Proc. NatL. Acad. Sci. USAVol. 80, pp. 4189-4193, July 1983Population Biology

Molecular area effects in Cepaea(land snails/enzyme polymorphism/genetic drift/natural selection/speciation)

HOWARD OCHMAN*, J. S. JONESt, AND ROBERT K. SELANDER**Department of Biology, University of Rochester, Rochester, New York 14627; and tDepartment of Genetics and Biometry, Galton Laboratory, University College,University of London, Wolfson House, 4 Stephenson Way, London NWI 2HE, United Kingdom

Contributed by Robert K. Selander, March 7, 1983

ABSTRACT Enzyme polymorphisms in the land snail Cepaeanemoralis in the central Pyrenees show concordant geographicpatterns of strong differentiation that are not correlated with thedistributions of characters of shell color and handing or with themajor pattern of variation in climate and vegetation type. Threeregions of relative genetic uniformity separated by steep dines inallele frequencies are designated as "molecular area effects." Amodel of allopatric differentiation of populations in temporarygeographic isolation during the last period of Pleistocene glacia-tion, followed by invasion of the Pyrenees and hybridization insecondary contact, is proposed to account for the present-day pat-tern of genetic differentiation. The genetic structure of the Pyr-enean populations of C. nemoralis is not interpretable in terms ofstasipatric or parapatric models of speciation.

In the apparent absence of environmental correlates of shellcolor and banding in the land snail Cepaea nemoralis, evolu-tionists (1-5) initially attributed geographic patterns of poly-morphic variation to random genetic drift in populations of smalleffective size. Subsequently, however, predation (6-9) and cli-mate (10-13) were shown to affect morph frequencies, at leastin some habitats and regions. Because the patterns of variationare complex and poorly predictable (10, 14-16), many evolu-tionary forces are believed to act on morphological characters,with the relative influence of each factor varying among pop-ulations (17).

In parts of Britain, frequencies of alleles at genes controllingshell polymorphisms in C. nemoralis are relatively homoge-neous over ecologically diverse areas much larger than thepanmictic unit; and these areas are separated by steep dinesfrom adjacent areas of very different allele frequencies (15, 18,19). Comparable "area effects" for morphological characters havealso been observed in other species of Cepaea (15, 20, 21) andin land snails of some other genera as well (22-24).

White (25) finds significant similarities between area effectsin Cepaea and patterns of distribution of geographic subspeciesin many groups of higher organisms. Expanding on earlier the-oretical and empirical work (26-29), he contends that area ef-fects denote the accumulation of major genetic differences, re-sulting in local coadapted gene complexes. White furtherproposes that area effects can lead to a form of parapatric orstasipatric speciation in the absence of geographic isolation.

If area effects are early stages of evolution to the species level,one would expect coincident patterns of spatial variation at ge-netic loci other than those controlling shell characters. At-tempts to test this prediction have yielded inconclusive results:local correlations between patterns of variation in allele fre-quencies at morphological loci and enzyme loci, assayed elec-trophoretically, have been observed in some areas but not inothers (30, 31).

Several recent studies have suggested that populations of C.nemoralis are differentiated on a geographic scale much largerthan that represented by morphological area effects (31-34).Attention has focused particularly on populations in the Pyr-enees (11, 12, 31, 34-36), where differences in genetic back-ground have been postulated to account for patterns of geo-graphic variation in shell banding (37, 38).We here compare molecular and morphological polymor-

phisms in C. nemoralis in an extensive region of the Pyrenees.Our analysis reveals concordant geographic patterns of strongdifferentiation at polymorphic enzyme loci that are not cor-related with the distributions of shell character morphs. Pat-terns of genetic variation in the Pyrenean populations are in-terpreted in relationship to environmental heterogeneity,probable demographic history, and potential for speciation.

MATERIALS AND METHODSArea Studied. We sampled 197 populations in a 180 x 60 km

region of the central Pyrenees in which elevation varies be-tween 250 and 3,400 m above sea level (Fig. 1A). Because C.nemoralis does not occur above 2,000 m and has rarely beenfound above 1,500 m (35), the total Cepaea population is di-vided by physical barriers into a series of subpopulations oc-cupying river valleys draining the north and south slopes. Theonly avenue of north-south contact across the crest of the Pyr-ene.es in this region is a pass between the Garonne and NogueraPalleresa valleys.

