Redescription of Orculella aragonica (Westerlund 1897), an Iberian species different from O. Bulgarica (Hesse 1915) (Gastropoda: Pulmonata: Orculidae)

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). 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