Biochemical Systematics and Ecology 28 (2000) 633}649

Cyanotypic frequencies in adjacent and mixed populations of Trifolium occidentale Coombe and L. are regulated by di!erent mechanisms Piet Kakes*, Agnes N. Chardonnens

Ecology and Ecotoxicology of , Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, Netherlands Received 27 May 1999; accepted 27 August 1999

Abstract

The cyanogenic polymorphism in Trifolium repens is caused by the variation in two genes, the interaction of which produces four distinct cyanotypes. Along the Atlantic coasts of Bretagne, T. repens is sometimes found in populations mixed with the related species Trifolium occidentale, although the latter species usually occurs only in a narrow fringe along the coast, whereas T. repens is a more inland species. No plants of T. occidentale have ever been reported to have linamarase activity. Indeed, of 763 T. occidentale plants studied, none contained linamarase activity. However, the variation in the proportion of cyanotypes in T. repens was enormous, even between sites less than 2 km apart. Our results con"rm the presumption that T. repens and T. occidentale are indeed separate species. Both the fact that T. occidentale plants never contain linamarase activity, and the di!erence in proportion of plants with cyanoglucosides in mixed stands show that gene #ow between the species must be rare. These dissimilar distributions strongly indicate that cyanotypic frequencies in adjacent and mixed populations of the very closely related species T. occidentale and T. repens are regulated by di!erent mecha- nisms ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Trifolium occidentale; Trifolium repens; ; Cyanogenesis; Cyanogenic glucosides; Geographic distribution; Ecology; Linamarin; Lotaustralin; Linamarase

* Corresponding author. Tel.: #31-20-4447063; fax. #31-20-4447123.

0305-1978/00/$- see front matter ( 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 1 0 - 6 634 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649

1. Introduction

Trifolium repens (white ) is one of many species that are cyanogenic, i.e. produce HCN when the leaves are damaged. Almost all populations of T. repens in Europe are polymorphic for cyanogenesis. The polymorphism is caused by variation in two genes. Li is the structural gene for linamarase: lili plants do not contain measurable amounts of linamarase. Ac regulates the presence of the cyanogenic glucosides linamarin and lotaustralin: acac plants do not contain cyanogenic gluco- sides. Both Li and Ac are incompletely dominant genes. The interaction of Ac and Li produces four of the so-called cyanotypes, which can be distinguished with a simple semiquantitative test. T. repens is an amphidiploid species, presumably originating from a hybrid between the diploid species Trifolium. nigrescens Viv. and a diploid form of T. repens (Gibson and Beinhart, 1969; Kakes and Hakvoort, 1994). A diploid species resembling T. repens has been described as T. occidentale by Coombe (1961). T. occidentale occurs on the Atlantic coasts of Cornwall, Bretagne and the Cotentin peninsula of Normandie. Populations of T. repens are generally found in less exposed situations, more inland than T. occidentale, which only occurs in a narrow fringe along the coast. However, mixed populations of T. repens and T. occidentale can be found in the cli! vegetation of L'Ile d'Ouessant and L'Ile Mole`ne, west of the coast of Bretagne (GeH hu, 1962). The distribution of the cyanotypes of T. repens has been the object of many studies (see Kakes, 1990 for a review). Daday (1954a) has found that the distribution of the genes Li and Ac in Europe is clinal, with high frequencies of the dominant alleles in the south, gradually decreasing with increasing latitude. As a corresponding altitudinal cline has been found by Daday (1954b) and by Till et al. (1988), one would suppose that the cline is related to temperature. The relation may be caused by pleiotropic e!ects of Ac and Li on frost resistance, or by genes closely linked to Ac and Li. Daday (1965) preferred the latter explanation. The Atlantic coast and the Channel islands are characterised by a very moderate climate with high winter temperatures. Therefore, if Daday's theory holds true, it is to be expected that the populations of T. repens in these localities would show a high proportion of cyanogenic plants. However, a preliminary study showed that this proportion is not particularly high (Kakes, unpublished). A later study on the origin of cyanogenesis (Kakes and Hakvoort, 1994) provided strong evidence that T. nigrescens is the donor of the Li gene in T. repens. Since there are maximally two active Li alleles in the amphidiploid genome of T. repens,itis unlikely that the other parent species contributed an active Li allele. Gibson et al. (1972) studied cyanogenesis in T. occidentale. They did not "nd plants with linamarase activity. Since Gibson et al. studied only 10 plants, the precise origin of which was not given, it appeared worthwhile to make a more thorough study of cyanogenesis in T. occidentale. In view of the presence of mixed populations, T. repens plants mixed with or adjacent to T. occidentale were also studied. In this paper, the following questions will be addressed:

1. What is the distribution of cyanotypes of the two species? 2. Is there gene #ow between the species? P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 635

A similarity between the distribution of the cyanotypes in the two species could be the result of a common (selective) mechanism causing the proportions at each site. If there is gene #ow between T. repens and T. occidentale, the low frequency of cyanogenic plants in T. repens could be explained by introgression of ac and li- alleles from T. occidentale. If there is no gene #ow, other factors must be responsible for the low frequency. Gene #ow could also be responsible for the occurrence of linamarase containing plants in T. occidentale, if such plants were found. On the other hand, the lack of linamarase containing plants in T. occidentale as a species would not only show that gene #ow between T. repens and T. occidentale is either absent or very rare, but would also be interesting in itself, as at the level of the species, cyanoglucosides and the corresponding b-glucosidase generally occur to- gether (HoK sel and Conn, 1982).

2. Materials and methods

Twenty to "fty stolons of one or of both species were collected by walking over the site (see the appendix for a description of the sites and Fig. 1 for the approximate positions) and cutting a stolon with at least a 2 m interval in the summer of 1992, 1994 and 1995. These samples will be indicated as `Adulta. Seed heads were collected in 1992 and 1995. At least 20 seed heads spread out over the site were taken and mixed. All sampling sites were, at the time of collection designated as `dunea (d), `falaisea (" rocky coast or cli!) (f), or `inlanda (b), according to the descriptions given by GeH hu (1963). A seed sample from Sicily that only contained AcAcLiLi plants was added for comparison. The stolons were rooted in the greenhouse and the seeds germinated as described earlier (Till et al., 1988). The cyanotype of each plant was determined with the Feigl}Anger test as described by Kakes (1991). Linamarase and total b-glucosidase activity were determined according to Boersma et al. (1983). The type and proportion of the cyanoglucosides in T. occidentale was determined using HPLC as described by Kakes and Hakvoort, 1999.

3. Results

3.1. Distribution of the cyanotypes

The cyanotypes of all the plants studied: 685 of T. repens and 763 of T. occidentale. are shown in Table 1. On the dune and falaise sites T. occidentale is the predominant species, but mixed populations (designated as TRO) do occur. On inland sites (never more than 500 m from the high watermark) T. occidentale is absent. Two properties of the distribution of the cyanotypes stand out: 1. The variation in the proportion of the cyanotypes in T. repens was enormous, even between sites less than 2 km apart. 2. No plants with linamarase activity were found in T. occidentale. 636 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649

Fig. 1. (a) Outline of the coast of S. England and NW France showing the approximate positions of the sites studied; (b) Outline of the coast of Bretagne showing the approximate positions of the sites studied in that area; (c) Schematic map of Ile d'Ouessant showing the approximate positions of the sites studied on that island. P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 637

