elec o ish-
ing in he A e Ri e (Table 1). The i s specimen (FRISK3481) was cap u ed in
2017 du ing a egula ish moni o ing campaign. Upon examina ion, he e was a
suspicion ha he specimen could be an un epo ed non-na i e species o Po u-
guese eshwa e s and hus a issue sample was kep o gene ic analysis. Fo he
same eason, in 2020, issue samples we e collec ed om se en addi ional indi id-
uals (PEI0456–PEI0462). The emaining 29 indi iduals we e cap u ed du ing sub-
sequen moni o ing campaigns be ween 2024 and 2025 a ou dis inc loca ions
along he A e Ri e (Table 1). Fin clips we e ex ac ed om hese specimens and
p ese ed in absolu e e hanol o gene ic analyses. Fo hese, he whole indi iduals
we e kep o mo phological analysis. The one specimen cap u ed in 2024 was
anspo ed ozen o he labo a o y and s o ed a -20 °C o pos e io mo pholog-
ical iden i ica ion. The 28 specimens cap u ed in 2025 we e eu hanized in he ield
using clo e oil and immedia ely ixed in 10% o malin. Following ixa ion o a
leas h ee days, hese specimens we e g adually ansi ioned o a 70% e hanol solu-
ion o long- e m p ese a ion, in ol ing exposu e o p og essi ely highe e hanol
concen a ions (30% o wo days, ollowed by 50% o h ee days). Addi ionally,
o se e as e e ence o he gene ic analysis, issue samples om six S. cephalus in-
di iduals om he na i e ange (50°01'55.6"N, 14°59'35.0"E), cap u ed in 2022,
and ou om S. ca oli e ii, cap u ed in 2017 in he A e Ri e , we e added.
Mo phological analysis
In he labo a o y, he specimens we e pho og aphed, and species iden i ica ion was
ca ied ou using gene al iden i ica ion keys (Doad io and Ca mona 2006; Ko ela
and F eyho 2007). Mo phological measu emen s we e conduc ed om poin o
poin o he nea es 0.1 mm wi h e nie calipe s on he le side o he speci-
men (Suppl. ma e ial 1), ollowing he me hodology desc ibed by Bogu skaya and
Zupanič (1999; 2010). The s anda d leng h was measu ed om he ip o he snou
o he end o he hypu al complex. The leng h o he caudal peduncle was measu ed
om he pos e io edge o he base o he las anal in ay o he end o he hypu al
complex, a he mid-heigh o he caudal in base. Bo h head leng h and in e o bi -
al wid h we e measu ed including he skin old. Pos -do sal leng h was measu ed
om he inse ion poin o he do sal in o he end o he hypu al complex. The
o al la e al-line scale coun included all po ed scales, ex ending om he i s scale
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Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
jus pos e io o he pos empo al bone o he mos pos e io scale loca ed a he
base o he caudal- in ays. Fin ays we e coun ed sepa a ely as simple and b anched
ays (Bogu skaya and Zupanič 2010; Suppl. ma e ial 1). The las wo b anched
ays a icula ing on a single p e ygiopho e in he do sal and anal ins we e no ed
as “1½”. P io o s a is ical analysis, all mo phome ic a iables we e s anda dized
o emo e he e ec s o size ollowing Ko ela and F eyho (2007). Head- ela ed
measu emen s we e exp essed as p opo ions o head leng h (HL), while all e-
maining body measu emen s we e exp essed as p opo ions o s anda d leng h (SL)
o body dep h. Di e ences in mo phome ic and me is ic cha ac e is ics be ween
he s udied g oups we e assessed using independen wo-sample (S uden ’s) - es ,
a e con i ming no mali y wi h Shapi o–Wilk es s (Za 1996). All da a p ocess-
ing and analyses we e pe o med in R e sion 4.4.3 (R De elopmen Co e Team
2025). Specimens and issues we e deposi ed in he zoological collec ions “Museu
Bocage” o he Museu Nacional de His ó ia Na u al e da Ciência, Uni e sidade de
Lisboa, Po ugal (MNHNC-MB05-004006 – MB05-004015) (Suppl. ma e ial 2).
