NORTH-WESTERN JOURNAL OF ZOOLOGY 8 (2): 292-299 ©NwjZ, O adea, Romania, 2012
A icle No.: 121126 h p://biozoojou nals.3x. o/nwjz/index.h ml
Redesc ip ion o O culella a agonica (Wes e lund 1897),
an Ibe ian species di e en om O. bulga ica (Hesse 1915)
(Gas opoda: Pulmona a: O culidae)
José ARRÉBOLA1, Oihana RAZKIN2,
Benjamín GÓMEZ-MOLINER2 and Ba na PÁLL-GERGELY3,*
1. Depa amen o de Zoología. Fac. Biología. Uni e sidad de Se illa. A da. Reina Me cedes, 6. 41012-Se illa, Spain.
2. Depa amen o de Zoología y Biología Celula Animal, Facul ad de Fa macia y Cen o de Es udios A anzados CIEA;
Uni e sidad del País Vasco, UPV/EHU, Vi o ia (Ala a), Spain.
3. Depa men o Biology, Shinshu Uni e si y, Ma sumo o 390-8621, Japan.
* Co esponding au ho , B. Páll-Ge gely, E-mail: [email p o ec ed]
Recei ed: 21. Feb ua y 2011 / Accep ed: 02. June 2012 / A ailable online: 11. June 2012 / P in ed: Decembe 2012
Abs ac . O culella bulga ica (Hesse 1915) has been eco ded om Bulga ia and wes e n Asia, including
Tu key, bu has also been epo ed om Spain by se e al au ho s. Mos s udies on his species ha e been on
sub ossil shells. Recen indings o li ing popula ions in Tu key and Spain ha e allowed us o epo on he
ep oduc i e sys em mo phology and m DNA sequences o his axon. Despi e he appa en lack o
conchological di e ences be ween specimens om hese wo geog aphical a eas, his new in o ma ion
e ealed he p esence o wo species. Re iew o he li e a u e pe aining o Ibe ian o culids, led us o
conclude ha he examined popula ion in Spain species mus be assigned o O culella a agonica (Wes e lund
1897), and all p e ious epo s o O. bulga ica om he Ibe ian Peninsula should be asc ibed o he o me .
This species is edesc ibed and diagnosed he ein, highligh ing di e ences be ween i and O. bulga ica. Some
no es abou i s conse a ion s a us and biogeog aphic o igin a e also p o ided.
Key wo ds: O culella a agonica, O culella bulga ica, dis ibu ion, Tu key, Spain, axonomy.
In oduc ion
O culella bulga ica was desc ibed by Hesse (1915)
om “Gebedsche bei Va na” (Bulga ia). Subse-
quen au ho s ex ended i s dis ibu ional ange o
wo dis an a eas, namely (1) Bulga ia and Wes -
e n Asia (A menia, Aze baijan, Russian Fede a-
ion, Tu key) (Hausdo 1996) and (2) Spain (Ga -
ido e al. 2005, Robles & Ma inez-O í 2009), wi h
no eco ds wi hin his b oad dis ibu ional hia us.
The s udies we e based on emp y shells ( ossil o
“sub ossil”), excep hose collec ed in Bulga ia by
U bański in 1960 (Gi enbe ge 1983). The geni al
ana omy emained unknown because o he lack
o li ing ma e ial. As a esul , Hausdo (1996)
ques ioned he gene ic posi ion o O. bulga ica, be-
cause O culella and Schileykula could only be dis-
inguished on he basis o he geni al s uc u e,
namely (mainly) he p esence (O culella) o ab-
sence (Schileykula) o a penial appendix (see also
Páll-Ge gely 2011).
The Ibe ian O culella has been p e iously as-
signed o Pupa dolium D apa naud 1801, Pupa
dolium a . plagios oma, Sandbe ge 1875, Pupa
dolium a . no a Wes e lund (in Calde on 1897),
O cula dolium o ma a agonica Wes e lund 1897
and O cula plagios oma (Sandbe ge ) (in B un-
nacke & Ložek 1969). The known locali ies we e
compiled by Gi enbe ge (1983) Gómez-Moline
(1988), Ga ido e al. (2005) and Robles & Ma i-
nez-O í (2009).
