Molecular Ecology (2003)

12

, 2731–2745 doi: 10.1046/j.1365-294X.2003.01943.x

© 2003 Blackwell Publishing Ltd

Blackwell Publishing Ltd.

Ecological and morphological differentiation among cryptic evolutionary lineages in freshwater limpets of the nominal form-group

Ancylus fluviatilis

(O.F. Müller, 1774)

MARKUS PFENNINGER, S ID STAUBACH, CHRISTIAN ALBRECHT, BRUNO STREIT and KLAUS SCHWENK

Abteilung Ökologie und Evolution, Zoologisches Institut der J.W. Goethe-Universität, Bio-Campus Siesmayerstraße, D-60054 Frankfurt/Main, Germany

Abstract

The phylogeny and potential mode of speciation of the river limpet

Ancylus fluviatilis

(Basom-matophora) was examined using mitochondrial DNA sequences from 16S ribosomal RNA,cytochrome

c

oxidase subunit I (COI) and nuclear DNA from internal transcribed spacer(ITS-1) regions from 103 populations across Europe. Four highly divergent lineages wereobserved within

Ancylus.

Clade 1, representing the nominal taxon

Ancylus fluviatilis

(O.F.Müller, 1774), is mainly found in central and northern Europe, Clade 2 is present in a singlePortuguese population, Clade 3 is distributed on the Canary islands, North Africa and theeastern Mediterranean region, whereas Clade 4 inhabits the Northern Mediterraneancoasts. Phylogenetic analyses revealed an overall consistent topology of nuclear and mito-chondrial gene trees. Based on a molecular clock, we estimated that the basic radiationoccurred in the late Pliocene. Although clades differ significantly in size independent shellshape, morphological differentiation of lineages is not feasible without genetic data.Environmental data related to climate (precipitation, temperature, etc.) showed a significantdifferentiation of clades. Clade 1 dwells in relatively colder and more stable habitats thanClades 3 and 4, whose habitats in turn differ in a low or high amount of precipitation duringspring and autumn, respectively. Based on the combined data sets on mitochondrial DNA,nuclear DNA, morphological and ecological differentiation, we conclude that

Ancylus

represents a cryptic species complex of reproductively and genetically isolated lineages. Inaddition, the joint analysis suggests that ecological speciation is probable to explain currentpatterns.

Keywords

: Basommatophora, cryptic species, Gastropoda, speciation

Received 12 March 2003; revision received 7 June 2003; accepted 24 June 2003

Introduction

Studies of speciation rely on the identification and delim-itation of the species in question. However, the taxonomicalassessment of existing biodiversity in freshwater invertebratesis often hampered either by a lack of morphological diver-sification or the confounding effects of high phenotypicplasticity (e.g. Lee 2000; Müller 2000; Gómez

et al

. 2002;Witt

et al

. 2003). This results potentially in a serious under-estimation of evolutionary divergence due to cryptic speciescomplexes (Knowlton 1993). In such cases, an integrative

approach, using molecular, morphometrical and environ-mental information can be useful to delimit species(Pfenninger

et al

. 2002; Wilke

et al

. 2002). Molecular markers,in particular the joint analysis of cytoplasmic and nuclearloci (Hare 2001), can be used in a first step to infer separ-ately evolving lineages. In a second step, information onbiogeography, environmental differences and adaptivetransitions that are significantly associated with lineagesnot only allow the delimitation of species, but also infer-ences about the processes that were involved during speci-ation (Templeton 1998).

The form-group

Ancylus fluviatilis

(O.F. Müller, 1774), afreshwater limpet, is distributed over large parts of thewestern Palaearctic, including parts of North Africa. In this

Correspondence: Dr Markus Pfenninger. Fax: + 49 69 79824910;E-mail: Pfenninger@zoology.uni-frankfurt.de

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Molecular Ecology

, 12, 2731–2745

area, the species occurs in almost all permanent lowlandrivers, creeks, the shore zone of lakes and even irrigationchannels or fountains, without demanding high water quality(Glöer & Meier-Brook 1998). Alpine mountain ranges likethe Alps or the Pyrenees are not colonized by these limpets(Turner

et al

. 1998; own observations), most likely becausethe considerable bed-load of high mountain streams doesnot allow the establishment of populations. The presenceof a hard substrate, from which the limpets graze thebiofilm and where the egg-clutches are deposited, is anecessary prerequisite for their occurrence. The life cycle isannual, with the reproduction in spring after which themajority of the individuals die. There are indicationsthat the species is at least tetraploid (Kraus 1988), if notoctoploid (Burch

et al

. 1960). Allozyme studies suggestedthat current

A. fluviatilis

lineages resulted from the hybrid-ization of two relatively divergent parental taxa (Städler

et al

. 1996) as is reported for other basommatophorans(Goldman

et al

. 1983).

Ancylus fluviatilis

is highly selfing(Städler

et al

. 1993) and local populations can consist ofreproductively more or less isolated strains (Städler 1997).

In the western Palaearctic, dozens of species have beendescribed on the basis of substantial variation in anatomyand shell morphology among populations. In a revision,Hubendick (1970) accorded no taxonomic value to thisvariation and declared these taxa to be synonymous to

A.fluviatilis,

except the endemic species in the ancient lakeOchrid (Macedonia). Indeed, shell variation in

Ancylus

hasbeen shown to be at least partially induced by environ-mental factors (McMahon & Whitehead 1987). However,Hubendick (1970) pointed out that his inclusion of all west-ern Palaeartic populations of

Ancylus

into the so-calledform-group

fluviatilis

is based solely on the absence of sep-arating characters and suggests further investigations withmore elaborate techniques. The absence of conspicuousconchological or anatomical variation does not necessarilyindicate a single evolutionary lineage. Cryptic radiation isnot uncommon in molluscs (Emberton 1995; Sanjuan

et al

.1997; Trewick 1998; Simison & Lindberg 1999; Collin 2000)as well as hidden speciation, where the presence of few dis-tinct evolutionary lineages is veiled by extensive variationin shell morphology (Pfenninger & Magnin 2001).