Gene ic analysis
DNA was ex ac ed om in clips o muscle issue p ese ed in absolu e e hanol
using comme cial ki s. Mo e speci ically, he se en samples collec ed in 2020 we e
p ocessed using EZ-10 Genomic DNA Ki (Bio Basic, Canada), while he emaining
we e p ocessed using he E.Z.N.A. Tissue DNA Ki (Omega Bio-Tek, No c oss, GA,
USA). Bo h app oaches ollowed he manu ac u e ’s p o ocols. DNA quali y was as-
sessed h ough 0.8% aga ose gel elec opho esis. Molecula assessmen o specimen
iden i y was based on a ia ion a h ee loci. The mi ochond ial cy och ome b (cy b)
gene was ampli ied h ough he p ime se Glu-F and Th -R (amplicon ~1140 bp,
Za doya and Doad io 1998). Two nuclea genes we e also a ge ed: ea ly g ow h
esponse 2b (eg 2b, ~747 bp; Waap e al. 2011) and be a-ac in (ac b, ~933 bp;
Robalo e al. 2006). PCR eac ions we e ca ied ou in a 10 µl olume con aining
5 µl o MyTaq Mix (Me idian Bioscience, Cincinna i, Ohio, USA) and 0.4 µl o
each p ime (10 µM). The ollowing empe a u e p o ile was implemen ed: ini ial
dena u a ion a 95 °C o 10 min, ollowed by 40 cycles o 95 °C o 1 min, 55 °C o
1 min (50 °C in he case o cy b) and 72 °C o 1 min, and a inal ex ension a 72 °C
o 10 min. PCR success was e alua ed h ough 2% aga ose gel elec opho esis. The
esul ing amplicons we e sequenced in bo h di ec ions using Sange sequencing, im-
plemen ed by he molecula analysis se ices om STAB Vida (Capa ica, Po ugal).
The ob ained ch oma og ams we e manually aligned using Geneious P ime
(Bioma e s L d., New Zealand, . 2025.1.2). Fo each locus, he sequences gen-
e a ed in his s udy we e joined o publicly a ailable sequences in NCBI o he
Table 1. De ails o Squalius specimens collec ed o gene ic analysis, including sample codes, num-
be o indi iduals (n), geog aphic coo dina es (la i ude and longi ude), and yea o collec ion. Sam-
ples ma ked wi h an as e isk (*) we e also examined o mo phological and me is ic cha ac e is ics.
Codes Samples (n) Coo dina es Yea
FRISK3481 1 41°21'05.0"N, 8°40'54.6"W 2017
PEI0456–PEI0462 7 41°20'48.9"N, 8°35'48.0"W 2020
Scep_1_13* 1 41°22'22.7"N, 8°42'07.6"W 2024
Squa1-Squa10* 10 41°24'43.8"N, 8°22'46.2"W 2025
Squa11-Squa13* 3 41°28'18.1"N, 8°20'48.0"W 2025
Squa14-Squa28* 15 41°26'57.1"N, 8°21'00.2"W 2025
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Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
genus Squalius (Suppl. ma e ial 3). Up o 10 sequences pe each species we e
downloaded wi h ca e o co e mul iple d ainages o widesp ead axa; o he wise,
10 andom sequences we e downloaded. Addi ionally, h ee sequences om he
genus Ibe ochond os oma Robalo, Almada, Le y & Doad io, 2007 we e added as
ou g oups. Sequences we e aligned using AliView (La sson 2014) implemen ing
he MUSCLE algo i hm. Fo he nuclea ma ke s (eg 2b and ac b) haplo ypes
we e phased in DnaSP . 5.10.1 (Lib ado and Rozas 2009). Gene ees o each
ma ke we e es ima ed using he maximum likelihood me hod implemen ed in
IQ ee (Nguyen e al. 2015). The same p og am was used o selec o he bes
subs i u ion model conside ing pa i ions based on codon posi ion. Node suppo
was ob ained h ough 1000 as boo s aps. IQ ee was un using he web se e
W-IQ-TREE (T i inopoulos e al. 2016). The ob ained ee was isualised and
cus omised using FigT ee e . 1.4.4 (h p:// ee.bio.ed.ac.uk/so wa e/ ig ee/).
Social media and ci izen science da a collec ion
The use o social media in o ma ion abou in asi e ishes has p o en aluable o de-
ec ing and iden i ying new in asi e ish (Ribei o and Ve íssimo 2014; Kalous e al.