In the central Pyrenees, C. nemoralis occurs in a large va-riety of habitats, ranging from sub-Mediterranean to Alpine (37).There are major differences in climate and vegetation betweenthe north and south slopes, caused in large part by a rainshadoweffect (39-42). The ecological contrast is greatest in the east,where, in the Segre and Valira valleys, on the south slope, lowrainfall and high temperatures produce a sub-Mediterraneanhabitat that is quite unlike the cultivated pasture land in theAriege valley to the north (40).

Collection Localities and Allele Frequencies. Each samplewas collected in an area less than 500 m2, which approximatesthe size of a panmictic unit in this region (35). Allele frequen-cies at loci determining yellow or pink shell color (C) and thepresence or absence of shell bands (B) were estimated from thephenotypes of all individuals (N 30) collected at each locality,whereas those for six polymorphic enzyme loci were derivedfrom 10 snails chosen randomly from each -sample. The en-zymes studied were malate dehydrogenase (Mdh-1), leucineaminopeptidase (Lap-1), indophenol oxidase (Ipo-1), two phos-phoglucomutases (Pgm-2 and Pgm-3), and phosphoglucoseisomerase (Pgi-1). Mendelian inheritance of these enzymepolymorphisms has been demonstrated (33, 43). Techniques ofstarch gel electrophoresis and enzyme staining were similar tothose described by Selander et aL (44).

Wright's (32) hierarchical F statistics were used to estimatestandardized variances of allele frequencies at three levels of

4189

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4190 Population Biology: Ochman et aL Proc. Natl. Acad. Sci. USA 80 (1983)

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i.._.FIG. 1. Map of the Pyrenees. (A) Collecting localities. Elevations above 1,500 m are shaded. Hatched area denotes the central region of geneticdifferentiation at enzyme loci. (Localities in this region are indicated by open circles in Fig. 2.) (B) Allele frequencies at the shell color locus. (C)Allele frequencies at the Ipo-1 locus.

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Proc. Natl. Acad. Sci. USA 80 (1983) 4191

3-population structure: FDR, demes (sample localities) within rivervalleys; FRS, valleys on the north and south slopes of the Pyr-enean chain; and FST, both slopes within the total region sam-pled.

RESULTSGeographic variation in allele frequencies at three levels ofpopulation structure is summarized in Table 1. The degree ofdifferentiation among demes within river valleys (FDR) is fairlyuniform over all six enzyme loci, which suggests that randomgenetic drift, rather than adaptive response to environmentalfactors, is the primary if not exclusive cause of variation at thisgeographic level. There is, however, a large difference be-tween the estimates of FDR for the two morphological charac-ters. The relatively large variance for shell banding (B) reflectsthe circumstance that in seven valleys the frequency of un-

banded shells increases (often to fixation) at higher elevations(11, 12, 31, 36-38). The correlation between frequency of shellbanding and elevation is most apparent in the eastern half ofthe region sampled.

Estimates of variance in allele frequencies among river val-leys on the north and south slopes (FRS) are highly variable over

loci. There is, for example, much differentiation among riversat the Ipo-l locus (Fig. 1C) but very little at the shell color locus(C) (Fig. 1B). A surprising result of our analysis is the virtualabsence of differentiation at any locus between the north andsouth slopes, notwithstanding the fact that the main crest of thePyrenees is a formidable barrier to gene flow between regionsof different climate and vegetation type. Note that for each lo-cus, FST is zero or nearly so (Table 1).

As exemplified by Ipo-l (Fig. 1C), the six enzyme loci showan east-west pattern of geographic differentiation that is notapparent in the distribution of allele frequencies for the mor-

phological characters (Fig. 1B). To study the genetic structureof populations at a multilocus level, we used a principal com-

ponents analysis (45) of the arcsine-transformed frequencies of24 alleles occurring at the six enzyme loci. As shown in Fig. 2,the 197 samples cluster into three groups in a plot of scores onthe first two principal axes, which together account for 38% ofthe total variance in allele frequencies. These groups corre-spond to three major geographic subdivisions of the total Pyr-enean population: western, central, and eastern, each of whichextends across the crest of the Pyrenees (Fig. 1A). (Samplesfrom each region are represented in Fig. 2 by a distinctive sym-

bol to show the extent of overlap in factor scores.) These resultsreflect a high degree of concordance of geographic patterns ofvariation in allele frequencies at the six enzyme loci. As a con-

sequence, there are in the Pyrenees three large regions of rel-ative genetic uniformity separated from one another by steepclines that are not coincident with physical or obvious ecologicalbarriers to migration.

