Biodi e si y Da a Jou nal 13: e168586
doi: 10.3897/BDJ.13.e168586
Da a Pape
DNA ba coding o Messo an s o Bulga ia wi h
insigh s in o hei axonomic di e si y
Albena Lape a-Gjono a , Monika P ama a o a , Lech Bo owiec , Ilia Gjono , Rumyana Kos o a ,
Ros isla Bekchie , Simeon Bo isso
‡ So ia Uni e si y, Facul y o Biology, So ia, Bulga ia
§ Na ional Museum o Na u al His o y, Bulga ian Academy o Sciences, So ia, Bulga ia
| Uni e si y o W oclaw, W oclaw, Poland
¶ Ins i u e o Biodi e si y and Ecosys em Resea ch, Bulga ian Academy o Sciences, So ia, Bulga ia
Co esponding au ho : Albena Lape a-Gjono a (gjono [email protected])
Academic edi o : F ancisco Hi a Ga cia
Recei ed: 12 Aug 2025 | Accep ed: 20 Oc 2025 | Published: 04 No 2025
Ci a ion: Lape a-Gjono a A, P ama a o a M, Bo owiec L, Gjono I, Kos o a R, Bekchie R, Bo isso S (2025)
DNA ba coding o Messo an s o Bulga ia wi h insigh s in o hei axonomic di e si y. Biodi e si y Da a Jou nal
13: e168586. h ps://doi.o g/10.3897/BDJ.13.e168586
Abs ac
Backg ound
Despi e ongoing e o s o ca alogue Eu opean an species, s udies ocusing on he
gene ic di e si y o Balkan an s emain limi ed. An in eg a i e app oach combining
mo phology, gene ics, ecology and biogeog aphy is p e e able o accu a ely iden i ying
species and esol ing axonomic unce ain ies, pa icula ly amongs challenging insec
axa, such as he an s in he genus Messo (Hymenop e a, Fo micidae).
New in o ma ion
In his s udy, we analyse an s o he genus Messo using DNA ba code sequences, wi h a
pa icula ocus on he Bulga ian auna. A o al o 85 COI sequences we e examined,
including 84 om Messo specimens and one om Aphaenogas e , which was used as
an ou g oup. O hese, 81 sequences we e newly gene a ed, while ou we e e ie ed
om GenBank. The majo i y o specimens we e collec ed in Bulga ia (61), wi h addi ional
‡ ‡,§ | ‡ ‡
§ ¶
© Lape a-Gjono a A 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.
samples om G eece (13), Tü kiye (4), Albania (1) and No h Macedonia (2), p o iding
b oade gene ic and geog aphic ep esen a ion.
Al hoge he , 11 Messo mo phospecies we e iden i ied, based on specimens used o
molecula analysis. To assess he deg ee o cong uence be ween mo phological and
molecula da a, six species delimi a ion analyses we e conduc ed: RESL, GMYC, ASAP,
ABGD, bPTP and mPTP. In addi ion, haplo ype ne wo k analysis o all sequences
iden i ied 35 dis inc and cohe en ly clus e ed haplo ypes, p o iding insigh s in o gene ic
di e si y.
The COI ba code egion success ully dis inguished Messo wasmanni K ausse, 1910, M.
oe zeni Fo el, 1910 and M. ibe icus San schi, 1931. In con as , species pai s, such as M.
a anasso ii A anasso , 1982 and M. c e icus Sala a & Bo owiec, 2019, as well as M.
pon icus S eine e al., 2018 and M. hellenius Agos i & Collingwood, 1987, could no be
eliably di e en ia ed using COI da a. Fu he mo e, Messo s uc o (La eille, 1798)
showed high in aspeci ic gene ic di e si y. Finally, he s uc o and ins abilis species
g oups we e eco e ed wi h mode a e o high suppo in bo h Maximum Likelihood and
Bayesian In e ence analyses, con i ming ha M. oe zeni and M. hellenius belong o he
s uc o g oup.
Ou esul s p o ide a e e ence o u u e esea ch and unde sco e he alue o
in eg a i e axonomic app oaches in an biodi e si y s udies.