2018), desc ibing spa ial in asion pa e ns (Banha e al. 2017b), and upda ing he
dis ibu ion o ecen ly a i ed in asi e species (Ma elo e al. 2021). To upda e he
dis ibu ion o alien chubs in he A e Ri e , ex ensi e da a collec ion was conduc ed
using a ious sou ces o in o ma ion as ecommended by T ica ico (2022), including
Facebook and ci izen science pla o ms. Publicly a ailable social media pos s ela ed
o ishing o indi iduals belonging o he genus Squalius we e collec ed om Face-
book, a popula pla o m among angle s in Po ugal (see Gago e al. 2016; Banha
e al. 2017b). The da a collec ion p ocess adhe ed s ic ly o e hical guidelines o
using social media in ishe ies esea ch, ensu ing ha no pe sonal in o ma ion was
eco ded and ha no o iginal use con en was sha ed publicly wi hou pe mission
(Monkman e al. 2018). Sea ches we e conduc ed wi hin ishing- ocused g oups
whe e ca ches p edominan ly occu in Po uguese wa e s ollowing he guidelines
desc ibed in Chowdhu y e al. (2024). The ime window o ou sea ch was om July
2019 ( he c ea ion da e o he oldes ishing g oup) o Decembe 2024. Keywo ds
used o iden i y ele an pos s included he species’ common name and i s a ian s,
such as “chub”, “escalo”, “leucisco”, “che esne” and he nickname “ u as do A e”.
Online sea ches we e also conduc ed on BioDi e si y4All (h ps://www.biodi e si-
y4all.o g/), a Po uguese biodi e si y ci izen science pla o m connec ed o he in-
e na ional p ojec iNa u alis . Species iden i ica ion was p ima ily based on he i-
sual examina ion o pho og aphs accompanying he pos s, using c i e ia es ablished
by Gago e al. (2016). Only pos s explici ly ela ed o ec ea ional ishing o ish
belonging o he genus Squalius we e analysed. These pos s ea u ed con en such as
ideos and pho og aphs o he ish specimens a e being caugh . Fo each pos , he
p ima y objec i e was o ex ac in o ma ion on he da e and loca ion o he ca ch.
Publica ions lacking a pho og aphic eco d o he specimen we e no conside ed o
p esence eco ds. All pos s we e manually e iewed o con i m species iden i ica ion
and e i y ca ch loca ions. Whe e possible, he backg ound o he pho og aphs was
c oss- e e enced wi h sa elli e image y a ailable on Google S ee View o inc ease
he accu acy o he loca ion da a ( ollowing he geo e e encing s eps on Chowdhu y
e al. 2024). The in o ma ion collec ed was compiled in o a da ase lis ing loca ions
and da es o he ca ches, se ing as he basis o u he analysis.
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Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
Resul s
Mo phological analysis
A e he isual examina ion o he 29 specimens, 10 we e iden i ied as S. cephalus
and 19 as S. squalus (Bonapa e, 1837). The S. cephalus indi iduals had s anda d
leng hs (SL) o 107–169 mm and body masses o 20.5–87 g. Fig. 1 illus a es wo
S. cephalus indi iduals: he specimen in Fig. 1a (S_cep_1_13), collec ed in 2024,
and he specimen in Fig. 1b (Squa13), collec ed in 2025. Bo h indi iduals (Fig.
1a, b) we e s eamlined, la e ally comp essed, and usi o m, lacking a en al keel
an e io o he anus. Each displayed a poin ed head, a sligh ly a ched do sal p o-
ile, and a mo e con ex en al p o ile, o ming a clea head- unk discon inui y.
Flanks showed a e icula e pa e n om black pigmen along scale ma gins. Ven al
and anal ins we e i id o ange- ed in Fig. 1a bu less in ensely colou ed in Fig. 1b,
which showed only ed-o ange pigmen s in he en al and pel ic ins.
a)
b)
c)
Figu e 1. Rep esen a i e hyb id specimens be ween Squalius cephalus and S. squalus cap u ed in he A e Ri e , illus a ing he obse ed
mo phological con inuum. a. hyb id indi idual wi h p edominan ly Squalius cephalus-like mo phological ai s (169 mm o al leng h,
87.0 g); b. hyb id indi idual wi h p edominan ly Squalius cephalus-like mo phological ai s (135 mm o al leng h, 41.9 g); c. hyb id ex-
hibi ing mo e Squalius squalus-like ai s (125 mm o al leng h, 30.1 g). All h ee indi iduals we e con i med as hyb ids by mi ochond ial
and nuclea ma ke s and ha e been deposi ed in he Museu Bocage collec ion, Museu Nacional de His ó ia Na u al e da Ciência (Uni e -
si y o Lisbon, Po ugal), unde accession numbe s MB05-004015, MB05-004008 and MB05-004012, espec i ely.