Fig. 1. continued

We consider the variation in T. repens "rst: The chi square and p-values for presence/absence of cyanoglucosides (gene Ac) and/or linamarase (gene Li) for all comparisons in which the sample size permitted a chi square test for homogeneity are given in Table 2. The following comparisons were made: 1. Between regions: Bretagne, Normandie and Cornwall. 2. Between sites: The areas of sampling, presumably representing populations. 3. Between vegetation types: inland, dune, falaise. 4. Between collections: seeds, adults. There is a highly signi"cant site e!ect for both Ac and Li in adult and in seed samples. If we group the sites according to vegetation type in d, f and b we see that this factor is responsible for a signi"cant part of the variation in Ac but not in Li. The plants grown from seed collected on Ile d'Ouessant show a remarkable di!erence (Table 3): Those collected on inland sites show the high frequency of the dominant alleles that we expected from the high winter temperature. However, in the falaise collections it is the other way around: here we "nd low frequencies of the dominant alleles. In adult populations, the di!erence between allelic frequencies is not signi"cant. In T. occidentale, no plants that contain linamarase were found: there is only variation in the proportion of plants containing cyanoglucosides. As Tables 2 and 3 show, this variation is far less than that in T. repens. The French sites do not show any signi"cant variation between sites, the Cornwall sites only for seed collections. However, there is a signi"cant di!erence between regions. This is caused by a lower frequency of plants with cyanoglucosides in the French regions, compared to the ones in Cornwall. Table 4 shows the linamarase and the b-glucosidase activity of T. repens and T. occidentale. In plants of T. repens containing at least one active Li-allele, there is clear, but very variable, linamarase activity. The b-glucosidase activity (of which the Table 1 638 Number of plants of the cyanotypes of Trifolium in Brittany, Normandy and Cornwall!

Species Adult(P)/ Exposition: Sample nr. Cyanotype Ac-lili acacLi- acaclili Total Chardonnens A.N. Kakes, P. Seed (Z) Dune (d) Ac-Li Falaise (f) Inland (b) Ile d'Ouessant: T. repens Pb TO1931333423 T. repens P b TR 314 14 0 1 2 17 T. repens P b TR 324 11 5 0 3 19 T. repens Pb TR325960520 T. repens P d TR 321 7 10 0 0 17

T. repens P d TR 323 22 1 0 0 23 / T. repens P d TR 327 10 2 4 0 16 633 (2000) 28 Ecology and Systematics Biochemical T. repens Pd TRO19102035 T. repens P f TR 320 33 2 0 0 35 T. repens Pf TR3261231521 T. repens Pf TR328750820 Total 127 38 11 40 216 T. occidedentale Pd TRO19107007 T. occidentale P f TO 192 0 14 0 0 14 T. occidentale Z f TO 199 0 34 0 4 38 Total0550459 T. repens Z b TR 177 21 2 1 0 24 T. repens Z f TR 178#1901736071 Total22946095 Ile Mole` neT. repens P b TR 330 10 3 0 1 14 T. repens P d TR 333 18 1 0 0 19 T. repens P d TR 334 20 2 0 0 22 T. repens P f TR 329 18 3 1 1 23 T. repens P f TR 331 11 0 0 0 11

T. repens P f TR 332 13 3 0 0 16 } Total901212105649 T. occidentale Pf TO3320900 9 Bretagne T. repens P b TR 196 24 4 1 0 29 T. repens Pf TR198730111 Total3171140 T. occidentale P d TO 194 0 11 0 4 15 T. occidentale Pd/fTO1950700 7 T. occidentale Z d TO 200 0 25 0 1 26

T. occidentale Z d/f TO 201 0 35 0 2 37 Chardonnens A.N. Kakes, P. T. occidentale Z f TO 202 0 38 0 5 43 T. occidentale Z f TO 203 0 38 0 3 41 Total 0 154 0 15 169 Normandie T. repens P f TR 405 10 4 0 0 14 T. repens Pf TRO4064100 5 T. repens Pf TRO40741005 Total1860024 T. repens Z f TR 415 67 21 2 2

T. occidentale P f TR 404 0 11 0 2 13 / T. occidentale Pf TR407090716 633 (2000) 28 Ecology and Systematics Biochemical T. occidentale Pf TR408080412 Total 0 28 0 13 41 T. occidentale Z f TO 414 0 42 0 14 56 T. occidentale Z f TRO416085041126 T. occidentale Z f TRO41703901150 Total 0 166 0 66 232 Cornwall T. repens Pf TRO4100632433 T. repens Pf TRO4112801323 T. repens Pf TRO4120004848 Total214385104 T. repens Zf TRO4200009 T. occidentale P f TRO41001301528 T. occidentale Pf TRO411050611 T. occidentale Pf TRO41301034 Total 0 19 0 24 43 }

T. occidentale Z f TRO41804402670649 T. occidentale Z f TRO41905801270 T. occidentale Z f TRO42105002070 Total 0 152 0 58 210 639 !Note:P.KakesandA.N.Chardonnens:CyanotypicfrequenciesinT. repens and T. Occidentale. 640 .Kks ..Chardonnens A.N. Kakes, P.