Gi enbe ge (1983) compa ed Ibe ian O culella
o O. bulga ica om Bulga ia, concluding ha hey
we e conspeci ic on he basis o conchological
cha ac e s. The shells om bo h a eas show a p o-
oconch mic osculp u e o i egula spi al idges
and he p esence o a pala al lamella in body
who l o ju eniles, bu lacking in adul s. Mo e e-
cen ly, Hausdo (1996) dis inguished O. bulga ica
lamella a (li ing in sou heas e n Tu key) om O. b.
bulga ica on he basis o well-de eloped lamella in
adul shells.
Ga ido e al. (2005) disco e ed ou ex an
popula ions o he Ibe ian O culella in G anada
P o ince (sou he n Ibe ian Peninsula). They de-
sc ibed he pe ios acum o li e-collec ed ju enile
shells ( hin, oblique ibs in he shape o pla ele s
which a e los in he adul shells), as well as he
ep oduc i e ana omy. The la e con i med he
gene ic posi ion o he species wi hin he genus
O culella.
Based on eco ds spanning se e al million
yea s in age (Plio-Pleis ocene o Recen ), Robles &
Ma inez-O í (2009) concluded ha he species
was o me ly widely dis ibu ed along he eas e n
hal o he Ibe ian Peninsula, bu i s geog aphical
Redesc ip ion o O culella a agonica
293
ange was signi ican ly educed ecen ly. Gi en-
be ge (1983) and P eece (1991) published de ailed
in e p e a ions o his dis ibu ional con ac ion.
A he p esen ime, h ee o he ou li ing popu-
la ions ecen ly disco e ed by Ga ido e al. (2005)
in Andalusia ha e become ex inc , bu se en new
popula ions ha e been disco e ed a e in ense
sea ching in he su ounding a eas (A ébola &
Ga ido 2008, A ébola in p ep.).
We ha e also ound a small li ing popula ion
o O. bulga ica bulga ica in no heas e n Tu key.
This allowed us o compa e specimens om he
wo dis an geog aphic loca ions h ough analysis
o shell and ep oduc i e sys em mo phology, and
DNA sequences in o de o cla i y he sys ema ic
posi ion o he Spanish popula ions.
Ma e ial and me hods
Samples
Samples om di e en locali ies in sou he n Spain (see
Ma e ial Examined and Table 1 o desc ip ions) and one
locali y in Tu key we e used o mo phological s udies o
he shell and ep oduc i e sys em. The Spanish locali y
habi a s we e desc ibed by Ga ido e al. (2005). The
Tu kish locali y was a we , ma shy bank o a s eam,
whe e specimens we e c awling on he g ound o on de-
caying plan s among dense ege a ion. The specimens a e
deposi ed in he collec ions o he Zoology Depa men o
Se ille Uni e si y (Se ille), Na u al His o y Museum o
Valencia and in he p i a e collec ion o he las au ho
(Mosonmagya ó á , Hunga y). Specimens o O. a agonica
(6) and O. bulga ica (2) we e p ese ed in 96% e hanol and
used o molecula s udies (Table 1). Fi e Spanish (P1, P5,
P6) and wo Tu kish specimes we e dissec ed.
Abb e ia ions used in he ex :
H: shell heigh
NHMW: Na u his o isches Musem, Wien (Vienna, Aus-
ia)
DNA isola ion
DNA was isola ed om he oo o each snail using he
Qiagen DNeasy Blood & Tissue Ki acco ding o he
manu ac u e ’s ins uc ions.
DNA Ampli ica ion and Sequencing
Polyme ase chain eac ion (PCR) was used o ampli y wo
mi ochond ial gene agmen s, cy och ome oxidase sub-
uni I (COI) and 16S RNA (16S) using uni e sal p ime s
(Folme e al. 1994, Palumbi e al. 1994, espec i ely).