Keywo ds
he Balkans, COI, species delimi a ion, Fo micidae, My micinae
In oduc ion
The genus Messo , commonly known as ha es e an s, consis s o ypically g ani o ous
species in ol ed in seed dispe sal, nu ien cycling and mic oclima e modi ica ion in
su ace soil laye s (Camme aa e al. 2002, Plowes e al. 2013, El Boukh issi e al. 2023).
These an s ypically inhabi a id and semi-a id en i onmen s, wi h 134 species cu en ly
ecognised wi hin he Palaea c ic, A o opical and O ien al biogeog aphic egions
(B ans e e e al. 2022, Sala a e al. 2023, Bol on 2025). While species ichness is
highes in No h A ica and he Middle Eas , he en i onmen al condi ions and
biogeog aphic his o y o he Sou he n Balkans also a ou he p esence o a subs an ial
numbe o Messo species (Bo owiec 2014, Janicki e al. 2016, Guéna d e al. 2017,
Wang e al. 2023, Ju é e al. 2025a).
Comp ehensi e mode n s udies on he species composi ion and dis ibu ion o Messo
species in Bulga ia emain insu icien . Two ecen species e isions ha e add essed
some species wi hin he genus ound in he coun y. One such e ision, ocusing on he
Eu opean species o he s uc o g oup (S eine e al. 2018), included Bulga ian ma e ial,
al hough samples o igina ed om only a ew locali ies. This s udy ecognised h ee
2Lape a-Gjono a A e al
species om he g oup in Bulga ia: Messo s uc o (La eille, 1798), M. pon icus S eine
e al., 2018 (wi h ype locali y in Bulga ia) and M. ibe icus San schi, 1931. Addi ionally,
wo o he species, M. mca hu i S eine e al., 2018 and M. hellenius Agos i &
Collingwood, 1987, we e ecen ly epo ed om he coun y by Lape a-Gjono a and
Bo owiec (2022). The la e was no included in he e ision o his g oup by S eine e al.
(2018), bu was ecognised as such by Bo owiec and Sala a (2025). Fu he mo e, a
phylogene ic analysis by Ju é e al. (2025a) e ealed Messo oe zeni Fo el, 1910, a
well-known species om Bulga ia, as he six h membe o he s uc o g oup in he
coun y. Messo wasmanni K ausse, 1910 and M. a anasso ii A anasso , 1982, he la e
wi h i s ype locali y in Bulga ia, a e he only ep esen a i es o he ins abilis g oup
cu en ly known om Bulga ia. This species g oup om he Eas e n Medi e anean
egion was ecen ly e ised (Sala a e al. 2023). The e ision included a de ailed e-
desc ip ion o M. a anasso ii, con i ming i s alidi y as a dis inc species and epo ing
addi ional locali ies in bo h Bulga ia and G eece. The la es s udies on Messo in he
Palaea c ic Region ha e e-e alua ed he ea lie eco ds and concluded ha ou
species p e iously epo ed om Bulga ia (M. ba ba us (Linnaeus, 1767), M. caducus
(Vic o , 1839), M. capi a us (La eille, 1798) and M. concolo San schi, 1927) do no
ac ually occu in he Balkans. Consequen ly, eigh Messo species a e cu en ly
ecognised in Bulga ia: M. a anasso ii, M. wasmanni, M. oe zeni, M. s uc o , M.
mca hu i, M. pon icus, M. hellenius and M. ibe icus ( Lape a-Gjono a and An ono a
2022).
The sca ci y o his o ical desc ip ions, coupled wi h high mo phological a iabili y wi hin
species and occu ences o hyb idisa ion and e en xenopa i y, makes he genus Messo
axonomically and biologically challenging (Schlick-S eine e al. 2006, S eine e al. 2011
, Romiguie e al. 2017, S eine e al. 2018, Saa e al. 2023, Ju é e al. 2025a, Ju é e al.
2025b). This necessi a es he applica ion o complex app oaches alongside he
mo phological one o esol e species delimi a ions.