Table 2 Distribution of the cyanotypes of T. repens and T. occidentale. Statistical treatment!

Species Origin Locality Grouping Character Chi'2df Signi"cance

T. repens Adults Ile d'Ouessant Site $Cyanoglucosides 76.35 10HHH $Linamarase 74.20 10HHH T. repens Seeds Ile d'Ouessant Site $Cyanoglucosides 58.35 1HHH /

$Linamarase 66.79 1HHH 633 (2000) 28 Ecology and Systematics Biochemical T. repens All Ile d'Ouessant, all b,d,f $Cyanoglucosides 9.74 2HH $Linamarase 2.35 2 ns T. repens All Ile d'Ouessant, inland Adult, seed $Cyanoglucosides 4.24 1H $Linamarase 6.55 1H T. repens All Ile d'Ouessant, falaise Adult seed $Cyanoglucosides 45.68 1HHH $Linamarase 45.59 1HHH T. repens Adults Cornwall Sites $Cyanoglucosides 22.87 2HHH T. repens All All Regions $Cyanoglucosides 172.5 2HHH $Linamarase 127.7 2HHH T. occidentale All Ile d'Ouessant site $Cyanoglucosides 2.37 2 ns T. occidentale Adults Normandie Site $Cyanoglucosides 2.69 2 ns T. occidentale Seeds Normandie Site $Cyanoglucosides 2.38 2 ns T. occidentale Adults Cornwall site $Cyanoglucosides 0.66 2 ns T. occidentale Seeds Cornwall site $Cyanoglucosides 7.05 2HH T. occidentale All All Regions $Cyanoglucosides 47.14 2HHH

!See Note to Table 1. } 649 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 641

Table 3 Comparison of the frequency of Ac and Li in adult and seed populations of T. repens on Ile d'Ouessant!

Ac- (%) acac (%) Li- (%) lili (%) Total

Adult Inland 61 77 18 23 51 65 28 35 79 Falaise 50 66 26 34 44 58 32 42 76 Seed Inland 23 96 1 4 22 92 2 8 24 Falaise 8 11 63 89 4 6 67 94 71

Total: 142 57 108 43 121 48 129 52 250

!See Note to Table 1. linamarase activity forms only a part) shows a similar pattern. For comparison, the data on LiLi plants of T. repens from Sicily have been added. In plants of T. repens recessive for li there is no linamarase activity, but there remains some b-glucosidase activity. In T. occidentale, no plants with linamarase activity were found. The b- glucosidase activity of T. occidentale also is low, but comparable, at least in the sample of adult plants, to that of T. repens without linamarase activity (lili). The b-glucosidase activity of T. occidentale plants raised from seeds is lower than that of adult plants. The proportion of linamarase to total cyanoglucosides is the same for T. occidentale and T. repens (Table 5). In T. nigrescens, the other presumed ancestor of T. repens, there is much more lotaustralin.