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Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
The S. squalus specimens anged om 46–134 mm in SL and weighed be ween
1.27 and 40.12 g. Fig. 1c shows a ep esen a i e S. squalus (Squa19), which exhib-
i ed an elonga ed body lacking he p onounced do sal discon inui y cha ac e is ic
o S. cephalus. A ow o dense black pigmen do s along he ou e ma gins o he
scales on he back and lanks p oduced a egula e icula e pa e n. Body colo -
a ion anged om b onze o b own, while he en al and anal ins we e ypically
black o hyaline, wi hou he dis inc i e ed pigmen a ion seen in S. cephalus.
Al hough all 39 mo phome ic a iables exhibi ed o e lapping alues be ween
S. cephalus and S. squalus, a subse o 12 ai s was ound o signi ican ly di -
e en ia e he body shape and p opo ional ana omy o he wo species (Suppl.
ma e ial 1). Squalius cephalus exhibi ed a signi ican ly longe pos o bi al dis ance
(47.3–51.7% HL s. 41.9–49.1% HL; p < 0.001) compa ed o S. squalus. The
head mo phology u he di e ed wi h S. cephalus ha ing g ea e head leng h ela-
i e o s anda d leng h (24.9–27.9% SL s. 24.2–26.8% SL; p < 0.05) and g ea e
head leng h as a p opo ion o p edo sal dis ance (45.7–52.2% s. 44.7–48.9%;
p < 0.05). Squalius cephalus exhibi ed a signi ican ly deepe body p o ile a he do -
sal in o igin (23.4–26.2% SL s. 20.6–25.2% SL; p < 0.01) and a g ea e dis ance
be ween he pec o al and pel ic in o igins (25.2–29.9% SL s. 23.7–29.6% SL;
p < 0.05). In con as , S. squalus displayed a hicke caudal peduncle ela i e o
body dep h (40.0–46.4% s. 35.3–43.2%; p < 0.01). Rega ding in mo phology,
S. squalus exhibi ed signi ican ly g ea e do sal and anal in dep hs ela i e o body
dep h (do sal in: 80.1–95.9% s. 78.2–89.2%, p < 0.01; anal in: 63.5–89.9%
s. 51.1–73.8%, p < 0.01), as well as a la ge anal in dep h ela i e o s anda d
leng h (15.5–19.1% SL s. 12.8–17.6% SL; p < 0.05). Squalius squalus had sig-
ni ican ly la ge eyes ela i e o bo h s anda d leng h and head leng h (6.2–8.4%
SL s. 4.9–6.9% SL; 23.3–29.9% HL s. 19.8–26.8% HL; p < 0.01 o bo h). I
also exhibi ed g ea e head dep h a he nape compa ed o S. cephalus (68.3–79.3%
HL s. 67.4–75.2% HL; p < 0.01). The numbe o b anched ays in he anal
in di e ed signi ican ly, wi h S. squalus showing mos ly 9½ ays ( a ely 8½) and
S. cephalus 8½ (Suppl. ma e ial 1). O he me is ic ai s did no di e signi ican ly.
Gene ic analysis
Sequence in o ma ion (in GenBank as PV686259–PV686389) was success ully
e ie ed o all samples o cy b and eg 2b, while o ac b sequencing ailed o
wo indi iduals (Table 2, Suppl. ma e ial 2). These we e added in o a inal align-
men con aining addi ional 217, 44, and 62 sequences om 37, 9, and 11 Squalius
species o cy b, eg 2b, and ac b, espec i ely. The inal ma ix o he cy b se-
quences consis ed o 1,156 cha ac e s, con aining 453 a iable cha ac e s wi h 401
pa simony in o ma i e si es. The alignmen s o eg 2b and ac b genes comp ised
638 and 859 cha ac e s, espec i ely, wi h 20 and 69 a iable si es, o which 17 and
64 we e in o ma i e. Fo cy b, he samples o he uniden i ied Squalius clus e ed
in o h ee g oups wi h ela i ely high suppo alues (>80%). Six indi iduals clus-
e ed wi h S. cephalus, one wi h he g oup con aining all S. ca oli e ii sequences,
while he emaining indi iduals g ouped wi h S. squalus (Fig. 2). This las g oup
also includes he Ad ia ic-Hellenic Squalius clade, comp ising S. z manjae Ka a-
man, 1928, S. lucumis (Bianco, 1983) and S. p espensis (Fowle , 1977). Unknown
Squalius sequences o he nuclea genes we e also clus e ed in o h ee g oups (Fig.
