RADIATIONIN BYTHINELLA MOQUIN-TANDON,1856 …yadda.icm.edu.pl/yadda/element/bwmeta1.element... ·...

RADIATION IN BYTHINELLA MOQUIN-TANDON 1856(MOLLUSCA GASTROPODA RISSOOIDEA)IN THE BALKANS

ANDRZEJ FALNIOWSKI1 MAGDALENA SZAROWSKA1 PETER GLOumlER2 VLADIMIR PEŠIAElig3DILIAN GEORGIEV4 MICHAL HORSAacuteK5 IOAN SIRBU6

1Department of Malacology Institute of Zoology Jagiellonian University Gronostajowa 9 30-387 KrakoacutewPoland (e-mail andrzejfalniowskiujedupl)2Schulstr 3 D-25491 Hetlingen Germany3Department of Biology University of Montenegro Cetinjski put bb 81000 Podgorica Montenegro4Department of Ecology and Environmental Conservation University of Plovdiv Tzar Assen St 24BG-4000 Plovdiv Bulgaria5Department of Botany and Zoology Masaryk University Kotlaacuteoslashskaacute 2 61137 Brno Czech Republic6Department of Ecology and Environmental Protection Lucian Blaga University 31 Oituz St 550337Sibiu Romaniacorresponding author

ABSTRACT 61 sequences of cytochrome oxidase subunit 1 (COI) 570 bp long from Central Europe (8)Slovenia (2) and from the Balkans (51 6 from Romania 15 from Greece 15 from Bulgaria 6 from Serbiaand 9 fromMontenegro) 33 of them new together with 61 sequences of the ribosomal 18S 450 bp long all ofthem new were analysed to infer the pattern of radiation of Bythinella in the Balkans Thirty two nominal taxaof Bythinella (22 nominal species B austriaca B calimanica B charpentieri B compressa B dacica B dispersa Bgrossui B hansboetersi B luteola B micherdzinskii B molcsanyi B nonveilleri B pannonica B pesterica Bradomani B rhodopensis B robiciana B schmidti B slaveyae B srednogorica B taraensis and B viseuiana onenominal subspecies B austriaca ehrmanni and nine Greek species not yet described) were included repre-sented mostly by paratypes or at least topotypes collected at 31 Balkan localities The phylogeny inferred onthe combined data set with the ML approach showed two large clades although they were weakly supportedOne of them comprised the Romanian and Montenegro populations and one Serbian population the other(less genetically diversified) consisted of one Serbian and all the BulgarianGreek populations The origin ofthose two clades was dated with the external data to be more than 4341plusmn0753 MYA old thus its origin was as-signed to isolation by the Dacic Basin (part of Paratethys) All the Bulgarian populations presumably belong toone species which may be assigned to the recent recolonisation of this territory from the south

KEY WORDS Rissooidea Balkan Peninsula DNA phylogeny taxonomy speciation

INTRODUCTION

The genus Bythinella Moquin-Tandon 1856 is dis-tributed from west Europe (the Iberian Peninsula) towest Asia These dioecious oviparous snails with mi-nute dextral ovoid shells inhabit freshwater springssmall brooks and groundwaters (GIUSTI amp PEZZOLI1977 FALNIOWSKI 1987) They are found in abun-dance on mosses and other aquatic plants fallenleaves dead wood etc The rich literature on

Bythinella (RADOMAN 1976 1983 1985 GIUSTI ampPEZZOLI 1977 FALNIOWSKI 1987 1992 GLOumlER 2002SZAROWSKA amp WILKE 2004) pertains mainly to west-ern southern and central Europe The characters ofeither the shell or the soft parts do not allow for cleardelineation of Bythinella species Recently BICHAIN etal (2007a b) HAASE et al (2007) FALNIOWSKI et al(2009b c) and BENKE et al (2009) applied molecular

Vol 20(1) 1ndash10

doi 102478v10125-012-0006-2

data and proved the distinctiveness of several speciesof the genus The ranges of some Bythinella speciesstudied molecularly so far (eg BICHAIN et al 2007ab FALNIOWSKI et al 2009b c BENKE et al 2009 FAL-NIOWSKI amp SZAROWSKA 2011) are restricted to a singlespring spring complex or local watershed whilethose of the other congeners stretch across one ormore drainage dividesThere were several species described and rede-

scribed in central Europe (JUNGBLUTH amp BOETERS1977 FALNIOWSKI 1987 1992 GLOumlER 2002SZAROWSKAampWILKE 2004 HAASE et al 2007) and theformer Yugoslavia (RADOMAN 1976 1983 1985GLOumlER 2008 GLOumlER amp PEŠIAElig 2010) Recently wefound six molecularly distinct species of this genus inRomania (FALNIOWSKI et al 2009a b) and ten molec-ularly distinct (9 of them not yet described) species inGreece (FALNIOWSKI amp SZAROWSKA 2011) Severalnominal species were recently reported from Bulgaria(GLOumlER amp PEŠIAElig 2006 FALNIOWSKI et al 2009aGLOumlER amp GEORGIEV 2009) and Montenegro (GLOumlERamp PEŠIAElig 2010)FALNIOWSKI et al (2009b c) and FALNIOWSKI amp