DNA ba coding using mi ochond ial cy och ome c oxidase I (COI) gene agmen s has
p o en o be an e icien me hod o species iden i ica ion and biodi e si y assessmen ,
including an s o Messo genus ( Schlick-S eine e al. 2006, S eine e al. 2018,
S ohmaie e al. 2025) and o he S enammini (Cen o ame e al. 2018, Gómez e al. 2018,
Galkowski e al. 2019, Schi ani e al. 2022, Zięcina e al. 2024). Howe e , COI is no
uni e sally eliable o an iden i ica ion and species delimi a ion due o biological ac o s,
such as incomple e lineage so ing, in og ession, hyb idisa ion, NUMTs and
endosymbion e ec s, as well as echnical issues like gaps in e e ence lib a ies and
h eshold inconsis encies (Hu s and Jiggins 2005, Da as and A on 2015, Romiguie e
al. 2017). Despi e hese limi a ions, i emains a apid, cos -e ec i e ool wi h easonable
species-le el esolu ion, suppo ed by widely-used p ime s and ex ensi e sequence
eposi o ies (deWaa d e al. 2019, Ma oni e al. 2024, Onyinyechi e al. 2025).
Acco dingly, expanding ba coding e o s in unde explo ed egions, such as he Balkans,
is c ucial o imp o ing ou unde s anding o species di e si y and e olu iona y
ela ionships. While a la ge-scale ba coding p ojec o Eu opean an s is unde way
DNA ba coding o Messo an s o Bulga ia wi h insigh s in o hei axonomic ... 3
(Menche i e al., unpublished), u he esea ch speci ically a ge ing he gene ic di e si y
o Balkan Messo an s will p o ide aluable insigh s in o axonomy and phylogeny.
Sampling me hods
Sampling desc ip ion:Specimens o DNA ba coding we e p ima ily selec ed, based on
mo phology and hei o igin om di e se collec ion si es ac oss he coun y.
Mo phological iden i ica ions ollowed S eine e al. (2018) and Bo owiec and Sala a
(2025).
Molecula analyses: DNA ex ac ion, ampli ica ion and sequencing o he s anda d 658
bp COI ba code egion we e pe o med by he Canadian Cen e o DNA Ba coding
(CCDB) using he p ime s LepF1 and LepR1 (Hebe e al. 2004). DNA was ex ac ed
om he hind legs o specimens p ese ed in e hanol. In o al, 84 COI Messo sequences
we e analysed, o which 80 we e newly gene a ed. The ollowing ou sequences we e
ob ained om GenBank and we e included in he phylogene ic analyses: KT184551
(Messo s uc o ), KT184569 (M. mca hu i), KT184511 (M. ibe icus) om S eine e al.
(2018) and DQ074353 (M. pon icus) om Schlick-S eine e al. (2006). The sequence o
Aphaenogas e es ae Eme y, 1915 gene a ed in he cu en s udy was selec ed as an
ou g oup in he phylogene ic analyses. All 81 sequences gene a ed in his s udy a e
deposi ed in he Ba code o Li e Da a Sys em (BOLD) unde he BGMES p ojec , whe e
collec ion in o ma ion and pho os o each specimen a e also p o ided. Vouche
specimens a e p ese ed in he Zoological Collec ion o So ia Uni e si y (BFUS).
To assess he deg ee o cong uence be ween mo phological iden i ica ion conduc ed
p io o he molecula da a, mul iple species delimi a ion app oaches we e applied o he
molecula da ase . Sequence alignmen and imming we e pe o med using MEGA .12
(Kuma e al. 2024). In he BOLD sys em, he sequences we e assigned o Ba code Index
Numbe s (BINs), an algo i hm-based app oach o delinea e ope a ional axonomic uni s,
which we e au oma ically calcula ed o eco ds by Re ined Single Linkage (RESL)
analysis. These BINs ha e a unique iden i ie and p o ide a good p oxy o species
(Ra nasingham and Hebe 2013). To es ima e gene ic dis ances and enable
compa ison, pai wise dis ances we e calcula ed unde he Kimu a 2-pa ame e (K2P)
model using MEGA .12 (Kuma e al. 2024), whe ea e species bounda ies we e es ed
wi h Assemble Species by Au oma ic Pa i ioning (ASAP) and Au oma ic Ba code Gap
Disco e y (ABGD). Subsequen ly, ul ame ic ees we e gene a ed in BEAST . 10.5.0
(Baele e al. 2025) wi h a s ic clock, coalescen ee p io and 100 million gene a ions,
sampling e e y 1000 ees. The e ec i e sample size (ESS) was moni o ed in T ace .