The PCR ampli ica ions we e conduc ed in 23 μl e-
ac ions con aining 2 μl dilu ed DNA, 2 μl dNTP (2.5 mM),
1.25 μl MgCl2 (50 mM), 2.5 μl NH4 bu e 10X, 0.5 μl o
each p ime (0.25 mM), 0.25 μl Taq Polyme ase (5 U/μl)
and 1 μl o BSA (10mg/ml). We used a Bio ad iCycle
he mal cycle wi h he ollowing cycling condi ions: an
ini ial dena u a ion s ep a 96 ºC o 1 min; 35 cycles o
dena u ing a 94 ºC o 30 s, annealing a 51 ºC o COI o
56 ºC o 16S o 30 s and ex ending a 72 ºC o 1 min;
and a inal ex ending s ep a 72 ºC o 10 min. Reac ions
we e held a 4 ºC.
PCR p oduc s we e un in 1.5% aga ose gels s ained
wi h GelRed, o e i y he ampli ica ions. Amplicons
we e sequenced using he BigDye Te mina o Ki 1.1
(Applied Biosys ems) in an ABI PRISM Model 3100 A an
Gene ic Analyze . The sequences we e accessioned in o
GenBank (Table 1).
DNA sequences Analysis
Sequences we e aligned using CLUSTALX 1.81 (Thomp-
son e al. 1997) and hen manually adjus ed o minimize
misma ches. The COI and 16S RNA sequences we e con-
ca ena ed in o a single da ase o phylogene ic analyses.
Pupilla musco um (Linnaeus 1758) was used as ou g oup
(sequences a ailable in GenBank wi h accession numbe s
GQ921664.1 o COI and GQ921551.1 o 16S RNA).
Phylogene ic econs uc ions using PAUP 4.0b3
(Swo o d 2002) we e pe o med by a dis ance me hod
using he neighbou -joining algo i hm (NJ) (Sai ou & Nei
1987). The HKY (Hasegawa e al. 1985) model was se-
lec ed as he bes - i model o nucleo ide subs i u ion o
he molecula da a se by he Akaike in o ma ion c i e ia
app oach using Model es 3.6 (Posada & C andall 1998).
The obus ness o he ees was assessed by boo s ap e-
sampling (1000 eplica ions o NJ analysis; Felsens ein
1985).
We also pe o med a Bayesian analysis (BA) using
M Bayes 3.0b4 (Huelsenbeck & Ronquis 2001). The
Ma ko Chain Mon e Ca lo (MCMC) sea ch was pe -
o med using ou chains o 2.5 million gene a ions and
using he mos sui able model de e mined by Model es .
Bayesian pos e io p obabili ies we e picked om he
50% majo i y ule consensus o ees sampled e e y 100
gene a ions ( he i s 2000 ees we e disca ded as “bu n
in”).
Unco ec ed pai wise p-dis ances we e calcula ed
wi h PAUP . 4.0b10 o he COI, 16S and conca ena ed
da ase (leng h 635, 486 and 1121 nucleo ides, espec-
i ely). These dis ances we e in e p e ed as pe cen ages.
Numbe o m DNA haplo ypes (H), haplo ype di e -
si y (Hd), nucleo ide di e si y (π) and numbe o nucleo-
ide di e ences (k) we e calcula ed o he combined
da ase using DnaSP 4.10.4 so wa e (Rozas e al. 2003).
Resul s - Sys ema ical pa
Family O culidae Pilsb y 1918
Genus O culella S eenbe g 1925
O culella a agonica (Wes e lund 1897)
Ma e ial Examined (P= popula ions o O. a a-
gonica) ( o al/dissec ed/DNA analyzed speci-
mens): P1 (393/03/00) Rambla de la Viña sp ing,
940 m asl, 30SVG8029. P3 (12/00/01): 1400 m
sou h om Ba io los Pa ales (Lope a) (Po e a
sp ing), 940 m asl, 30SVG7829. P5 (08/01/00): 325
m S-SE om Co ijo la Ca asca, 1417 m asl,
J. A ébola e al.
294
Table 1. Species abb e ia ions, geog aphical coo dina es (UTM g id e e ences)
and Genbank accession numbe s.