SZAROWSKA (2011) demonstrated that (1) themorphostatic mode of evolution as defined by DAVIS(1992) is common in Bythinella (2) it is impossible todistinguish Bythinella species if there are no moleculardata although the morphology must be considered as

well (BICHAIN et al 2007b HAASE et al 2007) In thisstudy we applied mitochondrial cytochrome oxidasesubunit I (COI) and ribosomal 18S to infer the gen-eral pattern of radiation in this genus in the BalkanPeninsula Most of the specimens sequenced wereparatypes or at least topotypes of the nominal taxaconsidering all the controversies concerning the spe-cies distinctness within Bythinella (GIUSTI amp PEZZOLI1977 FALNIOWSKI 1987 BICHAIN et al 2007a bHAASE et al 2007 FALNIOWSKI et al 2009b cFALNIOWSKI amp SZAROWSKA 2011) we leave unresolvedthe taxonomic status of those nominal taxaThe Balkans once harboured one of the main Eu-

ropean glacial refugia (HEWITT 1996 2000 VOGEL etal 1999 PETIT et al 2003 SCHMITT 2007 FALNIOW-SKI et al 2009b) Hence they provide an interestingarea for a study of radiation In Romania (FALNIOW-SKI et al 2009a b) we found greater genetic differ-ences between Bythinella species than in Greece (FAL-NIOWSKI amp SZAROWSKA 2011) However the data onthe genus inhabiting the zone between Romania andBulgaria are scarce (FALNIOWSKI et al 2009a) Thusthe aim of the present paper was to answer the follow-ing questions (1) Can the pattern of genetic differen-tiation decreasing from north to south be applied toall the Balkans (2) Does the pattern of (presumably)vicariant speciation reflect any palaeogeographic con-ditions in the Balkans

MATERIALS AND METHODS

MATERIAL COLLECTION AND FIXATION

We collected snails with a sieve or by hand at 30localities in the Balkans (Fig 1 Table 1) Snails werewashed twice in 80 ethanol and left to stand in it forca 12 hours after which the ethanol was changedtwice in 24 hours and finally after a few days the 80solution was replaced with a 96 one The materialwas stored at ndash20degC

MOLECULAR TECHNIQUES APPLIED

Snails were hydrated in TE buffer (3 times 10 min)and their DNA was extracted with the SHERLOCK ex-tracting kit (AampA Biotechnology) the final productwas dissolved in 20 microl of TE buffer The PCR reactionwas performed with the following primers LCOI490(5rsquo-GGTCAACAAATCATAAAGATATTGG-3rsquo) andCOR722b (5rsquo-TAAACTTCAGGGTGACCAAAAAATYA-3rsquo) for the COI gene (FOLMER et al 1994) andSWAM18SF1 (5rsquo-GAATGGCTCATTAAATCAGTCGAGGTTCCTTAGATGATCCAAATC-3rsquo) SWAM18SR1(5rsquo-ATCCTCGTTAAAGGGTTTAAAGTGTACTCATTCCAATTACGGAGC-3rsquo) for the 18S gene (PALUMBI1996) The PCR conditions were as follows

1 For CO1 4 min at 94degC followed by 35 cycles of 1min at 94degC 1 min at 55degC 2 min at 72degC afterall cycles an additional elongation step of 4 min at72degC was performed

2 For 18S 4 min at 94degC followed by 40 cycles of 45sec at 94degC 45 sec at 51degC 2 min at 72degC after allcycles an additional elongation step of 4 min at72degC was performedThe total volume of each PCR reaction mixture

was 50 microl To check the quality of the PCR products10 microl of the PCR product was run on 1 agarose gelThe PCR product was purified using Clean-Up col-umns (AampA Biotechnology) The purified PCR prod-uct was dye-terminator amplified in both directions(HILLIS et al 1996) using BigDye Terminator v31(Applied Biosystems) following the manufacturerrsquosprotocol and with the primers described above Thesequencing reaction products were purified using Ex-Terminator Columns (AampA Biotechnology) and thesequences were read using an ABI Prism sequencer

DATA ANALYSIS

The COI sequences were aligned by eye using Bio-Edit 500 (HALL 1999) and edited with MACCLADE

2 A Falniowski M Szarowska P Gloumler V Pešiaelig D Georgiev M Horsaacutek I Sirbu

405 (MADDISON amp MADDISON 2002) We examinedmutational saturation for the COI dataset with satura-tion test of XIA et al (XIA et al 2003) performed withDAMBE 529 (XIA 2000) In 18S alignment was per-formed using CLUSTALX 182 (THOMPSON et al1997)Phylogenies of COI and 18S haplotypes were in-

ferred using maximum-likelihood (ML) minimumevolution (ME) and maximum parsimonyThe maximum-likelihood technique of phylog-

eny reconstruction (SWOFFORD et al 1996 NEI ampKUMAR 2000 FALNIOWSKI 2003) although criticisedis commonly used for molecular data and consideredthe most reliable one so we decided to use the MLapproach for each of the two data sets For each max-imum likelihood analysis we tested different modelsof sequence evolution using Modeltest v306(POSADA amp CRANDALL 1998 POSADA 2003) Follow-ing the recommendations of POSADA amp BUCKLEY(2004) and SOBER (2002) the best model for eachdataset was chosen using the Akaike InformationCriterion (AKAIKE 1974) ML analyses were per-formed in PAUP40b10 (SWOFFORD 2002) using anheuristic search strategy with stepwise addition oftaxa 10 random-sequence addition replicates andtree-bisection-reconnection (TBR) branch swapping(SWOFFORD et al 1996) Nodal support was esti-

Radiation in Bythinella in the Balkans 3

Table 1 Localities and taxa studied N ndash number of COI sequences used for phylogenetic inferrence sequences not pub-lished earlier marked with aterisk