1.7.2 (Rambau e al. 2018). T ees we e summa ised ia T eeAnno a o (Sucha d e al.
2018) emo ing 10% as a bu n-in. Species delimi a ion analyses included Gene alised
Mixed Yule Coalescen App oach (GMYC) wi h a single h eshold (implemen ed on he
web se e h ps://species.h-i s.o g/gmyc/, accessed on 27 July 2025), he Poisson T ee
P ocesses (bPTP) (implemen ed on he web se e h p://species.h-i s.o g/p p/, accessed
on 28 July 2025) and, inally, he mul i- a e Poisson T ee P ocesses (mPTP)
(implemen ed on he web se e h p://mp p.h-i s.o g/#/ ee, accessed on 27 July 2025)
4Lape a-Gjono a A e al
(Fujisawa and Ba aclough 2013, Zhang e al. 2013, T i inopoulos e al. 2016, Kapli e al.
2017).
Phylogene ic econs uc ion was pe o med using bo h Maximum Likelihood (ML) and he
Bayesian In e ence (BI) analyses. ML analysis was pe o med in IQ-TREE (Nguyen e al.
2015) on he W-IQ-TREE in e ace (T i inopoulos e al. 2016). The in eg a ed
ModelFinde (Kalyaanamoo hy e al. 2017) was used o in e he bes subs i u ion model.
Nodal suppo was ob ained h ough a s anda d non-pa ame ic boo s ap wi h 1000
eplica es. BI analysis was un using M Bayes .3.2.7a (Ronquis e al. 2012).
Phylogene ic ees (BI and ML) we e isualised using iTOL .5 (Le unic and Bo k 2021).
Haplo ype analysis was conduc ed u ilising he DnaSP .6 so wa e (Rozas e al. 2017)
and he esul s we e isualised h ough he u ilisa ion o PopA employing TCS ne wo k
analysis (Clemen e al. 2002, Leigh and B yan 2015).
A map o sequence sampling si es was c ea ed in QGIS e sion 3.34.12-P iz en, based
on he C oss Blended Hypsome ic map laye (h ps://www.na u alea hda a.com).
Geog aphic co e age
Desc ip ion:The specimens used in his s udy we e ecen ly collec ed, p ima ily om
Bulga ia (61), wi h addi ional samples om G eece (13), Tü kiye (4), Albania (1) and
No h Macedonia (2) o ensu e b oade gene ic and geog aphic ep esen a ion (Fig. 1).
Coo dina es:La i ude: min. 34.931 max. 43.768; Longi ude: min. 19.577 max. 27.794.
Figu e 1.
Map o sequence sampling si es.
DNA ba coding o Messo an s o Bulga ia wi h insigh s in o hei axonomic ... 5
Taxonomic co e age
Taxa included:
Rank Scien i ic Name
sub amily My micinae Lepele ie de Sain -Fa geau, 1835
genus Aphaenogas e May , 1853
species Aphaenogas e es ae Eme y, 1915
genus Messo Fo el, 1890
species Messo a anasso ii A anasso , 1982
species Messo c e icus Sala a & Bo owiec, 2019
species Messo hellenius Agos i & Collingwood, 1987
species Messo ibe icus San schi, 1931
species Messo mca hu i S eine , Csősz, Ma kó, Gamisch, Rinnho e , Fol e baue , Hamme le, S au e ,
A ho e & Schlick-S eine , 2018
species Messo oe zeni Fo el, 1910
species Messo pon icus S eine , Csősz, Ma kó, Gamisch, Rinnho e , Fol e baue , Hamme le, S au e , A ho e
& Schlick-S eine , 2018
species Messo s uc o (La eille, 1798)
species Messo wasmanni K ausse, 1910
species Messo c . s uc o
species Messo sp. 1
species Messo sp. 2
Usage licence
Usage licence:Open Da a Commons A ibu ion License
Da a esou ces
Da a package i le:Collec ion o COI sequences om Bulga ian species o he genus
Messo
Resou ce link: h ps://doi.o g/10.5883/DS-BGMESSOR
Numbe o da a se s:1
6Lape a-Gjono a A e al
Da a se name:Towa ds delimi ing he di e si y o Messo an s in Bulga ia using
molecula da a
Da a o ma :dwc, xml, s , as a
Desc ip ion: The da ase cons i u es a collec ion o sequences pe aining o
Bulga ian species o he genus Messo (Hymenop e a, Fo micidae). This da ase
comp ises all a ibu es and me ada a in acco dance wi h he BOLD ules and a e
a ailable o he public ia a Digi al Objec Iden i ie (DOI).