Table 1. Hierarchical analysis of allele frequencies in C.nemoralis in the Pyrenees

Variance estimatesLocus FDR FRS FST

Color (Cr) 0.144 0.008 0.011Banding (BO) 0.345 0.113 0Mdh-1 0.170 0.341 0Lap-1 0.189 0.160 0Ipo-1 0.187 0.175 0Pgm-2 0.146 0.089 0Pgm-3 0.177 0.059 0.001Pgi-1 0.196 0.139 0

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FIG. 2. Factor scores of samples from the Pyrenees, based on allelefrequencies at six polymorphic enzyme loci. Western (x), central (o),and eastern (x) populations are shown (see Fig. 1A).

Allele frequencies and genetic distances for the three geo-graphic regions are presented in Table 2. Populations in thewestern region are more similar to those in the eastern regionthan to those in the adjacent central region. Although the cen-

Table 2. Allele frequencies at six enzyme loci of C. nemoralis inthree geographic regions in the Pyrenees

RegionLocus Allele West Center EastMdh-l 100 0.545 0.047 0.026

90 0.097 0.876 0.64260 0.358 0.077 0.332

Lap-1 110 0.008 0.007 0.144100 0.899 0.489 0.78690 0.041 0.364 0.04880 0.052 0.140 0.022

Ipo-1 140 0.115 0.001 0130 0.588 0.145 0.200100 0.071 0.626 0.26480 0.226 0.228 0.536

Pgm-2 200 0.001 0.017 0.028175 0.026 0.005 0.004150 0.142 0.091 0.010125 0.026 0 0100 0.805 0.887 0.958

Pgm-3 150 0.007 0.088 0.045100 0.018 0.086 0.03075 0.019 0.082 0.17750 0.940 0.740 0.69725 0.016 0.004 0.051

Pgi-1 100 0.812 0.849 0.97750 0.175 0.150 0.02310 0.013 0.001 0

Nei's D* (within) 0.032 0.046 0.037

Nei's D (between) 0

0.077

* Standard genetic distance (46).

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4192 Population Biology: Ochman etaLP

tral region spans the fewest river valleys, it shows the largestaverage genetic distance among its populations.

DISCUSSIONPopulations of C. nemoralis in the Pyrenees show marked geo-graphic variation in the frequencies of alleles at six poly-morphic enzyme loci but relatively little variation at loci con-trolling shell color and shell banding. Because each of the threeregions of genetic differentiation at the enzyme loci spans sev-eral river valleys and both slopes of the Pyrenees, and thus avariety of habitats, we designate these discontinuities as "mo-lecular area effects" in analogy to the morphological area effectsdescribed on the English downlands by Cain and Currey (15).

Three models have been proposed to account for the evo-lution of morphological area effects: (i) Environmental selec-tion, presumably by microclimatic or other as-yet-unmeasuredfactors (15, 47-50). This model does not require geographic iso-lation. (ii) Accumulation of genetic differences and the devel-opment of coadapted gene complexes, in situ and in the ab-sence of geographic isolation, as a result of the diffusion ofmodifier genes (27-29, 32, 51, 52). (iii) Accumulation of geneticdifferences in small, geographically isolated populations throughadaptive response to environmental factors, genetic drift, orboth, followed by expansion of populations and secondary con-tact (26, 31, 53).

In accounting for the molecular area effects in the Pyrenees,the first two models seem to be ruled out. The primary patternof variation-involving differentiation of western, central, andeastern groups of populations-is not concordant with the ma-jor pattern of variation in climate and vegetation type. Eachgroup occupies several river valleys on both slopes of the Pyr-enees, and the boundaries between adjacent groups are or-thogonal to the main crest of the mountain range. This situationcontrasts with the patterns of distribution of morphologicalpolymorphisms: the frequency of color morphs is relativelyuniform over the entire region, and banding varies for the mostpart with elevation in certain river valleys in a manner that isnot concordant with the pattern shown by the enzyme loci (31,34). Moreover, differentiation by diffusion of modifiers fromfocal points in a continuously distributed population cannot haveoccurred because of the barrier to migration imposed by thecrest of the Pyrenees. Our analysis suggests that the thirdmodel-differentiation in temporary geographic isolation, fol-lowed by immigration of genetically dissimilar populations intothe Pyrenees-most plausibly accounts for the observed pat-terns of variation.