Here, we report the presence of divergent crypticspecies, inferred with mitochondrial (16S, COI), nuclear(ITS-1) DNA markers, morphometrics and environmentalinformation in the from-group

A. fluviatilis

throughout thedistribution range.

Materials and methods

Populations sampled

Forty-nine populations from all over the distribution rangeof the form group

Ancylus fluviatilis

were sampled, including

the type locality in the river Ilm (Ger 19, Thüringen/Germany,Table 1). Additional information about frequencies andgeographical distribution of 16S haplotypes for 54 samplingsites were obtained from Bugert (2001; GenBank accessionnumbers AY238703-757). In total, 103 populations wereanalysed (Table 1).

DNA extraction, fragment amplification and sequencing

Individuals were extracted from their shells and grinded in10% w/v laundry detergent solution for storage at roomtemperature and tissue digestion following the protocol ofBahl & Pfenninger (1996). All samples were shaken for 24h at 37

°

C prior to phenol/chloroform extraction of totalDNA following a standard protocol (Sambrook

et al

. 1989).A 5

′

-fragment of the 16S target-DNA was amplified forall samples by PCR with universal primers of the sequence16S1 5

′

> CGC AGT ACT CTG ACT GTG C < 3

′

and 16S25

′

> GTC CGG TTT GAA CTC AGA TC < 3

′

. Amplificationwas performed with Boehringer

Taq

-polymerase in 12.5

µ

Ltotal reaction volume with standard reaction conditions.Samples were amplified for 10 cycles (92

°

C for 50 s, 44

°

Cfor 50 s and 72

°

C for 40 s) and 36 cycles (92

°

C for 30 s,48

°

C for 40 s and 72

°

C for 40 s) after initial incubation of90

°

C for 2 min 30 s. Both strands of the purified amplifica-tion products were cycle-sequenced with the Perkin Elmer

Taq

DyeDeoxy™ Terminator Cycle Sequencing Kit after theprotocol of the supplier and read automatically on the ABIPrism 377® sequencing device of the same manufacturer.Sequences were deposited in GenBank (AY350526–350568).

Sequences were initially aligned with

clustalw

andadjusted manually, following the molluscan 16S second-ary structure model of Lydeard

et al

. (2000). Obtained16S sequences were collapsed to haplotypes using the

collapse

1.0 tool provided by David Posada (http://bioag.byu.edu/zoology/crandall_lab).

After initial phylogenetic analysis (see below), three tofour individuals for each clade inferred with 16S sequenceswere chosen for additional analysis with partial COI andITS-1 sequences. The COI fragment was amplified withprimers described in Folmer

et al

. (1994) under the samereaction conditions as the 16S fragment. The nuclear ITS-1locus was amplified with primers obtained from Arm-bruster

et al

. (2000). Amplification was performed with 40cycles (92

°

C for 1 min, 48

°

C for 1 min, and 72

°

C for 1 min30 s). PCR products from both loci were processed andsequenced as described above. Sequences are availablefrom GenBank (AY350493–350525).

Phylogenetic analyses

Phylogenetic analyses were performed for three datasets: 16S rDNA fragment, both mitochondrial sequences16S and COI concatenated and the nuclear ITS-1 locus. The

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Table 1

List of sampling sites used in the study, their abbreviation, geographical position, number of molecular characterized individuals,method of characterization and number of individuals morphometrically characterized. Populations from Bugert (2001) are marked withan asterik