2), he main di e ence being ha he S. squalus g oup o ms a poly omy a he
256
NeoBio a 102: 249–268 (2025), DOI: 10.3897/neobio a.102.148337
Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
base o S. cephalus and S. ca oli e ii o eg 2b, and S. cephalus is pa aphyle ic
o S. squalus o ac b. Fu he mo e, in bo h gene ees S. ca oli e ii sequences do
no o m a monophyle ic g oup being in e mingled wi h o he Ibe ian Squalius
Table 2. Summa y o mo phological and molecula esul s. Mo phological species iden i ica ion was based on iden i ica ion keys (Doad-
io and Ca mona 2006; Ko ela and F eyho 2007). Measu emen s we e ounded o he nea es 0.1 mm wi h e nie callipe s. Molecula
analyses include esul s o he mi ochond ial cy och ome b gene (“cy b”; amplicon ~1140 bp, Za doya and Doad io 1998), and haplo-
ypes, labelled as “H1” and “H2”, o he wo nuclea genes, he ea ly g ow h esponse 2b gene (“eg 2b”; ~747 bp, Waap e al. 2011), and
β-ac in gene (“Be a ac in” ; ~933 bp, Robalo e al. 2006).
Sample code Fi s sc eening O igin Mo phological
ID cy b eg 2b Be a ac in Molecula ID
H1 H2 H1 H2
Squa1 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa2 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. cephalus S. cephalus S. cephalus S. cephalus S. squalus x cephalus
Squa3 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus Fail Fail S. squalus x cephalus
Squa4 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa5 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa6 Unknown Squalius sp. A e Ri e S. squalus S. cephalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
Squa7 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
Squa8 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. cephalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa9 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. cephalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa10 Unknown Squalius sp. A e Ri e S. cephalus S. cephalus S. cephalus S. cephalus S. squalus S. cephalus S. squalus x cephalus
Squa11 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. cephalus Ibe ian
lineage
S. squalus Ibe ian
lineage
S. squalus x cephalus x
ca oli e ii
Squa12 Unknown Squalius sp. A e Ri e S. squalus S. cephalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus
Squa13 Unknown Squalius sp. A e Ri e S. cephalus S. squalus S. squalus S. squalus S. cephalus S. cephalus S. squalus x cephalus
Squa14 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus
Squa15 Unknown Squalius sp. A e Ri e S. cephalus S. squalus S. squalus S. cephalus S. cephalus S. cephalus S. squalus x cephalus
Squa16 Unknown Squalius sp. A e Ri e S. cephalus S. ca oli e ii S. cephalus S. cephalus S. squalus S. cephalus S. squalus x cephalus x
ca oli e ii
Squa17 Unknown Squalius sp. A e Ri e S. cephalus S. cephalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
Squa18 Unknown Squalius sp. A e Ri e S. cephalus S. cephalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa19 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus
Squa20 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa21 Unknown Squalius sp. A e Ri e S. cephalus S. squalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus
Squa22 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. cephalus S. cephalus S. squalus S. squalus S. squalus x cephalus
Squa23 Unknown Squalius sp. A e Ri e S. cephalus S. ca oli e ii S. squalus S. cephalus S. squalus S. squalus S. squalus x cephalus x
ca oli e ii