Sample U.T.M._1km GenBank accession numbe s COI / 16S
1-O.bulga ica* JQ765693 / JQ765685
2-O.bulga ica* JQ765694 / JQ765686
3-O.a agonica P6a 30SVG6818 JQ765695 / JQ765687
4-O.a agonica P9 30SVG6523 JQ765700 / JQ765692
5-O.a agonica P6b 30SVG6818 JQ765696 / JQ765688
6-O.a agonica P8 30SVG6020 JQ765699 / JQ765691
7-O.a agonica P3 30SVG7829 JQ765697 / JQ765689
8-O.a agonica P7 30SWG2354 JQ765698 / JQ765690
*O. bulga ica specimens: Tu key, on he bo de s o il. E zu um and il. E zincan, s eam
nea Te can üneli ( unnel), 39°50'25.36"N, 40°34'0.56"E, leg. Páll-Ge gely, B., 05.07.2010.
Figu e 1. Shells o O culella species. A: O culella a agonica (Wes e lund 1897) 325 m S-SE om Co ijo la Ca asca,
1417 m asl, 30SWG0938, H= 7,94 mm B: O culella a agonica, labelled as O cula dolium plagios oma, Spain, P o . G a-
nada, Gal a, Coll. Klemm, leg. Falkne 1968, NHMW 60920, H= 6.5 mm; C: O culella bulga ica bulga ica (Hesse 1915)
Tu key, on he bo de o il. E zu um and il. E zincan, Te can üneli, s eam nea he unnel, 39°50'25.36"N,
40°34'0.56"E, leg. Páll-Ge gely, B., 5.7.2010, NHMW ALSV6606, H=6.6 mm; D: O culella bulga ica bulga ica (Hesse
1915), Tu key, Gaybi, 15km SW E egli, Coll. Klemm, leg. Ressl 10.6.1965, de . B. Hausdo 1989, NHMW 55500. H=
6.8 mm; E: O culella bulga ica lamella a Hausdo 1996, pa a ype, Tu key, Yesilyu , Coll. Klemm, leg. Ressl 2.6.1965,
de . B. Hausdo 1992, NHMW 55531, H= 7.7 mm.
30SWG0938. P6 (14/01/02): sp ing on Ba anco de
las Ramillas, 1431 m asl, 30SVG6818. P7
(21/00/01): 400 m NW om Co ijo del Oli a ,
730 m asl, 30SWG2354. P8 (18/00/01): Collado de
Pue o Blanco, 1400 m asl, 30SVG6020. P9
(13/00/01): La To e sp ing, 1364 m asl;
30SVG6523.
Shell (Fig. 1A): Conical-o al in shape, da k b own
in colou and somewha anslucen . P o oconch
wi h 1 ½ o 1 ¾ who ls and mic osculp u e o ine,
i egula , spi al idges on he su ace, mo e accen-
ua ed a he pe iphe y, bu absen in he eleo-
conch. 7 ¼ o 9 ¼ slowly and egula ly g owing,
sligh ly con ex who ls wi h shallow su u e. Las
who l sligh ly aised nea he pe is ome and la ge
han he p e ious one. Shell su ace pa ly co e ed
wi h dense, ine, ans e se ibs. The umbilicus is
Redesc ip ion o O culella a agonica
295
elonga ed, na ow and shallow. Ape u e mo e o
less ounded, somewha alle han wide. Pe is-
ome in e up ed, hickened, wi hou (o wi h a
e y weak) pa ie al callosi y and sligh ly e lec ed,
especially in he umbilical a ea. Pala al wall
sligh ly conca e wi h a c enula ed edge la e ally.
Adul s wi h a sho and small pa ie al lamella
o igina ing some dis ance om he edge, in e -
nally ex ended ½ who l. The deep columella la-
mellae a e no always isible in on al iew. Ju-
eniles wi h bo h hin columella and pa ie al la-
mella in he ape u e, a ounded and deep umbili-
cus and sculp u e made up o hin, oblique ibs in
he shape o pla ele s which a e los in he adul
s ages (see Ga ido e al. 2005).
Measu emen s: H=7.24 mm, SD= 0.38 mm (P5,
n= 57). H=6.73 mm SD= 0.37 (P1, n= 393).