Sampling site Latitude Longitude

N

clade characterization

Method of clade characterization

N

morphometrics

NOR1* 59

°

92 10

°

71 2 sequencingLET1 56

°

20 26

°

05 5 sequencingLET2 56

°

46 23

°

24 5 sequencingGB1* 50

°

96

−

4

°

28 2 sequencingGB2 57

°

90

−

5

°

16 4 sequencing 4GB3 57

°

47

−

6

°

99 5 sequencing 3GB4 58

°

21

−

6

°

38 3 sequencing 7GER1* 54

°

28 10

°

58 2 sequencingGER2* 50

°

55 9

°

19 2 sequencingGER3* 50

°

57 9

°

23 3 sequencingGER4* 50

°

54 9

°

27 1 sequencingGER5* 50

°

47 9

°

16 1 sequencingGER6* 50

°

44 9

°

20 1 sequencingGER7* 50

°

40 9

°

28 1 sequencingGER8* 50

°

27 9

°

41 1 sequencingGER9* 50

°

23 8

°

36 4 sequencingGER10* 49

°

99 8

°

29 1 sequencingGER11* 49

°

99 8

°

30 2 sequencingGER12* 49

°

62 9

°

65 1 sequencingGER13* 49

°

35 6

°

71 2 sequencingGER14* 49

°

15 7

°

04 2 sequencingGER15* 48

°

79 8

°

24 2 sequencingGER16* 48

°

53 9

°

05 2 sequencingGER17* 47

°

76 9

°

13 1 sequencingGER18* 48

°

95 11

°

73 2 sequencingGER19 50

°

48 11

°

09 5 sequencing 5GER20 51

°

29 13

°

01 4 PCR-RFLP 4GER21 48

°

42 9

°

27 5 PCR-RFLP 5GER22 50

°

54 10

°

52 3 PCR-RFLP 3POL1* 53

°

24 19

°

83 2 sequencingPOL2* 50

°

16 19

°

63 1 sequencingPOL3* 50

°

08 21

°

96 2 sequencingAUT1* 46

°

69 16°00 4 sequencingAUT2* 46°68 16°00 1 sequencing 1AUT3 46°41 15°59 5 PCR-RFLP 4AUT4 48°03 16°18 3 PCR-RFLP 4UKR1* 48°31 23°05 2 sequencingUKR2 48°16 23°11 5 sequencingSLO1* 46°65 16°09 2 sequencingFRA1 47°32 6°43 3 sequencingFRA2 47°06 6°22 2 sequencingFRA3 43°46 2°44 5 sequencingFRA4 43°53 5°29 3 sequencing 6FRA5 47°22 0°31 1 sequencingFRA6 47°04 0°19 5 sequencing 5FRA7 46°25 0°16 5 PCR-RFLP 10FRA8 45°01 0°12 5 PCR-RFLP 6FRA9 44°02 0°09 3 PCR-RFLP 9FRA10 43°47 1°14 5 PCR-RFLP 3FRA11 42°35 2°22 4 PCR-RFLP 7FRA12 43°28 2°21 5 PCR-RFLP 14FRA13 44°21 2°58 5 PCR-RFLP 8FRA14 46°08 3°54 5 PCR-RFLP 10FRA15 46°56 4°13 4 PCR-RFLP 2

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COR1 42°37 8°78 3 PCR-RFLP 2COR2 42°27 8°71 3 PCR-RFLPCOR3 41°62 9°22 4 sequencing 1ITA1* 38°04 13°30 2 sequencingITA2* 37°88 14°81 2 sequencingITA3* 37°79 15°20 2 sequencingITA4* 38°22 16°12 2 sequencingITA5* 38°22 16°14 2 sequencingITA6* 38°38 16°39 2 sequencingITA7* 38°49 16°44 2 sequencingITA8* 39°07 16°39 2 sequencingITA9* 40°54 15°64 2 sequencingITA10* 41°56 14°55 2 sequencingITA11* 43°14 11°20 1 sequencingITA12* 43°45 11°44 2 sequencingITA13* 45°63 7°73 2 sequencingITA14* 45°67 7°69 2 sequencingSPA1* 40°86 −3°46 2 sequencingSPA2* 40°68 −1°88 2 sequencingSPA3* 38°68 −0°10 4 sequencingSPA4 43°19 −1°45 5 sequencing 4SPA5 42°45 −3°48 5 sequencing 3SPA6 39°46 −6°26 5 PCR-RFLP 10SPA7 38°03 −6°14 4 PCR-RFLP 1SPA8 36°44 −5°09 3 PCR-RFLP 7SPA9 37°33 −4°01 3 PCR-RFLP 1SPA10 38°25 −2°01 3 PCR-RFLP 14SPA11 40°07 −1°16 5 PCR-RFLP 3POR1* 37°64 −8°246 6 sequencingPOR2* 37°64 −8°316 7 sequencingPOR3 40°55 −8°16 3 sequencingPOR4 40°22 −8°15 3 sequencing 4POR5 37°13 −8°32 4 PCR-RFLP 13POR6 40°25 −8°29 3 PCR-RFLP 4CRO1 43°44 16°70 2 sequencingCRO2 42°43 17°59 3 PCR-RFLP 3CRO3 43°26 16°45 3 PCR-RFLP 5TUR1* 37°85 30°82 1 sequencingTUR2* 37°34 39°74 2 sequencingMAR1* 31°52 −8°01 2 sequencingMAR2* 31°92 −4°42 2 sequencingCAN1* 28°74 17°83 2 sequencingCAN2* 28°72 −17°83 2 sequencingCAN3* 28°16 −17°18 8 sequencingCAN4* 28°13 −17°14 2 sequencingCAN5* 28°15 −16°62 3 sequencingCAN6 28°25 15°90 1 sequencing 3CAN7 28°10 16°30 3 sequencing 2CAN8 28°16 16°50 4 PCR-RFLP 30CAN9 28°07 16°42 5 sequencing 19

Sampling site Latitude Longitude

N clade characterization

Method of clade characterization

N morphometrics

Table 1 Continued

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mitochondrial data sets were used in a combined analysisbecause (i) mitochondrial genes are not independentlyinherited and represent thus a single gene in the phylo-genetic sense and (ii) a homogeneity partition test (Farriset al. 1995) indicated that there are no significant differencesbetween the data sets (P = 0.65, 200 replicates).

Maximum likelihood (ML) methods were chosen toreconstruct the phylogenies of the DNA sequences. Thebest-fit model of nucleotide substitution for the ML ana-lyses was selected using the hierarchy of likelihood ratio testsimplemented in modeltest 3.0 (Posada & Crandall 1998).The search for optimal trees was conducted with a heuristicsearch, TBR branch swapping and 10 random sequenceaddition replicates. Sequences from the basommatophorantaxon Planorbarius corneus (Linnaeus 1758: AY350493–350525)were used consistently as outgroup in all analyses. Planorbisplanorbis (Linnaeus 1758: AY350493–350525), and an un-specified Ferrissia species (T. Wilke: AY350493–350525) wereused as additional outgroups in varying compositions,according to the availability of sequences. Nodal supportof the phylogenies was estimated using the bootstrapapproach (Felsenstein 1985) with 100 replicates. All analyseswere performed with paup* 4.10b (Swofford 1998).

Estimation of clade ages

No calibrated molecular clock rate is available for Ancylus orother closely related species. We have nevertheless attemptedto obtain a rough estimate of divergence times using awide range of molecular clocks (1–5% sequence divergenceper one million years). Therefore, the estimates should betaken with caution; however, they may still allow the abilityto distinguish between ancient vs. recent divergence estimates.