No Locality descriptionGeographicalcoordinates

Heigthasl

TaxonCom-ments

N

1 Catildelimani Mts near Satildelard villageToplita-Dead Gorges of the MureordmRiver Romania

46deg5710N25deg0407E

620 m Bythinella calimanicaFalniowski Szarowska etSirbu 2009

paratype 1

2 Viordmeu River Valley near BistraRomania


362 m Bythinella viseuiana FalniowskiSzarowska et Sirbu 2009

paratype 1

3 Izvoare Resort (Igniordm Mountains)Mara River Basin Romania(sympatrically)


909 m Bythinella grossui FalniowskiSzarowska et Sirbu 2009Bythinella molcsanyi H Wagner1941

paratype

topotype

1

1

4 Bihor Mountains close to VacircrtopPass Criordmul Batildeithorna River BasinRomania


1142 m Bythinella radomani FalniowskiSzarowska et Sirbu 2009

paratype 1

5 Retezat Mts Natural Park La BeciButa River Valley Romania


1181 m Bythinella dacica Grossu 1946 1

6 Canyon of the River Tara streamLjevok Montenegro

42deg5929N19deg2553 E

835 m Bythinella taraensis Gloumler etPešiaelig 2010

paratype 3

7 Pester Plateau Djerekare villageStraegraveijevac Hill Straegraveijevac springSerbia

43deg00N20deg08E

1350 m Bythinella pesterica Gloumler 2008 paratype 3

8 National Park Biogradska GoraBjelasica Mt Montenegro


1119 m Bythinella luteola Radoman1976

3

9 Spring near Biogradsko LakeMontenegro Berane spring inPetnjik village Montenegro


988 m Bythinella dispersa Radoman1976

3

Fig 1 Sampled localities of Bythinella in the Balkans Local-ities numbers see Table 1 Dotted line ndash border betweentwo main clades (A and B) Figure produced usingCartografx Professional Software



Heigthasl

TaxonCom-ments

N

10 Rtanj Mt Vrmdaa gorge Vrmdaaspring Serbia

43deg42N21deg49E

600 m Bythinella nonveilleri Gloumler2008

paratype 3

11 Anton town below Bolovan HillBulgaria


1630 m Bythinella hansboetersi Gloumler etPešiaelig 2006

1

12 Anton town below the top ofBolovan Hill Bulgaria


1850 m Bythinella hansboetersi 1

13 Sredna Gora Mts W of Djulevo vil-lage S of Streltcha town Bulgaria


393 m Bythinella srednogorica Gloumler etGeorgiev 2009

paratype 3

14 Stara Planina spring of CherniOsam Bulgaria


2150 m Bythinella hansboetersi paratype 1

15 West Rhodopes Mts S of Lilkovo vil-lage near Modar peak Bulgaria


1516 m Bythinella rhodopensis Gloumler etGeorgiev 2009

paratype 3

16 Mugla village Bulgaria 41deg3743N24deg3108E


17 Belasits Mts S of Belasitsa villageBulgaria


687 m Bythinella slaveyae Gloumler etGeorgiev 2011

paratype 3

18 Smoljan town below SmoljanskeLake Bulgaria



19 Smoljan town near Amzovo Bulgaria 41deg3342N24deg4141E


20 Anthousa Pindos Mts EpirusGreece


967 m Bythinella sp G1 1

21 Sirakon Pindos Mts Epirus Greece 39deg3541N21deg0616E


22 Palaiokhorion W of KardhitsaThessalia Greece



23 between Kalivia Filaktis and PortitsaSW of Kardhitsa Thessalia Greece



24 Pilion Mt N of Dhrakia E of VolosGreece Pilion Mt N of Dhrakia E ofVolos Greece

39deg2336N23deg0233E39deg2435N23deg0454E

1082 m

860 m

Bythinella sp G5

Bythinella sp G6

(4M24)

(4M22)

1

1

25 Kerasias Spring Sivros Lefkas IslandGreece



26 Agtrini S of Eptalofos ParrnassosMts Greece


1017 m Bythinella charpentieri (Roth1855)

1

27 Kessariani Monastery Athens AtticaGreece


358 m Bythinella charpentieri 1

28 between Bouzion and Kalianci NPeloponnese Greece



29 Agio Theodori near Belitseka andKoutiveika Taigetos Mts Greece



30 NNW of Ghorani Taigetos MtsPeloponnese Greece



31 spring at Karavas N of Kithira IslandGreece



spring at macrelazno S of Ksup3odzkoSudety Mts Poland


306 m Bythinella austriaca ehrmanniPax 1938

topotype 4

Rawka Mt Bieszczady Mts Poland 49deg0718N22deg3516E

846 m Bythinella micherdzinskiiFalniowski 1980

topotype 1

Table 1 continued

mated using the bootstrap approach (FELSENSTEIN1985) Bootstrap values for ML trees were calculatedusing 1000 bootstrap replicates the ldquofastrdquo heuristicsearch algorithm and the same model parameters asused for each ML analysisMinimum evolution (ME) and maximum parsi-

mony (MP) were run on PAUP nodal support wasestimated using the bootstrap approach (full heuris-tic search) with 1000 replicates PAUP was used totest the molecular clock hypothesis for COI with alikelihood ratio test (LRT) upon the estimated treeand best-fit model (FELSENSTEIN 1981 NEI amp KUMAR2000 POSADA 2003)In the phylogeny reconstruction for COI five

central European Bythinella species (B austriaca B

compressa B pannonica B robiciana and B schmidtiTable 2) and B micherdzinskii (Table 1) were used asoutgroups Next the partition homogeneity test(FARRIS et al 1995) was performed (1000 repli-cates) with PAUP to check whether the two genescould be analysed together The maximum likeli-hood heuristic search was then run for the combinedmolecular data K2P (KIMURA 1980) distances forthe COI data were calculated using PAUP40b10(SWOFFORD 2002) There is a rich literature con-cerning the use of K2P distances for COI data thuswe chose this distance for the sake of comparison ofthe levels of differentiation


Table 2 GenBank Accession Numbers and references of COI sequences already published

Species GenBankAN References

Bythinella austriaca (Frauenfeld 1857) FJ545132 FALNIOWSKI et al (2009a)