Squa24 Unknown Squalius sp. A e Ri e S. squalus S. cephalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
Squa25 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus
Squa26 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. cephalus S. squalus S. cephalus S. squalus x cephalus
Squa27 Unknown Squalius sp. A e Ri e S. squalus S. squalus S. squalus S. squalus Fail Fail S. squalus
Squa28 Unknown Squalius sp. A e Ri e S. cephalus S. cephalus S. cephalus S. cephalus S. squalus S. cephalus S. squalus x cephalus
S_cep__1_13 Unknown Squalius sp. A e Ri e S. cephalus S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
FRISK3481 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
PEI0456 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus
PEI0457 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
PEI0458 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus S. squalus S. squalus S. squalus
PEI0459 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
PEI0460 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus S. squalus S. cephalus S. squalus x cephalus
PEI0461 Unknown Squalius sp. A e Ri e No a ailable S. squalus S. squalus S. squalus Fail Fail S. squalus
FRISK481 S. ca oli e ii A e Ri e (na i e) No a ailable S. ca oli e ii S. ca oli e ii S. ca oli e ii Fail Fail S. ca oli e ii
FRISK 477 S. ca oli e ii A e Ri e (na i e) No a ailable S. ca oli e ii S. ca oli e ii S. ca oli e ii Fail Fail S. ca oli e ii
FRISK 478 S. ca oli e ii A e Ri e (na i e) No a ailable S. ca oli e ii S. ca oli e ii S. ca oli e ii Fail Fail S. ca oli e ii
FRISK 480 S. ca oli e ii A e Ri e (na i e) No a ailable S. ca oli e ii S. ca oli e ii S. ca oli e ii Fail Fail S. ca oli e ii
SC133 5L S. cephalus Czechia (na i e) No a ailable Fail S. cephalus S. cephalus Fail Fail S. cephalus
SC73 5L S. cephalus Czechia (na i e) No a ailable Fail S. cephalus S. cephalus S. cephalus S. cephalus S. cephalus
SC77 5L S. cephalus Czechia (na i e) No a ailable S. cephalus S. cephalus S. cephalus S. cephalus S. cephalus S. cephalus
SC82 5L S. cephalus Czechia (na i e) No a ailable S. cephalus S. cephalus S. cephalus Fail Fail S. cephalus
SC67 5L S. cephalus Czechia (na i e) No a ailable S. cephalus S. cephalus S. cephalus Fail Fail S. cephalus
SC72 5L S. cephalus Czechia (na i e) No a ailable Fail S. cephalus S. cephalus Fail Fail S. cephalus
257
NeoBio a 102: 249–268 (2025), DOI: 10.3897/neobio a.102.148337
Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
Figu e 2. Recons uc ed phylogeny o he cy och ome b (cy b), ea ly g ow h esponse 2b (eg 2b), and be a-ac in (ac b) sequences o
Squalius samples included in p esen s udy oge he wi h sequences a ailable in NCBI. The new sequences we e colo ed based on he
g oupings hey o m: ed S. ca oli e ii / Ibe ian Squalius lineage, blue S. squalus, and g een S. cephalus.
Boo s ap alues:
>70%
>80%
>90%
0.03
S. o galensis
S. cephalus KU302640
S. illy icusOP728012
S. pla yceps
S. ca oli e ii AJ69845
S. illy icus AJ251094
S. illy icus MN166114
S. enellus
S. lepidusAJ252812
Squa23_Ap25
S. ca oli e iiFRISK481
S. o ien alis; S. u cicus
Squa6_Ap25
S. cephalus MG8067701
S. a da ensis MK482045
S. cephalus, S. o pheus
Remaining unknown Squalius
S. laie anus
S. o pheus
S. lepidus
S. pu sakensis
S. cephalus
S. cephalus KU302635
S. ellowesii OP7228023
S. ca oli e ii FRISK477
S. i ideus
S. cephalus MN166129
S. s allize
S. squalus OP728021
S. cephalus PP928778
S. py enaicus; S. albu noides
S. malaci anus