Because populations of Cepaea periodically experience dra-matic contractions and expansions in size and geographic range(54, 55), historical demographic events may profoundly affectpatterns of genetic differentiation. For morphological charac-ters, Cameron et al. (53) have shown that area effects in En-gland are most evident in regions of habitat instability, whereuntil fairly recently C. nemoralis is likely to have occurred insmall isolated populations. For example, agricultural recordssuggest that populations on the English downlands were se-verely reduced in size and distribution about 200 years ago asa result of vegetational changes induced by heavy grazing ofsheep. Expansion from refugia apparently has produced manyof the present-day area effects in shell characters.

During periods of Pleistocene glaciation, the most recentending about 10,000 years ago, Cepaea could not have occupiedits present range in the Pyrenees, because glaciers extendeddown to low elevations, reaching 340 m on the north slope (42).To account for the molecular area effects in the Pyrenees, wepropose that, in the last period of glaciation, Cepaea was iso-

lated in lowland areas to the south, east, and west of the Pyr-enees, where genetic differentiation occurred, presumably inpart as the result of random drift of neutral or nearly neutralalleles at the enzyme loci. With amelioration of the climate, thePyrenees were invaded by three already differentiated popu-lations. Because the central group of populations shows thelargest degree of differentiation north to south, we presumethat it first reached the Pyrenees, probably from the south, oc-cupied the south slope and subsequently invaded the north slopethrough the Noguera Palleresa-Garonne pass. It may once haveranged more extensively in the Pyrenees than it does at pres-ent. To account for the low level of north-south differentiationin both the western and eastern groups of populations, we sug-gest that they invaded along both slopes from the west and east,respectively, eventually contacting and replacing or hybridizingwith populations of the central region. Particularly extensivehybridization apparently occurred in the Flamisell and No-guera Palleresa valleys, where allele frequencies at enzyme lociare intermediate between those of the eastern and central re-gions (Fig. 1C). Genetic distance between the central and east-ern groups of populations is less than between the western andcentral groups, in part because of this extensive region of in-termediate allele frequencies.

It is noteworthy that differentiation of populations in thePyrenees does not involve the occurrence of unique major al-leles. With the exception of some rare variants (frequencies lessthan 1%), all alleles detected in the Pyrenean populations arealso present in other parts of Europe or in the British Isles (un-published data). That the western Pyrenean populations andthose from Cantabria, 300 km to the west, have had a commonevolutionary or demographic history is suggested by the pres-ence in both regions of three alleles (Mdh-11°, Ipo-1140, andPgm-2125) that are rare or absent elsewhere in the Pyrenees.

Because of evidence that shell banding in C. nemoralis issubject to selection by climatic factors in the Pyrenees (36-38),it is likely that any differentiation in this character that had oc-curred in the ancestral populations invading the Pyrenees hasbeen obscured through adaptive response to contemporary en-vironments. In the present-day distribution of shell charactersin the Pyrenean populations, there is no suggestion of the un-derlying pattern of differentiation at the enzyme loci, the con-cordance of which permits recognition of three groups of pop-ulations that are, in a sense, expanded relicts from an earlierperiod of evolution.

Are C. nemoralis populations sufficiently differentiated bythe molecular area effects to qualify as semispecies or species?Species status seems to be ruled out by our failure to find anyregion in which populations belonging to different groups occureither sympatrically or parapatrically without intergrading, andby laboratory evidence of an absence of behavioral or other bar-riers to interbreeding between Pyrenean snails and those fromEngland (56, 57). Whether or not partial genetic incompati-bility has developed between individuals of the three groupsof populations in the Pyrenees remains to be determined bydetailed studies in areas of rapid transition in allele frequen-cies. But whatever the results, molecular area effects in C. ne-moralis in the Pyrenees do not seem to be interpretable in termsof White's area effects model or other models of stasipatric orparapatric speciation, whereas they can readily be explained interms of allopatric differentiation and secondary contact.

We acknowledge the contributions of Chris Jackson, who helpedthroughout but did not live to see the work completed. We thank P.Slepokura, R. White, and E. Wright for assistance in the field; T. S.Whittam for comments on the manuscript; and P. E. Pattison for aid inpreparing the manuscript. This research was supported by grants from

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the National Science Foundation (DEB 78-23263), the National Insti-tutes of Health (GM 22126), and the Royal Society.

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