B compressa (Frauenfeld 1857) AF367653 SZAROWSKA amp WILKE (2004)

B pannonica (Frauenfeld 1865) AY222660 SZAROWSKA amp WILKE (2004)

B robiciana (Clessin 1890) AY273998 SZAROWSKA amp WILKE (2004)

B schmidti (Kuumlster 1852) AY222649 SZAROWSKA amp WILKE (2004)

B dacica Grossu 1946 FJ545011 FALNIOWSKI et al (2009a)

B calimanica Falniowski Szarowska et Sirbu 2009 FJ545084 FALNIOWSKI et al (2009a)

B molcsanyi H Wagner 1941 FJ545062 FALNIOWSKI et al (2009a)

B grossui Falniowski Szarowska et Sirbu 2009 FJ545106 FALNIOWSKI et al (2009a)

B radomani Falniowski Szarowska et Sirbu 2009 FJ545069 FALNIOWSKI et al (2009a)

B viseuiana Falniowski Szarowska et Sirbu 2009 FJ545097 FALNIOWSKI et al (2009a)

B hansboetersi Gloumler et Pešiaelig 2006 locality 11 GQ152534 FALNIOWSKI et al (2009b)

locality 12 GQ152530 FALNIOWSKI et al (2009b)




B charpentieri (Roth 1855) locality 26 JF314213 FALNIOWSKI amp SZAROWSKA (2011)

locality 27 JF314160 FALNIOWSKI amp SZAROWSKA (2011)

Bythinella sp G1 locality 20 JF314305 FALNIOWSKI amp SZAROWSKA (2011)










Bithynia tentaculata (Linnaeus 1758) AF367643 WILKE et al (2001)

Marstoniopsis insubrica (Kuumlster 1853) AY027813 FALNIOWSKI amp WILKE (2001)

RESULTS

In total we analysed 63 sequences of COI 570 bplong Two of the sequences represented the outgroupspecies (Bithynia tentaculata and Marstoniopsisinsubrica Table 2) six came from Romania(FALNIOWSKI et al 2009b) 12 from Greece(FALNIOWSKI amp SZAROWSKA 2011) five from Bulgaria(FALNIOWSKI et al 2009a) 33 sequences were new(GenBank Accession Numbers JQ639854-JQ639886)All the 61 sequences of 18S 450 bp long were new(GenBank Accession Numbers JQ639793-JQ639853)

Saturation test of XIA et al (2003) showed little sat-uration in our COI data Iss = 0309 Issc = 0707 for sym-metrical topology df = 190 p = 0000 and Issc = 0385for asymmetrical topology df = 190 p = 00079For the COI the Akaike Information Criterion

(AIC) with ModelTest selected the model GTR+I+Atildewith base frequencies A = 03409 C = 02163 G =01225 T = 03204 substitution rate matrix [A-C] =2728169 [A-G] = 42221479 [A-T] = 2062349 [C-G]= 4383951 [C-T] = 27151609 [G-T] = 10000 pro-


Fig 2 Maximum likelihood phylogram of the studied Bythinella Bootstrap supports ML (in bold)MPMENJ are given foreach clade locality number in bold for each terminal branch Arrow indicates the bifurcation between the two main Bal-kan clades (A and B) of Bythinella whose border is marked with dotted line in Fig 1 Shaded rectangle indicates propor-tional shortening of the branches connecting the ingroup with the outgroup taxa

portion of invariable sites (I) = 06519 and Atilde distribu-tion with the shape parameter = 25072 The Likeli-hood Ratio Test (LRT) for the COI resulted in insig-nificant values (p = 0023) so we rejected the molecu-lar clock hypothesisThe partition homogeneity test (p=0732) showed

that the two genes could be analysed together For thecombined data set the Akaike Information Criterion(AIC) with ModelTest selected the model HKY+I+Atildewith base frequencies A = 03077 C = 02397 G =01821 T = 02704 substitution rate matrix titv ratio52693 proportion of invariable sites (I) = 07340 andAtilde distribution with the shape parameter = 10465Figure 2 shows the ML tree computed for the com-

bined data in which Bythinella pannonica from Slovakiaforms a distinct Bythinella clade closest to the root Bmicherdzinski B austriaca and B austriaca ehrmannifrom Poland form another clade The Balkan taxa

form two clades that are distinct but weakly supported(596489 depending on the technique) clade A(Fig 2) consisting of populations 1ndash9 (Table 1) Brobiciana and B schmidti although population 4 repre-senting B radomani (marked with asterisk in Fig 2)falls between the clade of the Polish Bythinella and theother Balkan clade (Fig 2 Arsquo) The second large Bal-kan clade (B in Fig 2) clusters all the other Balkanpopulations 10ndash31 (Table 1) (this bifurcation ismarked with arrow in Fig 2 the dotted line in Fig 1 in-dicates the geographic border between the two largeclades) Within clade A the differences between thenominal taxa reflected in branch lengths (Fig 2) aswell as in K2P distances (within group mean 01004)are greater than in clade B (within group mean00499) The pairwise K2P distances between the twoclades are 009ndash01744 mean 01224 net mean 00473