S. ca oli e ii FRISK480
Squa17_Ap25
S. lepidus
Squa28_Ap25
S. z manjae
S. aphipsi
S. ca oli e ii PP928777
S. illy icus MG806702
Squa24_Ap25
S. a adensis
S. ca oli e ii AJ698452
S. illy icusOR791606
S. illy icus HM560183
S. cii
S. ca oli e iiAJ698453
S. illy icusOR791605
Squa12_Ap25
S. a da ensis
S. mic olepis
S. squalus KU302634
S. cephalus MN166128
S. illy icus MN166111
I. lemmingii OR381970
Squa16_Ap25
Squa10_Ap25
S. be akOQ797999
S. alen inus
S. ca oli e ii
S. squalus PP928780
S. pam o icus
S. lepidus
I. lusi anicumKU368953
S. illy icus OR791604
S. squalus KU302627
S. squalus JQ652368
S. peloponensis
S. cephalus EU791877
S. cas ellanus
S. cephalus KU302632
S. agdamicusOQ798019
S. squalus, S. z manjae
S. illy icus HM560184
S. squalus, S. lucumonis
S. keadicus
S. cephalus OP728027
S. ca oli e iiMT817384
S. ca oli e ii FRIS478
S. lucumonis
S. cephalus SC675L
Squa18_Ap25
S. s allize
S. p espensis
Squaliussqualus clade
Squaliuscephalusclade
Squaliusca oli e i clade
0.002
Squa19_H1
I. lusi anicum KC836287.1_H2
S. cephalus SC825L_H2
Squa4_H2
Squa20_H1
PEI_458_H2
Squa16_H1
S. cephalus SC675L_2L_H2
F isk3481_H1
S. cephalusJN413311.1_H2
PEI_459_H1
Squa7_H2
Squa14_H1
S. alen inus
I. lemmingi
Squa8_H2
S. cephalus SC775L_H2
PEI_462_H2
Squa12_H2
S. py enaicus; S. cas ellanus
Squa28_H2
Squa19_H2
Squa15_H1
S. cephalusSC133_5L_Dyje_B ecla _H2
S. cephalus SC133_5L_Dyje_B ecla _H1
S. squalus JASFWY011386779_H2
PEI_456_H1
Squa7_H1
S. py enaicus JN413320.1_H2
Squa12_H1
S. cephalus SC775L_H1
S. ca oli e ii MT051823.1_H1
Squa6_H1
Squa22_H2
S. cephalus SC675L_H2
S. ca oli e ii MT051824.1_H1
S. cephalus SC825L_H1
S. ca oli e ii MT051823.1_H2
Squa1_H2
Squa14_H2
S. ca oli e ii
Squa11_H2
Squa17_H2
Squa3_H1
PEI_457_H2
S. ca oli e ii
Squa28_H1
Squa4_H1
Squa2_H2
Squa5_H1
Squa6_H2
PEI_458_H1
S. malaci anus; S. py enaicus; S. alen inus
Squa27_H2
PEI_459_H2
S. ca oli e ii; S. py enaicus
S. py enaicus JN413320.1_H1
S. py enaicus KC836325.1_H1
S. squalus JASFWY011386779_H1
Squa2_H1
S. ca oli e ii JN413314.1_H1
Squa22_H1
Squa25_H1
S_cep__1_13_H2
Squa1_H1
Squa13_H2
Squa26_H2
S. cephalusSC735L_H1
Squa25_H2
Squa21_H1
Squa5_H2
S. cephalusSC735L_H2
Squa21_H2
S. cephalus SC675L_H1
Squa9_H2
S. malaci anusMT051748.1_H2
Squa8_H1
Squa16_H2
S. cephalusJN413311.1_H1
Squa13_H1
S. ca oli e ii MT051840.1_H1
S. malaci anusMT051747.1_H2
Squa23_H2
Squa17_H1
Squa24_H2
Squa23_H1
S. o galensis; S. alen inus
S. py enaicus
Squa18_H2
Squa9_H1
I. lusi anicum KC836287.1_H1
Squa15_H2
S. ca oli e ii MT051824.1_H2
Squa18_H1
PEI_457_H1
PEI_456_H2
F isk3481_H2
Squa24_H1
Squa10_H1
Squa3_H2
PEI_460_H1
PEI_460_H2
S_cep__1_13_H1
S. cephalus
Squa10_H2
S. cephalus SC675L_2L_H1
PEI_462_H1
S. malaci anusMT051747.1_H1
Squa11_H1
Squa26_H1
S. py enaicus MT051791.1_H1
S. ca oli e ii MT051818.1_H1
Squa27_H1
Squa20_H2
Squalius squalus clade
Squalius cephalusclade
Ibe ian Squali
clade
0.003
S. ca oli e ii
Squa28_H2
S. py enaicus
Squa24_H2
Squa13_H1
S. albu naides
S. a adensis; S. o galensis
Squa6_H2
I._lusi anicum_DQ061941.1_H1
F isk3481_H2
S. squalus
Squa19_H2
S. cephalus_SC735L_H1
S. cephalus_OX439247_H2
S_cep_1_H2
S. ca oli e ii_MT051720.1_H2
Squa26_H2
Squa2_H2
Squa13_H2
PEI0460_H2
I._lusi anicum_DQ061941.1_H2
Squa17_H2
A. hispanica
S. cephalus_OX439247_H1
S. malaci anus
PEI0457_H2
PEI0459_H2
Squa11_H2
Squa10_H2
S. py enaicus; S. cas ellanus
Squa15_H2
Squa15_H1
S. idus_DQ061947.1_H1
Remaining unknown Squalus
Squa21_H2
Squa7_H2
Squa12_H2
S. cephalus_SC675L_H1
S. ca oli e ii
S. alen inus