Addi ional in o ma ion
Species delimi a ion and gene ic di e si y
A o al o 84 COI sequences, ep esen ing 11 mo phospecies and 10 o 15 molecula
lineages (depending on he species delimi a ion me hod used), we e analysed, including
80 newly-gene a ed sequences. The leng hs o he DNA ba codes anged om 579 o
658 bp, wi h he majo i y (59 sequences) being 658 bp long (Fig. 2). Haplo ype ne wo k
analysis o all sequences e ealed 35 dis inc haplo ypes, which clus e ed cohe en ly
(Fig. 3).
Figu e 2.
Resul s o species delimi a ion me hods, based on DNA ba coding. Each e ical colou ba
ep esen s di e en delimi a ion schemes ob ained wi h ASAP, RESL, ABGD, bPTP and mPTP
me hods, wi h he co esponding numbe o specimens. The ee is based on ASAP analysis,
wi h nodes colou coded depending on hei p- alue (black: p < 0.001, ed: p < 0.05, yellow: p
> 0.1, g ey: no applicable).
DNA ba coding o Messo an s o Bulga ia wi h insigh s in o hei axonomic ... 7
Messo ins abilis species g oup
The mo phological simila i y be ween Messo a anasso ii and M. c e icus is suppo ed by
low gene ic dis ance obse ed in he delimi a ion analyses (K2P 1.92%) (Suppl. ma e ial
1). These indings may indica e a ela i ely ecen di e gence be ween he wo species,
ollowed by geog aphic isola ion and ecological di e en ia ion. Howe e , despi e hei
o e all closeness, M. a anasso ii and M. c e icus consis en ly di e in s able
mo phological ai s. Speci ically, in M. a anasso ii, he occipi al a ea and e ex o he
head bea 12–20 la ge se ae, whe eas in M. c e icus, he numbe is always lowe , ne e
exceeding nine. In addi ion, unlike M. c e icus, which is es ic ed o he moun ain egions
o C e e, M. a anasso ii is a he mophilous lowland species ound in sou he n Bulga ia,
Cen al Macedonia and some o he Ionian Islands (Bo owiec and Sala a 2025). An
ongoing esea ch in o he e olu iona y his o y o his di e gence will cla i y he iming and
mechanisms unde lying his pa icula e en .
Specimens om ou nes samples — one om Cen al Macedonia in G eece and h ee
om sou h-wes e n Bulga ia — designa ed in his s udy as Messo sp. 1, exhibi ed
mo phological ai s cha ac e is ic o bo h Messo a anasso ii and M. wasmanni,
speci ically he se osi y o he o me and he la ge size o he la e . Howe e , all species
delimi a ion analyses s ongly suppo ed hei sepa a ion om bo h species and indica ed
Figu e 3.
TCS haplo ype ne wo k, based on COI sequences o Messo species. Each ci cle ep esen s a
unique haplo ype; size co esponds o he numbe o indi iduals. Lines indica e single
mu a ional s eps. Species-speci ic colou and le e coding ollow he phylogene ic ee.
8Lape a-Gjono a A e al
a close gene ic a ini y o M. wasmanni, wi h K2P dis ances o 6.28% and 5%,
espec i ely (Suppl. ma e ial 1). Cu en ly, M. a anasso ii and M. wasmanni a e he only
known ep esen a i es o he ins abilis g oup in his egion. Whe he he specimens
designa ed as Messo sp. 1 ep esen cases o hyb idogenesis o belong o a dis inc
species will be in es iga ed in a u u e s udy.