A log likelihood ratio test for the presence of a molecularclock was performed for each data set (Huelsenbeck &Crandall 1997). Uncorrected pairwise sequence diversityand divergence within and among clades were calculatedfor all data sets. Divergence times among inferred cladeswas estimated from the mitochondrial data set using theapproach of Nei (1987) with correction for ancestral poly-morphism (Edwards & Beerli 2000). The calculations wereperformed with mega2 (Kumar et al. 2001).

RFLP clade identification and characterization of populations

The COI alignment was used to identify differentialrestriction sites for clade 1, 3 and 4. Such sites were foundfor the restriction enzymes BamHI (Clade 1), BsaAI (Clade3) and MnI1 (Clade 4). RFLP-analysis of the PCR-fragmentwere carried out according to the recommendations of therestriction enzyme supplier, always including a positiveand negative control. Between three and five living adultindividuals per site were PCR-RFLP typed in cases whereno individuals were sequenced (Table 1).

Analysis of Molecular Variance (AMOVA)

amova was performed on a matrix of pairwise Euclideandifferences between haplotypes, hierarchically groupedafter clades and populations nested within clades. Separateanalyses were performed for each clade. To estimate thedegree of differentiation on the smaller spatial scale, aregion with higher sampling site density (Germany) wasanalysed separately. Only German populations from areasnot affected by Pleistocene glaciations were incorporatedin the analysis. The calculations were performed usingamova 1.55 (Excoffier & P.E. Smouse 1992).

Environmental characterization of sampling sites

Key climatic variables for each sampling site were extractedfrom global climate maps made available by the Food andAgriculture Organization of the United Nations at http://www.fao.org/waicent/faoinfo/sustdev/eidirect/climate/eisptext.htm. The maps provide mean monthly data, gainedby spatial extrapolation of neighbouring weather stationsfor grid cells of 0.5 degrees assembled over the period of1961–90 and represent thus the long-term climate in thisarea (Leemans & Cramer 1991). The following variables wereused to characterize the sampling sites: Rain_JAN (averageprecipitation in January in mm), Rain_APR (average preci-pitation in April in mm), Rain_JUL (average precipita-tion in July in mm), Rain_OCT (average precipitation inOctober in mm), Rain_DIV (coefficient of variation of monthlyprecipitation), Rain_AnnTOT (total amount of annualpreciptiation in mm), Sun_AnnTOT (average duration ofannual sunshine in percent of possible), Temp_AnnAV(average annual temperature in degree Celsius), Temp_JUL(average July temperature in degree Celsius), Temp_JAN(average January temperature in degree Celsius), Temp_MAX(average maximum temperature in hottest month in degreeCelsius), Temp_MIN (average minimum temperature incoldest month in degree Celcius) and Temp_NUM (numberof months with temperatures above 10 °C).

Morphometric characterization of shell variability

Shells were photographed with a digital camera from thetop, from the right side and from the back, always includ-ing a ruler on the image. The program tpsDIG (Rohlf 1999)was used to apply 300 equidistantly spaced points on theoutline of each of the three shell views. These points wereused to produce a closed outline curve. The shape of theshells was quantified by elliptic Fourier approximation asdescribed by Kuhl & Giardana (1982). This techniqueconsists of decomposing a closed contour curve in atwo-dimensional plane into a sum of harmonically relatedsequences. Fourier decompositions are sensitive to location,size and orientation of objects. We have consequently used

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the longitudinal, latitudinal and vertical axes of the shellsto rotate them into the same orientation. The images werethen centred and normalized for size. The decompositioninto Fourier series was computed with efawin (Isaev et al.1995), using the algorithms of Ferson et al. (1985). Theapplication of 3, 8 and 6 harmonics for top, side and backview was sufficient to reproduce the outline of each viewwith high accuracy. This resulted in 59 Fourier variables.Additionally, measurements of apex position were takenas relative deviations from the longitudinal, latitudinaland vertical axis, respectively.

Statistical treatment of the data

The morphometric and environmental variables were usedseparately to perform principal component (PCA) anddiscriminant analyses (DA), with statistica (StatSoft Inc.,1995). The inferred clade of the respective individual orpopulation was applied as grouping variable in DA. Apopulation was assigned to a clade when all geneticallytyped specimen belonged to a single clade. Populationswhere individuals from more than one clade were found(ITA 10 and ITA 13) were excluded from the analysis.

Results

Phylogenetic analysis

16S analysis yielded 64 unique haplotypes. The 16S dataset contained 341 positions. The sequence evolutionmodel best fitting the data was GTR. ML search found asingle best tree with four major clades, termed Clade 1 toClade 4. The nodes separating these clades had a bootstrapsupport higher than 90%. Clade 1 + 2 were resolved assister taxa, with Clade 3 as next relative and Clade 4 asmost basal lineage (Fig. 1).

Concatenating the COI and 16S data sets (Mito) for theindividuals for which both information was availableresulted in a data matrix of 1128 characters. A GTR + G + Imodel was chosen for this data set by modeltest. Again, asingle best tree was found by ML search. The same cladeswere identified with high bootstrap support as in 16S ana-lysis alone; however, the branching order differed. In thisanalysis, Clade 1 + 2 and Clade 3 + 4 were sister taxa to eachother (Fig. 2). This result is in general congruence with theresults for the nuclear ITS-1 locus. The ITS-1 data matrix con-tained 502 characters, which evolved most likely followingthe JC69 + G model. The ML search recovered four best trees,which differed only in the shallow branches of Clade 1. How-ever, Clade 3 and Clade 4 were not reciprocally monophy-letic; one haplotype (H30) appeared basal to Clade 4 (Fig. 2).