S. ca oli e ii
S. cephalus
Squa16_H2
S. py enaicus
Squa2_H1
S. py enaicus
S. idus_DQ061947.1_H2
I._lemmingii_DQ061940.1_H2
S. cephalus_SC675L_H2
S. py enaicus
S. malaci anus
I._lemmingii_DQ061940.1_H1
Squaliussqualus
clade
Squalius cephalus clade
Ibe ian Squaliusclade
cy b eg 2b
ac b
species. In e es ingly, se e al indi iduals ca ied he e ozygous geno ypes o haplo-
ypes g ouping wi h di e en species (Suppl. ma e ial 3). These included 13 indi-
iduals o eg 2b and 17 o ac b, wi h he mos common combina ion in ol ing
264
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Supplemen a y ma e ial 1
Resul s o S uden ’s - es s compa ing mo phome ic and me is ic a iables
be ween hyb id specimens om he A e Ri e wi h p edominan ly Squalius
cephalus-like ai s and hose wi h p edominan ly Squalius squalus-like ai s
Au ho s: Ch is os Gkenas, So ia Noguei a, Manuel Cu o, Diogo Dias, Diogo Ribei o, Rui Ri aes,
Joana Ga ido Noguei a, Manuel Lopes-Lima, Ma ia Judi e Al es, Filipe Ribei o
Da a ype: xlsx
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License (h p://openda a-
commons.o g/licenses/odbl/1.0/). The Open Da abase License (ODbL) is a license ag eemen
in ended o allow use s o eely sha e, modi y, and use his Da ase while main aining his same
eedom o o he s, p o ided ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/neobio a.102.148337.suppl1
Supplemen a y ma e ial 2
Vouche specimen in o ma ion and GenBank accession numbe s
Au ho s: Ch is os Gkenas, So ia Noguei a, Manuel Cu o, Diogo Dias, Diogo Ribei o, Rui Ri aes,
Joana Ga ido Noguei a, Manuel Lopes-Lima, Ma ia Judi e Al es, Filipe Ribei o
Da a ype: xlsx
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License (h p://openda a-
commons.o g/licenses/odbl/1.0/). The Open Da abase License (ODbL) is a license ag eemen
in ended o allow use s o eely sha e, modi y, and use his Da ase while main aining his same
eedom o o he s, p o ided ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/neobio a.102.148337.suppl2
268
NeoBio a 102: 249–268 (2025), DOI: 10.3897/neobio a.102.148337
Ch is os Gkenas e al.: In eg a i e axonomy iden i ies non-na i e Squalius
Supplemen a y ma e ial 3
Sequences downloaded om NCBI o complemen molecula analysis
Au ho s: Ch is os Gkenas, So ia Noguei a, Manuel Cu o, Diogo Dias, Diogo Ribei o, Rui Ri aes,
Joana Ga ido Noguei a, Manuel Lopes-Lima, Ma ia Judi e Al es, Filipe Ribei o
Da a ype: xlsx
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License (h p://openda a-
commons.o g/licenses/odbl/1.0/). The Open Da abase License (ODbL) is a license ag eemen
in ended o allow use s o eely sha e, modi y, and use his Da ase while main aining his same
eedom o o he s, p o ided ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/neobio a.102.148337.suppl3
Supplemen a y ma e ial 4
Loca ion o eco ds o he non-na i e Squalius hyb ids in he A e Ri e , de i ed
om social media and ci izen science pla o ms
Au ho s: Ch is os Gkenas, So ia Noguei a, Manuel Cu o, Diogo Dias, Diogo Ribei o, Rui Ri aes,
Joana Ga ido Noguei a, Manuel Lopes-Lima, Ma ia Judi e Al es, Filipe Ribei o
Da a ype: xlsx
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License (h p://openda a-
commons.o g/licenses/odbl/1.0/). The Open Da abase License (ODbL) is a license ag eemen
in ended o allow use s o eely sha e, modi y, and use his Da ase while main aining his same
eedom o o he s, p o ided ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/neobio a.102.148337.suppl4