The mos widesp ead species o he ins abilis g oup, Messo wasmanni, is ep esen ed in
his s udy by a la ge numbe o specimens (13) om he wides geog aphical ange —
spanning Bulga ia, Tü kiye and G eece (including C e e). I exhibi s an in aspeci ic
gene ic dis ance up o 0.81% (mean: 0.18%) (Suppl. ma e ial 1).
Messo s uc o species g oup
Species delimi a ion analyses we e consis en in suppo ing he dis inc i eness o Messo
oe zeni, M. mca hu i and uniden i ied species close o M. oe zeni (named Messo sp.
2), as well as one molecula lineage wi hin Messo s uc o ep esen ed by ou
sequences — h ee om wes e n Bulga ia and one om No h Macedonia. Fu he
e alua ion is also needed o a single sequence (BGANT032-23) ob ained om a nes
sample in he wes e n Balkan Moun ains (V achanski Balkan). Al hough his specimen is
mo phologically simila o M. s uc o and clea ly sepa a ed om all ecognised axa in
he analyses, he small sample size and absence o ep oduc i e specimens make i s
axonomic a ini y s ill unclea .
Messo ibe icus was consis en ly ecognised in dis ance-based me hods (ASAP, RESL,
ABGD) and he coalescen -based me hod GMYC, exhibi ing an in aspeci ic gene ic
dis ance up o 0.46% (mean 0.2%), bu no in he ee-based me hods bPTP and mPTP.
This disc epancy can be a ibu ed o di e ences in me hodological assump ions and
sensi i i y o gene ic a ia ion (Hube e al. 2024). I should be no ed ha ou s udy
analysed only he wo ke cas e o M. ibe icus, which, as ecen ly shown by Ju é e al.
(2025b), a e hyb ids wi h M. s uc o . Ne e heless, since hey inhe i he COI ma ke om
he ma e nal lineage, he gene ic pa e ns obse ed in ou s udy emain consis en wi h
hei ma e nal iden i y.
P e ious s udies in es iga ing Messo s uc o ac oss i s b oad geog aphic dis ibu ion —
spanning Aus ia, Bulga ia, Czechia, F ance, Hunga y, Romania and Slo enia —
e ealed he exis ence o mul iple mi ochond ial lineages wi hin he species (Schlick-
S eine e al. 2006, S eine e al. 2018, S ohmaie e al. 2025). These ea lie indings
align closely wi h he esul s o he p esen s udy, which de ec ed high in aspeci ic
gene ic di e si y (wi h K2P dis ance om 0 o 5.07%, mean 2.58%) and iden i ied en
haplo ypes (Fig. 3, Suppl. ma e ial 1). While se e al species delimi a ion me hods (ASAP,
ABGD, bPTP, mPTP) ecognised wo molecula lineages, he RESL algo i hm
dis inguished i e BINs wi hin M. s uc o , u he suppo ing he p esence o deep gene ic
s uc u ing, wi h mul iple lineages and haplo ypes sugges ing po en ial c yp ic di e si y
ac oss i s ange o long- e m popula ion isola ion wi hin he species.
DNA ba coding o Messo an s o Bulga ia wi h insigh s in o hei axonomic ... 9
• Zięcina D, Menche i M, Bo owiec L, B ačko G, Lape a-Gjono a A, Villa R, Sala a S
(2024) Taxonomic e ision o he Aphaenogas e sub e anea species g oup
(Hymenop e a: Fo micidae). Annales Zoologici 74 (2): 237‑282. h ps://doi.o g/
10.3161/00034541ANZ2024.74.2.002
Supplemen a y ma e ial
Suppl. ma e ial 1: Dis ance analyses, GMYC summa y and Bayesian In e ence
ee
Au ho s: Albena Lape a-Gjono a, Monika P ama a o a, Lech Bo owiec, Ilia Gjono , Rumyana
Kos o a, Ros isla Bekchie , Simeon Bo isso
Da a ype: genomic, phylogene ic
Download ile (3.42 MB)
16 Lape a-Gjono a A e al