In total, 310 individuals were typed for their mito-chondrial haplotype clade. Geographical distribution ofhaplotypes (Fig. 3) suggests that clades can be found in

more or less distinct areas with partial overlaps. Clade 1haplotypes were found in sampling sites ranging fromSouthwest Spain over the British Isles to Norway and theUkraine. This clade was not found in Italy. Clade 2 has anextremely localized distribution; it was found so far only ina single location in Portugal (Por1). Because of this uniqueoccurrence and the lack of shells, this clade was notincluded into the environmental and morphologic ana-lyses. Clade 3 showed the widest distribution, ranging fromthe Canary islands over North Africa, Italy and Austria tothe Balkans and Turkey. The distribution thus overlappedthe area of Clade 4 in Italy and of Clade 1 in Austria. InItaly, there were two syntopic occurrences of Clade 3 and4 haplotypes within the same sampling site (ITA 10, ITA13). Other populations of Clade 4 were collected near thecoast in Portugal, Spain, France, Italy, Croatia and theislands of Sicily and Corsica.

Age of the clades

The log likelihood ratio test failed to reject the molecularclock hypothesis for the combined mitochondrial data set.Uncorrected sequence diversity within clades ranged from

Fig. 1 Maximum likelihood phylogeny of 16S haplotypes. Italicnumbers at nodes represent boostrap values larger than 50%.

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0.2% in Clade 4–2.2% in Clade 3. The uncorrected sequencedivergence among clades varied between 5.4% and 7.9%.The divergence time estimates among clades with the cor-rection for ancestral polymorphisms according to (Nei 1987)ranged from 0.9 to 1.6 Ma for 5% sequence divergence per 1million years to 4.5–7.8 Ma for a molecular clock rate of 1%.

Partition of 16S variation

Most of the variation was found among the clades (77.90%).There was little variation within populations (2.96%), leav-ing 19.14% of the variation among populations within theclades (Table 2). The inter/intra population relation did

Fig. 2 Maximum likelihood phylogeny ofmitochondrial (16S + COI) and nuclear(ITS-1) data sets. Italic numbers at nodesrepresent boostrap values larger than 50%.

Fig. 3 Distribution of haplotype diversity. Sampling sites where exclusively individuals of Clade 1 were found are represented by opencircles, of Clade 2 by a solid circle, of Clade 3 by triangles and of Clade 4 by solid squares. The two sampling sites where Clade 3 and Clade4 haplotypes were identified are marked by a half-filled square. Sites, where the morphometric variation was assessed are in italic bold face.

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not change much when each clade was analysed separately.In Clade 1, 88% of genetic variation was distributed amongpopulations, in Clade 3, 72% and in Clade 4, 94% (data notshown). However, analysed on a regional scale (German popu-lations only), a considerably weaker population structurewas observed (16% among populations), which indicates aconsiderable amount of past and/or present gene-flow.

Ecological differentiation of the clades

In PCA, axis 1, representing 51.7% of total variation, wasdetermined mainly by the variables Temp_Num (r =0.922), Temp_Jul (r = 0.905) and Rain_Oct (r = 0.902). Thisaxis ordinated the sampling sites mainly according to theirlatitudinal position. The second axis (26.5% of total vari-ation) was determined by Temp_Ann_Avenue (r = 0.887),Temp_Max (r = 0.878) and Ann_Rain_Tot (r = −0.875). Axis2 opposed more wet oceanic vs. rather dry continentalclimates. This ordination separated Clade 1 populationsfrom the other two clades reasonably well, however, nodistinction between Clade 3 and 4 populations was pos-sible. Clade 1 sampling sites covered more of the climatespace spanned by the PCA axes than either Clade 3 or 4(Fig. 4).

The DA, however, found variables that covaried signifi-cantly with all the clades. Eighty-one percent of the sam-pling points were a posteriori correctly categorized. This isreflected in a Wilk’s γ of 0.220 and an approximate F(24,168)of 8.029 (P < 0.0001). The first root is mainly determinedby the variables Sun_Ann_Tot (r = −0.80), Rain_Jul (0.74),Rain_Division (−0.67), Temp_Num (−0.63), Temp_JAN(−0.61), and Temp_MIN (−0.60). This root separates Clade 1populations on the positive end from sampling sites ofClade 3 and 4. The second root is mainly correlated to thevariables Rain_Apr (−0.46) and Rain_Oct (−0.42) withClade 3 sampling sites preferentially on the positive andClade 4 on the negative side (Fig. 5).

Morphological differentiation of the clades

The first two axes of the PCA accounted for 39.9% of totalmorphometric variation. However, the resulting ordinationof the individuals did not distinguish the clades. Despitethe thorough inspection of the shells projected to theextreme ends of the axes, no obvious shape trend could beidentified for either axis (results not graphically shown).

A Wilk’s γ of 0.195 and a highly significant approxi-mate F(68,420) of 7.80 (P < 0.0001) indicated a good

Source of variation d.f. SSVariance components

Percent variation P

Among clades 2 822 9.11 77.90 < 0.0001Among populations within clades 77 325 2.27 19.14 < 0.0001Within populations 123 34 0.35 2.96 < 0.0001Total 202 1181 11.69

Table 2 amova results of 16 S haplo-type sequence variation. Populations weregrouped according to their clade. Signi-ficance of variance components was assessedwith 9999 randomizations

Fig. 4 Plot of principal component scoresderived from PCA on environmentalvariables. Individuals of Clade 1 arerepresented by circles, of Clade 3 bytriangles and of Clade 4 by solid squares.