4. Conclusions

T. occidentale is treated as a subspecies of T. repens both in the Flora Europea and in Zohary and Heller's monograph on the genus Trifolium (Zohary and Heller, 1984). e present study shows that Coombe, the author of T. occidentale was right in his assumption that T. repens and T. occidentale are separate species: Although the di!erences between T. repens and T. occidentale are not very conspicuous, they are constant. No plants with intermediate morphology were found by us, either in the "eld, or in plants raised from seeds. Moreover, the di!erence in the proportion of plants with cyanoglucosides in mixed stands of the two species, together with the lack of linamarase containing plants in T. occidentale, show that gene #ow between the species, if existent at all, must be rare. The data for enzyme activity T. repens con"rm what was reported in earlier studies (Boersma et al., 1983; Kakes and Eeltink, 1985): linamarase and b-glucosidase activity (the latter including the former) are variable and dependent on genotype for Li and on genetic background. The presence of b-glucosidase activity in lili plants is caused by another gene, possibly related to Li (Hughes et al., 1988). The average linamarase activity for Li- plants from L'Ile d'Ouessant is less than half of the average for plants from Sicily. As the latter, coming from a monomorphic population, presumably are homozygous LiLi, there must be other di!erences (di!erent alleles, di!erent genetic background) between the populations. 642 .Kks ..Chardonnens A.N. Kakes, P.

Table 4 Linamarase and b-glucosidase activity of T. repens and T. occidentale!.

Sample: n linamarase activity b-glucosidase activity of Speci"c linamarase Speci"c b-glucosidase (mUnit) (mUnit) activity activity (mUnit/mg prot.) (mUnit/mg prot.)

Samples raised from seed:

T. repens,Iled'Ouessant,Li- 4 / iceia ytmtc n clg 8(00 633 (2000) 28 Ecology and Systematics Biochemical Average: 0.53 1.93 7.25. 29.48 standard dev. 0.28 0.61 1.95 5.63 T. repens,Iled'Ouessant,lili 4 Average: 0.00 1.13 0.00 14.03 standard dev. 0.00 0.49 0.00 5.98 T. repens, Sicily, LiLi 4 Average: 2.15 5.15 24.00 68.68 standard dev. 1.15 2.93 10.12 24.61 T. occidentale,Iled'Ouessant 4 Average: 0.00 0.50 0.00 6.75 standard dev. 0.00 0.18 0.00 2.49 Samples of adult plants: T. occidentale,Iled'Ouessant 6 Average: 0.00 1.08 0.00 13.57 standard dev. 0.00 0.24 0.00 2.05

!See Note to Table 1. } 649 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 643

Table 5 Proportion of linamarin to linamarin#lotaustralin in leaf extracts of the species studied!

Species Genotype/identity lin/lin#lot 95% conf. int.

T. occidentale Ile d'Ouessant 0.39 0.02 T. repens AcAc 0.46 0.01 T. repens AcAc 0.40 0.01 T. repens Acac 0.48 0.02

T. repens Acac 0.42 0.01 T. repens Acac 0.41 0.00 T. nigrescens Southern France 0.13 0.03

!See Note to Table 1.

The lack of linamarase activity in T. occidentale as a species forms an exception to the rule that species containing cyanoglucosides always contain enzyme(s) able to hydrolyse them (HoK sel and Conn, 1982). The b-glucosidase activity in adult plants of T. occidentale is comparable to that in plants of T. repens recessive for li, showing that, in the former species as well this activity is regulated by another gene or genes. We have no explanation for the low activity of T. occidentale plants raised from seeds. As the latter were grown in a di!erent time of the year, environmental di!erences may have played a role. In T. repens, plants containing cyanoglucosides, but not linamarase can be found in polymorphic populations, often forming an appreciable part of the population. In that species, plants containing only cyanoglucosides are partly protected against grazing by snails and slugs Kakes (1989). It cannot be concluded whether the same holds true for T. occidentale. It may be of importance, however, that snails are abundant in places where T. occidentale occurs. Both T. occidentale and T. repens are polymorphic for presence/absence of cyanog- lucosides, but as the proportions between the two cyanotypes are di!erent, even in adjacent or mixed populations of the two species, the mechanism regulating the frequencies is probably di!erent. In T. repens, the gene Ac that regulates the production of cyanoglucosides, has been implied to play a role in drought tolerance: acac plants survive better than Ac-plants under sub lethal moisture stress (Foulds and Grime, 1972). These authors further mention that under moisture stress #owering is completely inhibited in all cyanotypes except for acaclili. These observations may explain the di!erence between falaise and inland populations: The falaise populations grow on very shallow soil, where signs of moisture stress were observed during collection trips in dry spells (see also Coombe, 1961). The seed collections were made in summer, after a particular dry and hot period. If the seeds collected on the falaise were produced mainly by acaclili plants, because these were the only ones in #ower during that period, this would explain the high proportion of acaclili plants in the progeny. T. occcidentale is a species that is much better adapted to dry conditions than T. repens (Coombe, 1961). Its habitat is more uniform than that of T. repens. These circumstances may explain the lack of di!erentiation regarding Ac in the former 644 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 species. Our observations are in line with that of Coombe (1961), who noted that the variation in morphological characters is much less in T. occidentale than in T. repens. The two key questions addressed in the introduction can now be answered: 1. The distributions of the cyanotypes in T. repens and T. occidentale are dissimilar, showing that these distributions, in closely related plants, have di!erent causes. 2. There is no gene #ow of any importance between the species. The lack of linamarase containing plants in T. occidentale has several implications: It shows that the cyanogenic glycosides may have some function not related to their hydrolysis in the plant [as shown for T. repens by Kakes (1989)]. It lends support to the hypothesis of Kakes and Hakvoort (1994) that T. occidentale did not donate an active Li-allele in the amphidiploid T. repens. More generally, the evidence presented in this paper shows that it is dangerous, maybe impossible to extrapolate ecological "ndings from one species to another one, even if they are closely related.