DISCUSSION

FALNIOWSKI amp SZAROWSKA (2011) demonstratedthat in the Greek Bythinella there was much lessinterspecific differentiation than in the RomanianBythinella and the diversity decreased from north tosouth In this study we observed the same general pat-tern throughout the East Balkans The somewhatlarger distances between the studied Romanian popu-lations may contribute to this pattern However thiscannot be the main explanation as between the twosympatric species at locality 4 in Romania the distancewas not smaller than between the allopatric Romanianspecies Thus another explanation should be invokedThe Romanian Pleistocene refugium or rather a

set of small territories with a mild climate was situatedfarther to the north than the other European refugiaIn fact it was rather a group of nunatak-like habitatsthe fauna of which was probably under a strong influ-ence of unstable climatic conditions Across the coun-try there were several scattered glacial refugia whichduring the interglacials and especially in thepostglacial served as distribution centres (FALNIOWSKIet al 2009b) The expansions and contractions of theglacier proceeded by way of climatic changes and werefollowed by the contractions and expansions of thenunatak refugia thus Bythinella ranges in the studiedterritory may have promoted genetic differentiationwithin the genus (HEWITT 1989) This contrasts withthe south Balkan (Greek) refugium which was rathercontinuous geographically and characterised by rela-tively stable conditions Perhaps those more stable con-ditions during the Pleistocene are reflected in smallerdifferences between the species of Bythinella in thesouth of the BalkansThe two large clades in the inferred phylogeny are

well-defined geographically The dotted line in the

map (Fig 1) marks the border between these twoclades The first (clade A localities 1ndash9) includes thesix Romanian species (localities 1ndash5) the three Monte-negro populations (localities 6 and 8ndash9) and one oftwo Serbian populations of Bythinella (locality 7) Thisgroup is closer to B pannonica from Slovakia and Hun-gary B austriaca and B micherdzinskii from Poland Bcompressa from Germany as well as B schmidti and Brobiciana from Slovenia and to the outgroup In fact Bradomani from locality 4 (Arsquo in Fig 2) clusters betweenthe Polish taxa and the second large clade This may beexplained in part by the location of that species in thenorthernmost part of Romania thus not far from thePolish localities One can speculate that B radomani in-vaded the locality from the northnorthwestThe second clade (clade B) clusters all popula-

tions from Greece and Bulgaria together with onepopulation (10) from Serbia As stated above the ge-netic diversity as marked in COI was lower within thisclade It is noteworthy that presumably only one spe-cies occurs across Bulgaria As could be seen in themap (Fig 1) there are unfortunately no samplesclose to the border marked on the map thus its local-isation is a very rough estimate only Moreover wecannot prove the real distinctness of those two cladessince their bootstrap supports are rather low (Fig 2)Thus we can only hypothesise about this part of thehistory of Bythinella in the BalkansThere are no data on the fossil occurrence of

Bythinella in the studied part of Europe Fossil Bythinellawere found in the Italian PliocenePleistocene bound-ary (southern margin of the Alps north of BergamoESU amp GIANOLLA 2009) In the Balkans ndash their south-ern part at least ndash the history of this genus cannot beshorter As we rejected the molecular clock hypothesis


ndash and had not any precise point of callibration thatwould enable us to apply the techniques of relaxingmolecular clock assumptions ndash we could not put a pre-cise time frame on our tree However we can use withsome caution the estimate of FALNIOWSKI ampSZAROWSKA (2011) dating the origin of the SE clade(containing all the Greek and Bulgarian populations)thus the time of cladogenesis marked with the arrow inFig 2 certainly was before 4341plusmn0753 MYA (middlePliocene) This coincides in time with the existence ofthe Dacic Basin a vast water body that separated thepresent Carpathians from the present middle Bulgaria(POPOV et al 2004 2006 POPESCU et al 2009) TheDacic Basin part of the Paratethys connected with thePannonian Basin in the west the Euxinian Basin in theeast and directly with the present Aegean Sea in thesouth eventually decreasing in size was still present tillthe latest Pliocene 18 MYA The Dacic Basin mostprobably separated the ancestors of the two largeclades Later in the Pleistocene the unstable fluvio-

lacustrine system in SW Bulgaria and northernGreece with glaciers present in the Pirin and Rila Mts(ZAGORCHEV 2007) probably formed effective tempo-rary barriers for Bythinella and may have caused its ex-tinction in the vast part of Bulgaria Considering thedata known so far the small differences among thenine Bulgarian populations may reflect the short his-tory of Bythinella in the area that most probably wasrecolonised from the south not earlier than in the latePleistocene

ACKNOWLEDGEMENTS

The study was supported by a grant from the PolishMinistry of Science and Higher Education (PB2443P01200631) to ANDRZEJ FALNIOWSKI Wewould like to thank the two anonymous reviewers fortheir valuable comments on the previous version ofthe manuscript

REFERENCES

AKAIKE H 1974 A new look at the statistical model identifi-cation IEEE Transactions of Automatic Control 19716ndash723 doi 101109TAC19741100705

BENKE M BRAumlNDLE M ALBRECHT C WILKE T 2009 Pleis-tocene phylogeography and phylogenetic concordancein cold-adapted spring snails (Bythinella spp) MolecEcol 18 890ndash903doi 101111j1365-294X200804073x

BICHAIN J-M GAUBERT P SAMADI S BOISSELIER-DUBAYLEM-C 2007a A gleam in the dark Phylogenetic speciesdelimitation in the confusing spring-snail genus Bythi-nella Moquin-Tandon 1856 (Gastropoda RissooideaAmnicolidae) Mol Phylogenet Evol 45 927ndash941doi 101016jympev200707018

BICHAIN J-M BOISSELIER-DUBAYLE M-C BOUCHET P SA-MADI S 2007b Species delimitation in the genus Bythinella(Mollusca Caenogastropoda Rissooidea) a first attemptcombining molecular and morphometrical dataMalacologia 49 293ndash311doi 1040020076-2997-492293

DAVIS G M 1992 Evolution of prosobranch snails transmit-ting Asian Schistosoma coevolution with Schistosoma a re-view Prog Clin Parasitol 3 145ndash204

ESU D GIANOLLA D 2009 The malacological record fromthe Plio-Pleistocene Leffe Basin (Bergamo Northern It-aly) Quatern Int 204 11ndash19doi 101016jquaint200808001