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separation of the clades according to the morphometricvariables. The posterior classification categorized 85.8%of the individuals correctly to the observed genetic clade.Plotting the canonical scores of root 1 against 2 and linkingsome of the most extreme points with the three analysedviews of the respective individuals (Fig. 6) revealed thatClade 4 is more similar to Clade 1 than to the sister-clade 3. The main difference between Clade 1 and 4 is the

rounder vs. the more elongated shell shape. Clade 3 differsfrom these two clades by the relatively lower apex posi-tion, resulting in a more curved back. Additionally,individuals from Clade 3 show a pronounced shell sculp-ture with prominent radial ridges. However, there is anoverlap in the morphospace among all clades that makesan unequivocal assignment difficult, if not impossible(Fig. 6).

Fig. 5 Plot of canonical scores derived fromDA on the environmental variables forsampling sites grouped after haplotypeclades. The encircled numbers indicate thecentre of the respective clade. Samplingsites where exclusively individuals ofClade 1 were found are represented bycircles, of Clade 3 by triangles and of Clade4 by solid squares.

Fig. 6 Plot of canonical scores derived fromDA on the morphometric variables groupedafter haplotype clades. The encircled num-bers indicate the centre of the respectiveclade. The three analysed views are givenfor some of the most extreme individualsin morphospace. Individuals of Clade 1 arerepresented by circles, of Clade 3 by trianglesand of Clade 4 by solid squares.

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Discussion

Phylogenetic analyses

All phylogenetic analyses of the mitochondrial data revealedthe presence of four highly divergent clades, similar, butnot identical to those inferred by Bugert (2001). The topologyof the gene-trees of independently inherited mitochondrialand nuclear markers (Fig. 2) were overall congruent. How-ever, Clade 3 and Clade 4 are not completely reciprocallymonophyletic in ITS-1 analysis, due to a single individual(H30). Three explanations for this conflict can be considered:(i) the nuclear data set has not enough variable positions toresolve the clades; (ii) there was gene-flow among the cladesafter the separation (secondary contact); or (iii) the lineagesorting process in the slower evolving nuclear genome isnot yet completed. Although we cannot exclude the firsttwo explanations, we favour the latter. The ‘three timesrule’ (Palumbi et al. 2001) predicts that, due to the theoret-ically 4-fold larger effective population size of the mito-chondrial genome, the majority of the nuclear loci shouldhave reached monophyly when the interclade divergence/intraclade diversity ratio is larger than 3 in mitochondrialgenes. Even though this is the case, a poly or paraphyleticnuclear locus is not beyond the expectations (Hudson &Turelli 2003). The integration of additional nuclear lociwould be necessary to solve this question. However, thegood overall congruence of mitochondrial and nucleargene-trees suggested that the inferred phylogenies reflectindeed the organismic history (Nichols 2001).

The most recent common ancestor of the extant haplo-types within each lineage, however, lived apparently quiterecently. This is suggested by the short branches within cladesand the long branches leading to the nodes of their origin (Figs1 and 2). Such a tree topology is indicative for long lastingsubdivision with a lack of gene-flow (Avise 1989). The in-ference of absence of gene-flow among populations of dif-ferent clades is strengthened by the amova results (Table 2).Within clades, the populations are highly structured, indi-cating little gene-flow among populations within the clades.This indicates that the distributional patterns within cladesare relatively ancient, because the populations had the timeto differentiate genetically. However, the sampling sitesespecially of Clades 3 and 4 are on average very distantlylocated from each other. Therefore, the overall pattern ofisolation among populations may not represent the situ-ation on a more regional scale, as was suggested by the analysisof the German populations that showed little differentiationand, hence, indicated substantial past or present gene-flow.

Age of the clades

In comparison to other morphostatic radiations of fresh-water invertebrates, the cryptic Ancylus lineages seem to be

the product of comparatively recent divergence events. Ifwe apply substitution rates typical for invertebrates (Avise2000), ranging from 2%–5%, a divergence of the majorclades in the late Pliocene is suggested. Even under theconservative assumption of a molecular clock rate of only1%, the deepest estimated divergence time does not exceed7.8 Ma. This contrasts the findings in freshwater crustaceans,where several cryptic species complexes were found withestimated ages of up to 50 Myr (e.g. Schwenk et al. 2000;Gómez et al. 2002; Witt et al. 2003).

Calibrated clock rates for molluscs are rare. A substitu-tion rate of 2% per million years was calibrated for themudsnail genus Hydrobia (T. Wilke, pers. communication)and the mitochondrial genomes of land snails evolve inthe same time span perhaps as fast as 5–10% (Thomaz et al.1996). The sequence divergence of maximum 2.2% betweenputative cryptic patellogastropod species of the Notoacmeafascicularis (Menke, 1851) complex suggests that the diver-gence is comparatively recent, too (Simison & Lindberg1999). Even though snails have a comparatively good fossilrecord (Goodfriend 1992), the calibration of divergenceevents is difficult, because, as in the presented case, con-generic species tend to be morphologically too similar todistinguish reliably between intra- and interspecific vari-ation (Pfenninger & Magnin 2001).

Geographical distribution of inferred clades

A distinct geographical association of the inferred cladesis apparent (Fig. 3). For two exceptions (Ita 10, 13), only asingle clade was found at each sampling site. The moststriking spatial pattern is their unbalanced distributionrange. Clades 1, 3 and 4 are widespread, while Clade 2 isextremely localized. All clades occur either on the Iberianpeninsula or in Morocco which indicates that this areacould have been the origin of the radiation, because commonpresence of lineages in a particular region suggests thatthey originated within this area (Avise 1994).