Geni al s uc u e (Fig. 2B): Re ac o muscle o
igh omma opho e unning be ween penis and
agina. Vagina sho and hick, he penis ela i ely
sho , gene ally he p oximal po ion hicke , bu
in some specimens he en i e penis is cylind ical
(o same wid h). The penial appendix ela i ely
long, he i s po ion hicke , he second g adu-
ally ape ing o he end. The “penial caecum” is
no a ue “caecum”, bu o ms a loop (only isible
a high magni ica ion) wi h weak ib es on p oxi-
mal po ion (nea dis al end o penis). Re ac o
muscle a ached o dis al end o penis, nea base o
he “penial caecum”. Epiphallus sligh ly (na -
owe ) han penis, na owes a p oximal po ion
and slowly ape ing owa ds as de e ens. Bu sa
copula ix long and he bu sa i sel is no con-
spicuously hickened.
Di e en ial diagnosis: O. a agonica has a loop-
like “penial caecum” whe eas O. bulga ica has a
ue penial caecum and he penis and he epiphal-
lus is sho e in O. bulga ica han in O. a agonica
(see Fig. 2 and Table 2).
Habi a : Gene al da a ob ained om locali ies
whe e o adjacen o whe e li ing specimens o O.
a agonica we e collec ed sugges ed ha his species
has a na ow he moclima ic ole ance, being as-
socia ed o humid/sub-humid en i onmen s. The
species p e e s hyg ophilous habi a s such as
small ma shes; pe manen ly mois and wa e -
logged soil habi a s in pe manen eshwa e
sp ing a eas associa ed wi h limes one. This spe-
cies would be es ic ed o he mois es habi a s in
semi-a id a eas du ing he summe mon hs (Ga -
ido e al. 2005; A ébola & Ga ido 2008; A ébola
in p ep.).
Geog aphic ange: Fossil and sub ossil shells o
O. a agonica ha e been ound in he eas e n hal o
he Ibe ian Peninsula (Robles & Ma inez-O í
2009), bu li ing specimens a e only known om
G anada P o ince (Andalusia, sou he n Ibe ian
Peninsula).
Figu e 2. Male geni al s uc u es o O culella species. A:
O culella bulga ica (Hesse 1915) (Tu key, on he bo de
o E zu um and E zincan ilaye s, bank o he Te can
s eam close o he Te can üneli [ unnel]); B: O culella
a agonica (Wes e lund 1897) (Spain, 325 m S-SE om
Co ijo la Ca asca). Abb e ia ions: E: epiphallus; P: pe-
nis; Pa: penial appendix; Pc: penial caecum.
Table 2. Measu emen s (in mm) o some sec ions o he
geni alia o O culella a agonica (Wes e lund 1897). (P5:
325 m S-SE om Co ijo la Ca asca; P6: sp ing on Ba -
anco de las Ramillas) and O culella bulga ica (Hesse
1915) (Tu key, on he bo de o E zu um and E zincan
ilaye s, bank o he Te can s eam close o he Te can
üneli [ unnel]). Abb e ia ions: P1: penis un il he junc-
ion wi h he penial appendix; P2: penis un il he junc-
ion wi h he epiphallus; BC: bu sa copula ix. O he
abb e ia ions co espond wi h Figu e 3.
P1 P2 E Pc Pa BC
O. a agonica P5 1.5 2.4 3.5 0.5 7.7 6.2
O. a agonica P6 1.3 1.9 3.5 0.6 7.0 6.8
O. bulga ica 0.7 1.5 1.6 0.2 6.0 4.2
DNA sequence cha ac e is ics
The aligned COI da ase consis ed o 635 bp and
31 (4.88%) cha ac e s we e pa simony in o ma i e.
The sequences had a nucleo ide composi ion o : T
(38.5%), C (14.7%), A (28.3%) and G (18.5%).
J. A ébola e al.
296
Figu e 3. Neighbou -joining ee cons uc ed ollowing he HKY model o he combined da ase
o he COI and 16S genes. NJ boo s ap alues and Bayesian pos e io p obabili ies a e shown
a each node (NJ/BA). The as e isk indica es a node whe e he BA opology was di e en .
The aligned 16S RNA agmen consis ed o
486 bp and 27 (5.56%) cha ac e s we e pa simony
in o ma i e. The nucleo ide composi ion was: T
(36.4%), C (14.1%), A (33.1%) and G (16.4%).
The combined leng h o he wo aligned ag-
men s was 1121 bp and 58 (5.17%) cha ac e s we e
pa simony in o ma i e. The sequences had a nu-
cleo ide composi ion o : T (37.8%), C (14.2%), A
(30.4%) and G (17.6%).