FALNIOWSKI A 1980 Bythinella micherdzinskii n sp ndash new spe-cies from the family Bythinellidae s Radoman (Gastro-poda Prosobranchia) Bull Acad Pol Sci Ser sci biolCl 2 28 225ndash230

FALNIOWSKI A 1987 Hydrobioidea of Poland (Prosobran-chia Gastropoda) Folia Malacol 1 1ndash122

FALNIOWSKI A 1992 Genus BythinellaMoquin-Tandon 1855in Poland (Gastropoda Prosobranchia Hydrobiidae In

GITTENBERGER E GOUD J (eds) Proc 9th Int MalacCongr Edinburgh 31 Augustndash6 September 1986 UnitasMalacologica Leiden 135ndash138

FALNIOWSKI A 2003 Metody numeryczne w taksonomiiWydawnictwo Uniwersytetu Jagiellontildeskiego Krakoacutew

FALNIOWSKI A HORSAKM SZAROWSKAM 2009a Bythinellahansboetersi Gloumler et Pešiaelig 2006 (Gastropoda Risso-oidea) in Bulgaria its morphology molecular distinct-ness and phylogeography Folia Malacol 17 11ndash20doi 102478v10125-009-0002-3

FALNIOWSKI A SZAROWSKA M 2011 Radiation and phylo-geography in a spring snail Bythinella (Mollusca Gastro-poda Rissooidea) in continental Greece Ann ZoolFennici 48 67ndash90

FALNIOWSKI A SZAROWSKA M SIRBU I 2009b BythinellaMoquin-Tandon 1856 (Gastropoda Rissooidea Bythi-nellidae) in Romania species richness in a glacial refugi-um J Nat Hist 43 2955ndash2973doi 10108000222930903359636

FALNIOWSKI A SZAROWSKA M SIRBU I 2009c BythinellaMoquin-Tandon 1856 (Gastropoda RissooideaBythinellidae) in Romania its morphology with descrip-tion of four new species Folia Malacol 17 21ndash36doi 102478v10125-009-0003-2

FALNIOWSKI A WILKE T 2001 The genus Marstoniopsis(Gastropoda Rissooidea) intra- and intergeneric phylo-genetic relationships J Moll Stud 67 483ndash488doi101093mollus674483

FARRIS J S KAumlLLERSJOuml M KLUGE A G BULT C 1995 Test-ing significance of incongruence Cladistics 10 315ndash319doi 101111j1096-00311994tb00181x

FELSENSTEIN J 1981 Evolutionary trees from DNA se-quences a maximum-likelihood approach J MolecEvol 17 368ndash376 doi 101007BF01734359


FELSENSTEIN J 1985 Confidence limits on phylogenies anapproach using the bootstrap Evolution 39 783ndash791doi 1023072408678

FOLMER O BLACK M HOEH W LUTZ R A VRIJENHOEK RC 1994 DNA primers for amplification of mitochondrialcytochrome c oxidase subunit I from diverse metazoan in-vertebrates Mol Mar Biol Biotechnol 3 294ndash299

GIUSTI F PEZZOLI E 1977 Primo contributo alla revisionedel genere Bythinella in Italia Natura Bresciana Annu-rario del Museo Civico di Storia Naturale di Brescia 143ndash80

GLOumlER P 2002 Die Suumlsswassergastropoden Nord- und Mit-teleuropas Bestimmungsschluumlssel Lebensweise Ver-breitung Die Tierwelt Deutschlands 73 Teil Conch-Books Hackenheim

GLOumlER P 2008 Three new hydrobioid species from Serbia(Mollusca Gastropoda Hydrobiidae) In PAVIAEligEVIAElig DM PERREAU (eds) Advances in the studies of the subter-ranean and epigean fauna of the Balkan Peninsula Vol-ume dedicated to the memory of Guido NonvellierMonograph 22 Institute for Nature Conservation of Ser-bia pp 349ndash356

GLOumlER P GEORGIEV D 2009 New Rissooidea from Bulgaria(Gastropoda Rissooidea) Mollusca 27 123ndash136

GLOumlER P PEŠIAElig V 2006 Bythinella hansboetersi n sp a newspecies from Bulgaria Heldia 6 11ndash15

GLOumlER P PEŠIAElig V 2010 The freshwater snails of the genusBythinella Moquin-Tandon (Gastropoda RissooideaHydrobiidae) from Montenegro Arch Biol Sci Bel-grade 62 441ndash447

HAASE M WILKE T MILDNER P 2007 Identifying species ofBythinella (Caenogastropoda Rissooidea) A plea for anintegrative approach Zootaxa 1563 1ndash16

HALL T A 1999 BioEdit a user-friendly biological sequencealignment editor and analysis program for Windows9598NT Nucleic Acids Symp Ser 41 95ndash98

HEWITT G M 1989 The subdivision of species by hybridzones In OTTE D ENDLER J A (eds) Speciation and itsconsequences Sinauer Associates Inc PublishersSunderland Massachusetts pp 85ndash110

HEWITT G M 1996 Some genetic consequences of ice agesand their role in divergence and speciation Biol J LinnSoc 58 247ndash276

HEWITT G M 2000 The genetic legacy of the Quaternaryice ages Nature 405 907ndash913 doi 10103835016000

HILLIS D M MABLE B K LARSON A DAVIS S K ZIMMERE A 1996 Nucleic acids IV Sequencing and cloning InHILLIS D M MORITZ L MABLE B K (eds) MolecularSystematics Second Edition Sinauer Associates Inc Pu-blishers Sunderland Massachusetts pp 321ndash381

JUNGBLUTH J H BOETERS H D 1977 Zur Artabgrenzungbei Bythinella dunkeri und bavarica (Prosobranchia) Ma-lacologia 16 143ndash147