Clade 1 has its distribution in Northwest, Central Europeand Eastern Europe (Fig. 3). A part of this area (the North-ern European plane, including North Germany, Polandand Lettland, Scotland and Scandinavia) was ice coveredduring the Pleistocene and therefore most likely colonizedpostglacially. However, a larger part of the present dayclade range on the Iberian peninsular, France, South ofGermany and southeast Europe were not affected by thePleistocene in a way that would have prohibited the sur-vival of freshwater snails. The contemporary presence ofAncylus in Scandinavia up to 64° North (Hubendick 1970)shows that this species can survive climatic conditionssimilar to the periglacial areas in the Pleistocene. Thesister Clade 2 was found exclusively in a single popu-lation in Portugal. It is possible that this clade is regionallymore widespread, but it is very unlikely that we have

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missed a continent wide distribution due to inadequatesampling.

Clade 3 appears in two distribution areas: in the West theCanary islands and Morocco, and in the East Italy, the Bal-kans and Turkey. However, the large unsampled areas inNorth Africa, the Balkans and Greece do not allow to drawdefinite conclusions about the distribution. Clade 4 has adistribution along the northern Mediterranean coasts. Anoccurrence on the unsampled southern coasts can not beexcluded.

The distribution areas of the widespread clades 1, 3 and4 are apparently not limited by physical dispersal barrierswith respect to each other. On the Iberian peninsular, Sicilyand Italy, sampling sites of different clades are in closeproximity, sometimes within the same drainage system(Fig. 3). In general, unsuitable habitat, like alpine mountainranges or the sea, does not seem to limit the distribution ofthe clades. Clade 1 has crossed the Pyrenees and colonizedthe British Isles and Clade 4 can be found on Mediterra-nean islands. The most striking case is Clade 3, distributedover disparate areas with little common geological history,for example the Canary islands that are of volcanic originand were never in contact with a continent. Only the Alpsseem to have a certain isolating effect (Fig. 3). As the lim-pets are not able to cross land or sea water barriers actively,colonization of islands or other drainage systems musthave happened via passive dispersal. It was shown thatinsects can act as vectors for Ancylus (Rosewater 1970) andwas suspected for waterfowl (Boag 1986).

Ecologic differentiation of the clades

The variables identified by DA to discriminate signific-antly between the sampling sites of each clade are notidentical to those representing the most important climaticgradients over the investigated area in the PCA. Thisindicates that the significant covariation of environmentalvariables with clade distributions is not due to the con-siderable amount of climatic variation that can be expectedover a continent. Corroboration for this view arises fromthe larger overlap of the clades in the environmentalspace defined by the first two axes of the PCA (Fig. 4) incomparison to the DA (Fig. 5).

The climatic variables represent averages over a largearea (approximately 60 × 60 km). They are neverthelesscorrelated with clade ranges, which may be due to thefact that the local conditions in flowing water bodies aremore influenced by the conditions in the catchment areaupstream the sampling site than by the local climaticconditions.

The environmental variables that separate Clade 1 fromClade 3 and 4 in DA represent an environmental gradienton which the stations of Clade 1 are determined by wetsummers with little evaporation, constant water supply

throughout the year and, hence, little risk of running watersfalling dry. The many cold winter months affect snailactivity, which is not possible below a certain water tem-perature. Sampling sites where Clade 3 and 4 can be foundare mostly reigned by dry summers, high evaporation anda highly unbalanced precipitation regime, with rain fallingmainly in the moderately cold winter months. In this time,limpet activity is not restrained due to cold. On thesesampling sites, the risk of flowing water bodies falling dryor at least ceasing to flow during the summer is very high.Clade 3 is separated from Clade 4 by an even lower amountof precipitation in spring and autumn. Indeed, the samplingsites of Clade 3 were mainly temporally flowing waterbodies.

Ultimate factors influencing environmental variabilityas the investigated climatic variables were shown to havesubtle, indirect influence on the life history (Arad & Avivi1998), behaviour (Desbuquois & Madec 1998) and evenparasite infestation (Cabaret et al. 1997) of snails. For rheo-phile freshwater snails, especially the presence of runningwater with a certain amount of available oxygen can belimiting for the distribution (Glöer & Meier-Brook 1998).The variables distinguishing between the clades can all berelated to the water regime the limpets experience. It canthus be assumed that the climate patterns covarying withclade distributions present distinct selective regimes forthe respective Ancylus lineages. Differential life-historyand physiological adaptations may lead to the observedmutual exclusion. To test this hypothesis, fitness studiesbetween the Ancylus lineages on differences in physiologyand life-history traits, such as the timing of mating, eggdeposition or hatching to avoid unfavourable environ-mental conditions would be required. Additionally, an inspec-tion of the fine scaled distribution of the clades in one ofthe transitions zones could provide further clues for thereasons of the mutual exclusion.

Morphologic stasis

The clades showed a remarkably similar shell shape,considering that the lineages have evolved independentlyfor a considerable time. Even though the genetically inferredclades could be statistically distinguished a posteriori, theshape differences were only slight and it is virtually impos-sible to assign a given shell of unknown origin unambigu-ously to a clade without genetic and/or morphometricinformation. An explanation for this morphological stasisis not straightforward, because selective factors limitingmorphological diversification are not easily distinguishedfrom developmental constraints (Schlichting 1989). The limpetshell shape of Ancylus is believed to be an adaptation tolife on hard substrate exposed to flowing water. The largeaperture allows a large adhesion surface of the foot andthe shell has a hydro-dynamically favourable shape (Basch

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1963; Denny 2000). Even though there are large differencesin water regime between the habitats of the clades, resist-ance ability against removal from the substrate at somestages is certainly required in all the clades, preventingperhaps more pronounced morphological diversification.Another possible, not mutual exclusive factor seems to bethe reduction of predation risk due to enhanced attachmentto the substrate and therefore extended removal costs forthe predators of Ancylus, such as leeches, crayfish andmolluscivoran fishes.