Phylogene ic analyses
The wo me hods o phylogene ic in e ences (NJ
and BA) yielded almos he same ee opology.
The NJ ee o he conca ena ed da ase is p e-
sen ed in Figu e 3 indica ing bo h NJ boo s ap
and BA pos e io p obabili ies.
The phylogene ic analyses showed wo mo-
nophyle ic lineages ha we e s ongly suppo ed.
They a e indica ed as A (specimens om Tu key)
and B (specimens om Spain). Pai wise sequences
di e gence based on unco ec ed p-dis ance be-
ween hese clades anged om 5.27% o 5.45% in
he conca ena ed da ase .
The clade A joined he wo specimens om
Tu key and his monophyly was suppo ed by
high boo s ap alues and pos e io p obabili y
(100%, 100% o NJ, BA). The haplo ype di e si y’s
alue (Hd) was 1, because he wo specimens di -
e ed om each o he by 1 bp.
Clade B g ouped he six specimens collec ed
om i e si es in G anada P o ince (sou he n
Spain). The numbe o haplo ypes (H) in his clade
was 4 and he haplo ype di e si y’s alue (Hd)
0.8. The nucleo ide di e si y’s alue (π) was
0.00328 and he numbe o nucleo ide di e ences
(k) 3.67. The specimen O. a agonica P7 was he
mos di e en wi hin clade B and he gene ic dis-
ance be ween his sample and he o he s om
G anada anged om 0.80% o 0.89% in he con-
ca ena ed da ase . Collec ion si e o O. a agonica P7
was 67 km away om he o he ou collec ing
si es.
Discussion
Taxonomy
Due o he ema kable conchological simila i y be-
ween he Ibe ian O culella specimens and O. bul-
ga ica (Fig. 1), Gi enbe ge (1983) conside ed hem
o be conspeci ic. Pupa/O cula dolium D apa naud
was mos commonly employed by p e ious au-
ho s o he Ibe ian O culella un il Gi enbe ge
(1983). Howe e , O cula dolium is an Alpine-
Ca pa hian species (Ke ney e al. 1983) absen
om he Ibe ian Peninsula. Pupa dolium a . pla-
gios oma (‘B aun’ Sandbe ge 1875) (in Pilsb y
1922-1926, Gi enbe ge 1983) is a smalle o m o
O. dolium wi h one o no lamella on he columella
(Fig. 1B), ypically ound in loess in Cen al
Eu ope (Alsace o Aus ia, Hunga y, Lomba dia,
Swi ze land and F ance). Due o simila shell
mo phology (Gi enbe ge 1983) and habi a s,
B unnacke and Ložek (1969) e oneously em-
ployed he name O cula plagios oma o e e o
some shells o he Ibe ian O culella ound in he
loess o Alme ía. Robles and Ma inez-O í (2009)
examined o iginal ma e ial o Pupa dolium a .
no a ci ed by Calde on (1897) om Alhama de
A agón (no heas e n Ibe ian Peninsula) deposi ed

Redesc ip ion o O culella a agonica
297
in he Na ional Museum o Na u al His o y (Ma-
d id). These au ho s elega ed his name o he
synonymy o O. bulga ica because al hough
Calde on (1897) men ioned ha i was a new
(“no a”) a ie y o O. dolium he did no o mally
name i : “Plica pa ie ali imme se, b e i, enui e plicis
columella ibus p o undissimis obsole es dis inc a” (de-
ined by Wes e lund 1897). Howe e , Robles and
Ma inez-O í (2009) o e looked ha in he same
yea Wes e lund (1897: 69) desc ibed and named
his axon as a new “ o ma” o O. dolium as ol-
lows: “a agonica n., . enuiss. s ia ula, lu escen i-
co nea, ap. callo pala ali angus o anslucen e, ube -
culo ang. e lam. pa . enui, b e i . b e issima,
cae e um eden a e (Hisp., A agonia)”. This desc ip-
ion dis inguishes O cula dolium om he Ibe ian
O culella ( he only o culid species ac ually known
in his a ea), which consequen ly mus be named
as O culella a agonica (Wes e lund 1897). Al hough
Wes e lund (1897) did no speci y any locali y o
a agonica (only he geog aphic egion “A agonia”)
he e is no doub ha he ma e ial on which he
desc ip ion was made o igina ed om Alhama de
A agon and was collec ed by Calde on (1897).