KIMURA M 1980 A simple method for estimating evolution-ary rate of base substitutions through comparative stud-ies of nucleotide sequences J Mol Evol 16 111ndash120doi 101007BF01731581

MADDISON D R MADDISON W P 2002 MACCLADE Ver405 [Computer software and manual] Sinauer Associ-ates Inc Publishers Sunderland Massachusetts

NEI M KUMAR S 2000 Molecular evolution and phylo-genetics Oxford University Press Oxford

PALUMBI S R 1996 Nucleic Acids II The Polymerase ChainReaction In HILLIS D M MORITZ C MABLE B K(eds) Molecular Systematics Second Edition SinauerAssociates Inc Sunderland Massachusetts pp 205ndash247

PETIT R J AGUINAGALDE I DE BEAULIEU J-L BITTKAU CBREWER S CHEDDADI R ENNOS R FINESCHI S GRIVETD LASCOUX M MOHANTY A MUumlLLER-STARCK G DE-MESURE-MUSCH B PALMEacute A MARTIacuteN J P RENDELL SVENDRAMIN G 2003 Glacial refugia hotspots but notmelting pots of genetic diversity Science 300 1563ndash1565doi 101126science1083264

POPESCU S-M DALESME F JOUANNIC G ESCARGUEL GHEAD M MELINTE-DOBRINESCU M-C SUumlTO-SZENTAIM BAKRAC K CLAUZON G SUC J P 2009 Galeacystaetrusca complex dinoflagellate cyst marker of Parate-thyan influxes to the Mediterranean Sea before and afterthe peak of the Messinian Salinity Crisis Palynology 33105ndash134

POPOV S V ROumlGL F ROZANOV A Y STEININGER F FSHCHERBA I G KOVAC M 2004 Lithological-paleogeo-graphic maps of the Paratethys 10 maps Late Eocene toPliocene Courier Forschungsinstitut Senckenberg 2501ndash46

POPOV S V SHCHERBA I G ILYINA L B NEVESSKAYA L APARAMONOVA N P KHONDKARIAN S O MAGYAR I2006 Late Miocene to Pliocene palaeogeography of theParatethys and its relation to the Mediterranean Palaeo-geogr Palaeoclimatol Palaeoecol 238 91ndash106doi 101016jpalaeo200603020

POSADA D 2003 Selecting models of evolution In SALEMIM VANDAMME A- M (eds) The phylogenetic handbookA practical approach to DNA and protein phylogenyCambridge University Press Cambridge pp 256ndash282

POSADA D BUCKLEY T R 2004 Model selection and modelaveraging in phylogenetics Advantages of Akaike Infor-mation Criterion and Bayesian approaches over likeli-hood ratio tests Syst Biol 53 793ndash808doi 10108010635150490522304

POSADA D CRANDALL K A 1998 Modeltest testing themodel of DNA substitution Bioinformatics 14 817ndash818doi 101093bioinformatics149817

RADOMAN P 1976 Speciation within the family Bythinelli-dae on the Balkans and Asia Minor Z Zool System Evol14 130ndash152 doi 101111j1439-04691976tb00522x

RADOMAN P 1983 Hydrobioidea a superfamily of Proso-branchia (Gastropoda) I Systematics Monographs De-partment of Sciences Serbian Academy of Sciences andArts 547(57) 1ndash256

RADOMAN P 1985 Hydrobioidea a superfamily of proso-branchia (Gastropoda) II Origin zoogeography evolu-tion in the Balkans and Asia Minor Monographs Facultyof Science Department of Biology Institute of ZoologyBeograd 1 1ndash173

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SOBER E 2002 Instrumentalism parsimony and the Akaikeframework Philos Sci 69 112ndash123 doi 101086341839


SWOFFORD D L 2002 PAUP- Phylogenetic analysis usingparsimony ( and other methods) Ver 4 [Computersoftware and manual] Sinauer Associates Inc Sunder-land Massachusetts

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Received May 11th 2011Revised August 2nd 2011Accepted August 4th 2011


data and proved the distinctiveness of several speciesof the genus The ranges of some Bythinella speciesstudied molecularly so far (eg BICHAIN et al 2007ab FALNIOWSKI et al 2009b c BENKE et al 2009 FAL-NIOWSKI amp SZAROWSKA 2011) are restricted to a singlespring spring complex or local watershed whilethose of the other congeners stretch across one ormore drainage dividesThere were several species described and rede-

scribed in central Europe (JUNGBLUTH amp BOETERS1977 FALNIOWSKI 1987 1992 GLOumlER 2002SZAROWSKAampWILKE 2004 HAASE et al 2007) and theformer Yugoslavia (RADOMAN 1976 1983 1985GLOumlER 2008 GLOumlER amp PEŠIAElig 2010) Recently wefound six molecularly distinct species of this genus inRomania (FALNIOWSKI et al 2009a b) and ten molec-ularly distinct (9 of them not yet described) species inGreece (FALNIOWSKI amp SZAROWSKA 2011) Severalnominal species were recently reported from Bulgaria(GLOumlER amp PEŠIAElig 2006 FALNIOWSKI et al 2009aGLOumlER amp GEORGIEV 2009) and Montenegro (GLOumlERamp PEŠIAElig 2010)FALNIOWSKI et al (2009b c) and FALNIOWSKI amp