In general, gastropods in freshwater habitats occupy amuch smaller range of possible gastropodan morphospacethan in the marine realm (Hubendick 1952). This supportsthe observation that in freshwater organisms, the morpho-logic similarity of sibling species is not a product ofgene-pool cohesion, but instead of slow morphologicaldivergence, perhaps due to the stability of the habitat(Hebert 1998).

Species delimitation

The inferred evolutionary lineages are significantly associatedwith environmental habitat differences representing mostlikely distinct selective regimes. The inference of threedifferent species is therefore justified (Templeton 1989).Clade 1 sampling sites included the type locality (river Ilm,Thüringen) of A. fluviatilis (O.F. Müller, 1774) which allowsto designate this specific name to the lineage. The Canarianpopulations were described as A. striatus (Quoy & Gaimard,1833), whose description matches more or less the shellsof Clade 3 sampled from there. However, shells fromNorth African or European Clade 3 populations werenot available, so that the extension of the specific name tothe entire clade requires additional studies. Despite theplethora of taxonomic names given to various Ancylusshell types (Hubendick 1970), no existing description ordistribution pattern could be unequivocally matched tothe Clade 4 species.

Potential reasons for speciation

The information collected to delineate the species allowsto speculate about the potential reason for divergence ofthe lineages (Templeton 1998). Schluter (2001) classified thereasons for speciation, regardless of spatial arrangement,into four categories, along which we will discuss potentialspeciation scenarios.

1 Chromosomal rearrangements as the primary reasonfor divergence can not be excluded, given the suspicionthat Ancylus is of allopolyploid origin (Burch et al. 1960).However, cursory chromosome preparations in the courseof this study gave no hint on differences in chromosomenumber among the clades.

2 Genetic drift is another potential explanation, giventhe observed largely allopatric distribution pattern. Tomake this scenario probable, a geological scenario witheffectively isolated long time refugia — the basis for driftbased speciation — has to be identified. The present daydistribution of genetic variation is not congruent withthose of freshwater organisms for which Pleistocenerefugia were identified on the Iberian peninsular, Italyand the Balkans (Englbrecht et al. 2000; Kontula & Vainola2001; Kotlik & Berrebi 2001; Koskinen et al. 2002;Triantafyllidis et al. 2002). In these taxa, the inferredlineages have either an eastern or western distribution,which is compatible with the hypothesis of a recoloniza-tion from different Pleistocene refuges. The Ancylusclades, however, are all distributed over at least twodifferent potential refugial areas and the clades are stag-gered from North to South. Therefore, it is unlikely thatthe Pleistocene glaciations had an isolating effect on thelineages. Moreover, even though they must be treatedwith caution, the molecular clock estimates point to adivergence in the late Pliocene. The late Pliocene wasa period marked by drastic and rapid sea level changes(Hsü et al. 1997). These changes isolated parts of theIberian peninsula and North Africa and, hence, thefreshwater habitats on separate islands (Krijgsman et al.1999), triggering speciation in other taxa (e.g. Beerli et al.1996; Gili & Martinell 2000). However, present day dis-tributions of the clades are not limited by such dispersalbarriers, so it is difficult to imagine why this shouldhave been the case in the past. Overall, the lack of a plau-sible geological scenario makes drift based speciation inallopatry improbable for Ancylus.

3 Speciation due to adaptations to different selective regimesappears to be a more probable option. As we have shown,the clade distributions covary with climates that repres-ent most likely different selective regimes for Ancylus.Additional support for this hypothesis arises from theestimated divergence time. The late Pliocene was markedby the appearance of the Mediterranean climate as a partof a global trend towards cooler and dryer climates (Suc1984). This period of global change spurred speciationevents in gastropods (e.g. Wilke & Falniowski 2001;Pfenninger et al. 2003) and other taxa (Hewitt 1996; Avise& Johns 1999). As it is likely that the clades are at presentlimited in their distribution mainly by the water regime,this climate change surely exerted according selectivepressure on the precursor populations of contemporaryAncylus lineages. Even though the respective climatezones have shifted during the Pleistocene, the exampleof Clade 1 shows that Ancylus could follow these shifts.

4 The last category, speciation due to differential adapta-tion to a similar selective regime, is not very probable,because the clades are not likely to experience a similarselective regime.

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Conclusions

The explicitly spatial approach, including molecular genetic,morphometric and environmental information allowed todetect and disentangle a cryptic species complex and madeecological speciation as cause for the observed divergenceprobable. The findings of this study seem thus to contradictthe notion of Gittenberger (1991) that cryptic radiations aregenerally the product of drift-based divergence in allopatry.

Acknowledgements

We are grateful for comments of the three referees and editorHansen which greatly improved the manuscript. We thank HolgerGeupel and Monika Bugert for technical assistance. J. Økland, D.Telnov, F. Naggs, H. Nesemann, A. Falniowski, A. Korniushin, P.Bunje, A. Bahl, F. Reinhardt, R. Araujo, C. Meier-Brook, P. Reischütz,R. Sesen, M. Ghamizi, K. Groh, F. Pulido and M. Ibanez kindly pro-vided samples.

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Markus Pfenninger is interested in phylogeography and mechan-isms of speciation in molluscs. This study is based on the diplomathesis of Sid Staubach. Christian Albrecht is concerned with phylo-geny and character evolution of freshwater limpets. Bruno Streit islong since interested in all aspects of the biology of A. fluviatilis.The main emphasis of Klaus Schwenk’s research is in ecologicalgenetics and interspecific hybridization of aquatic organisms, focus-ing on Daphnia.