Consequen ly, his mus be assigned he ype lo-
cali y. On he o he hand, he specimens om he
Calde on collec ion deposi ed in Na u al Sciences
Museum o Mad id (MNCN): MNCN-15.05/37017
(11 shells) and MNCN-s/n (col. Azpei ia nº 2481)
(5 shells) may no ha e been seen by Wes e lund
and he e o e can no be conside ed ypes.
Since he Ibe ian specimens a e clea ly di e -
en om O. bulga ica (Hess 1915) and he co ec
name o hese popula ions has been shown o be
O. a agonica we p opose o emo e O. bulga ica
om he aunal lis o he Ibe ian Peninsula and
conside all p e ious eco ds o i in his a ea e -
e able o O culella a agonica (Wes e lund 1897).
Shell and ep oduc i e sys em mo phology
The inding o li e specimens o O. bulga ica om
Tu key made he conchological, ana omical and
gene ic compa ison o he wo species possible.
Acco ding o ou obse a ions, he shell o O. a a-
gonica is indis inguishable om O bulga ica bulga-
ica (Fig. 1), as i was s a ed by Gi enbe ge (1983).
O culella bulga ica lamella a Hausdo 1996 (Fig. 1E)
has a much s onge de eloped lamella han O.
a agonica and O. bulga ica bulga ica. Some di e -
ences can be seen in he ep oduc i e o gans o O.
a agonica and O. bulga ica. The la e species has a
ue penial caecum, whe eas O. a agonica has a
loop-like s uc u e (see Fig. 2). Ga ido e al. (2005)
desc ibed he la e ’s ep oduc i e ana omy, bu
ailed o men ion he loop-like “penial caecum”.
This is unique in O culella and may be o he en-
i e O culidae. All o he ana omically known o cu-
lids ha e ue penial caeca. We conside his di -
e ence subs an ial and ind i su p ising ha no
conchological di e ences ha e been ound.
The penis and he epiphallus a e sho e in O.
bulga ica han in O. a agonica. The measu emen s
o some sec ions o he geni alia o he wo species
a e compiled in Table 2.
Gene ic backg ound
Gene ic dis ances wi hin pulmona e gas opod
species can p esen a iable alues. Maximal in-
aspeci ic 16S RNA gene sequences abo e 10%
ha e been epo ed se e al imes: Wa anabe &
Chiba 2001: 14% o Euad a quaesi a; Goodac e
2002: 10% o species o he genus Pa ula; Pa -
makelis e al. 2003: 21.4% o species o he genus
Mas us; Teshima e al. 2003: 23% o Ainohelix
edi ha; Pinceel e al. 2005: 21% o species o he
genus A ion). Mos es ima es o di e gence a es
o s ylomma opho ans anged om 5% o 14%
o 16S RNA (Chiba 1999, Hayashi & Chiba 2000,
Thacke & Had ield 2000, Van Riel e al. 2005). Bu
among closely ela ed species mean gene ic dis-
ances can be e y di e en . Fo example a e age
COI gene ic dis ances in he wo sis e species
Candidula uni ascia a and C. ugosiuscula was 12.5%
(P enninge e al. 2003). In Ve igo gouldii COI se-
quence di e gences anged om 1.6% o 11.2%
(Nekola e al. 2009).
In he p esen s udy gene ic dis ances be ween
he wo monophyle ic lineages we e 4.88%-5.35%
o COI, 5.38%-5.80% o 16S and 5.18%- 5.45% o
he conca ena ed da ase . Al hough he wo mo-
nophyle ic g oups we e s ongly suppo ed,
knowing he gene ic a iabili y ha exis s be ween
gas opod species, we canno de e mine i he ob-
ained wo clades belong o di e en species bas-
ing exclusi ely on gene ic dis ances. Bu aking he
ana omical and biogeog aphical c i e ia in o ac-
coun , we can conclude ha O culella popula ions
om Tu key (and p obably o he neighbou ing
a eas) and om he Ibe ian Peninsula (clades A
and B o he phylogene ic analysis) a e wo di e -
en species.