SZAROWSKA (2011) demonstrated that (1) themorphostatic mode of evolution as defined by DAVIS(1992) is common in Bythinella (2) it is impossible todistinguish Bythinella species if there are no moleculardata although the morphology must be considered as

well (BICHAIN et al 2007b HAASE et al 2007) In thisstudy we applied mitochondrial cytochrome oxidasesubunit I (COI) and ribosomal 18S to infer the gen-eral pattern of radiation in this genus in the BalkanPeninsula Most of the specimens sequenced wereparatypes or at least topotypes of the nominal taxaconsidering all the controversies concerning the spe-cies distinctness within Bythinella (GIUSTI amp PEZZOLI1977 FALNIOWSKI 1987 BICHAIN et al 2007a bHAASE et al 2007 FALNIOWSKI et al 2009b cFALNIOWSKI amp SZAROWSKA 2011) we leave unresolvedthe taxonomic status of those nominal taxaThe Balkans once harboured one of the main Eu-

ropean glacial refugia (HEWITT 1996 2000 VOGEL etal 1999 PETIT et al 2003 SCHMITT 2007 FALNIOW-SKI et al 2009b) Hence they provide an interestingarea for a study of radiation In Romania (FALNIOW-SKI et al 2009a b) we found greater genetic differ-ences between Bythinella species than in Greece (FAL-NIOWSKI amp SZAROWSKA 2011) However the data onthe genus inhabiting the zone between Romania andBulgaria are scarce (FALNIOWSKI et al 2009a) Thusthe aim of the present paper was to answer the follow-ing questions (1) Can the pattern of genetic differen-tiation decreasing from north to south be applied toall the Balkans (2) Does the pattern of (presumably)vicariant speciation reflect any palaeogeographic con-ditions in the Balkans

MATERIALS AND METHODS

MATERIAL COLLECTION AND FIXATION

We collected snails with a sieve or by hand at 30localities in the Balkans (Fig 1 Table 1) Snails werewashed twice in 80 ethanol and left to stand in it forca 12 hours after which the ethanol was changedtwice in 24 hours and finally after a few days the 80solution was replaced with a 96 one The materialwas stored at ndash20degC

MOLECULAR TECHNIQUES APPLIED

Snails were hydrated in TE buffer (3 times 10 min)and their DNA was extracted with the SHERLOCK ex-tracting kit (AampA Biotechnology) the final productwas dissolved in 20 microl of TE buffer The PCR reactionwas performed with the following primers LCOI490(5rsquo-GGTCAACAAATCATAAAGATATTGG-3rsquo) andCOR722b (5rsquo-TAAACTTCAGGGTGACCAAAAAATYA-3rsquo) for the COI gene (FOLMER et al 1994) andSWAM18SF1 (5rsquo-GAATGGCTCATTAAATCAGTCGAGGTTCCTTAGATGATCCAAATC-3rsquo) SWAM18SR1(5rsquo-ATCCTCGTTAAAGGGTTTAAAGTGTACTCATTCCAATTACGGAGC-3rsquo) for the 18S gene (PALUMBI1996) The PCR conditions were as follows

1 For CO1 4 min at 94degC followed by 35 cycles of 1min at 94degC 1 min at 55degC 2 min at 72degC afterall cycles an additional elongation step of 4 min at72degC was performed

2 For 18S 4 min at 94degC followed by 40 cycles of 45sec at 94degC 45 sec at 51degC 2 min at 72degC after allcycles an additional elongation step of 4 min at72degC was performedThe total volume of each PCR reaction mixture

was 50 microl To check the quality of the PCR products10 microl of the PCR product was run on 1 agarose gelThe PCR product was purified using Clean-Up col-umns (AampA Biotechnology) The purified PCR prod-uct was dye-terminator amplified in both directions(HILLIS et al 1996) using BigDye Terminator v31(Applied Biosystems) following the manufacturerrsquosprotocol and with the primers described above Thesequencing reaction products were purified using Ex-Terminator Columns (AampA Biotechnology) and thesequences were read using an ABI Prism sequencer

DATA ANALYSIS

The COI sequences were aligned by eye using Bio-Edit 500 (HALL 1999) and edited with MACCLADE








Heigthasl

TaxonCom-ments

N




paratype 1




paratype 1




paratype

topotype

1

1




paratype 1





42deg5929N19deg2553 E


paratype 3


43deg00N20deg08E





3




3




Heigthasl

TaxonCom-ments

N


43deg42N21deg49E


paratype 3




1







paratype 3







paratype 3






paratype 3



















1082 m

860 m

Bythinella sp G5

Bythinella sp G6

(4M24)

(4M22)

1

1







1



















topotype 4



topotype 1

Table 1 continued






































RESULTS










DISCUSSION










ACKNOWLEDGEMENTS


REFERENCES





































































Heigthasl

TaxonCom-ments

N




paratype 1




paratype 1




paratype

topotype

1

1




paratype 1





42deg5929N19deg2553 E


paratype 3


43deg00N20deg08E





3




3




Heigthasl

TaxonCom-ments

N


43deg42N21deg49E


paratype 3




1







paratype 3







paratype 3






paratype 3



















1082 m

860 m

Bythinella sp G5

Bythinella sp G6

(4M24)

(4M22)

1

1







1



















topotype 4



topotype 1

Table 1 continued






































RESULTS










DISCUSSION










ACKNOWLEDGEMENTS


REFERENCES

































































Heigthasl

TaxonCom-ments

N


43deg42N21deg49E


paratype 3




1







paratype 3







paratype 3






paratype 3



















1082 m

860 m

Bythinella sp G5

Bythinella sp G6

(4M24)

(4M22)

1

1







1



















topotype 4



topotype 1

Table 1 continued






































RESULTS










DISCUSSION










ACKNOWLEDGEMENTS


REFERENCES




































































































RESULTS










DISCUSSION










ACKNOWLEDGEMENTS


REFERENCES



































































DISCUSSION










ACKNOWLEDGEMENTS


REFERENCES

































































ACKNOWLEDGEMENTS


REFERENCES































































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