O igin
The genus O culella is composed o mo e han 25
species dis ibu ed in he Ibe ian Peninsula, Magh-
eb, Sicilia and he eas e n Medi e anean egion
J. A ébola e al.
298
(Balkan Region, Ana olia, I an). Ana olia and
C e e (12 and 8 species, espec i ely) ha e he
highes di e si y (B and 1956, Hausdo 1988,
1996, Schü & Şeşen 1998, Gi enbe ge & Haus-
do 2004, Robles & Ma ínez O i 2009, Zilch
1960). This sugges s ha he cen e o o igin o
O culella can be placed in he Aegean-Ana olia e-
gion, whe e he genus shows he g ea es gene ic
di e si y. The ances o o O. bulga ica and O. a a-
gonica may ha e li ed in his egion. The O culella
dis ibu ional expansion wes o he Ibe ian Penin-
sula could ha e occu ed ac oss Eu ope. Ne e -
heless, he e a e no ossil eco ds wes o he Bal-
kans and, hus, paleon ological da a canno con-
i m his hypo hesis (Mania 1995, Esu 1995). By
con as , he p esence o O culella in Libya, Sicilia
and no he n A ica (B and 1956, Gi enbe ge &
Hausdo 2004, Hausdo 1988), sugges s ha he
coloniza ion o he Ibe ian Peninsula could ha e
occu ed ia he sou he n ma gin o he Medi e -
anean Sea. One possibili y is ha his coloniza-
ion occu ed a he end o he Pliocene, when he
Medi e anean Sea expe ienced a pe iod o in ense
desicca ion (Messinian Salini y C isis, 5-6 My BP)
(Hsü e al. 1977). The oldes ossils o O culella a a-
gonica a e da ed in he Pliocene-Pleis ocene (Al-
mena a-Casablanca-1 deposi , 1.8 MY, Robles &
Ma ínez-O í 2009, Agus í e al. 2010). Molecula
dis ances ob ained in he p esen wo k (abou 5%)
also sugges ed a mo e ecen sepa a ion be ween
O. a agonica and O. bulga ica. Al hough he e is no
calib a ed molecula clock a e a ailable o O -
culella o o he closely ela ed gene a, land snails
a e suspec ed o ha e as -e ol ing mi ochond ial
genome wi h an es ima ed di e gence ime o 2-
5% sequence di e gence pe one million yea s
(P enninge e al. 2003).
Wha e e i s ou e o dispe sal, h ough
Eu ope o om No h-A ica, O. a agonica is now
an endemic species o he eas e n hal o he Ibe-
ian Peninsula. The educ ion o i s ange o he
cu en s a us could ha e occu ed some ime in
he Holocene, when he ack o he popula ions is
los in he sou he n hal o he “Sis ema Cen al”
and Valencia.
Conse a ion S a us
The conse a ion s a us o O. a agonica was as-
sessed o he IUCN Species Red Lis (as O. bulga-
ica) by A ébola and Páll-Ge gely (2011) (see also
Cu elod e al. 2011). Acco ding o he me hodol-
ogy and c i e ia o he IUCN Red Lis P og am, i
was conside ed as a c i ically endange ed species.
Taking in o accoun ha O. a agonica is now e-
s ic ed o a ew, agmen ed loca ions in sou he n
Spain, his ca ego y is ap o his species.
Acknowledgemen s. This esea ch was suppo ed by he
Andalusia Regional Minis y o he En i onmen as pa
o he “P og am o Conse a ion and Sus ainable Snail
Exploi a ion in Andalusia”. This wo k also ecei ed
inancial suppo om he Uni e si y o he Basque
Coun y ( e . GIU06/09) and om he Spanish
“Minis e io de Ciencia e Inno ación” ( e . CGL2008-
01131). The au ho s wish o exp ess hei g a i ude o
Jose Ha l (NHMW) o aking pho os o O culella shells
deposi ed in he NHMW and o aluable ad ices, J.A.
Ga ido, and E. Gi enbe ge o making commen s on he
manusc ip , and o Ku Au enbe g (Flo ida Museum o
Na u al His o y) o his e iew o English g amma .
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