Acknowledgements

The senior author wishes to thank Mr. R. de Vries for his help during the collecting trip to Normandie and Cornwall.

Appendix A

Description of the sampling sites of Trifolium repens and Trifolium occidentale. The bold numbers correspond with those on the map. Codes used: TR: Samples containing only T. repens. TO: Samples containing only T. occidentale. TRO: Samples which contain both T. repens and T. occidentale. On l'Ile d'Ouessant: 1. Along the path North of the Baie de Lampoul, over a distance of $200 m. Among high grasses. Type: Falaise. Sample: TR 320. 2. Approximately 200 m E of the `Ancien Forta. Among short grasses. Grazed by sheep. Type: Dune. Sample: TR 321. 3. 20 m E of the `Ancien Forta. Among short grasses. Grazed by sheep. Type: Dune. Sample: TR 322. Remark: In the tables site 2 and 3 are lumped under TR 321. P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 645

4. Along the road from Point de Pern to the old mill. Type: Dune. Sample: TR 323. 5. Around the old mill. On a hay "eld. Type: Inland. Sample: TR 324. 6. Along the road from the old mill to the EcomuseH e. A relatively wet site. Type: Inland. Sample: TR 325. 7. On the N coast, near the Baie de Kergadou. NE exposition, gentle slope with short vegetation. Type: Falaise. Sample: TR 326. 8. In a valley, along a small brook that runs into the Baie de Kergadou. Less than 100 m from the coast. Type: Dune. Sample: TR 327. 9. Along the coast of the Pointe de Cadoran, towards the Phare du Sti!. On very dry, shallow soil. T. repens occurs only in the wetter places. Type: Falaise. Sample: TR 328. 10. 1 km E of the Phare de Creac'h. Short turf at the edge of the cli!. Mixed populations of T. repens and T. occidentale. Type: Falaise. Samples: TR 178, TR 190, TRO 191. 11. Along the road between the hamlet of Niou and the Phare de Creac'h. 1 km from the shore line. Type: Inland. Sample: TR 177. 12. Along the road from Cadoran to the Phare de Sti!. Near 9. but more inland. Only T. repens. Type: Inland. Sample: TR 193. On Ile Mole%ne: 13. On the SE side of the isle, along the path to the coast. Type: Falaise. Sample: TR 329. 14. In the same area, but 200}300 m from the coast line. Type: inland. Sample: TR 330. 646 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649

15. On the N coast of Ile Mole`ne, near the waste dump. Only T. repens and grasses. Type: Falaise. Sample: TR 331.

16. On the Northermost point of Ile Mole`ne, a dry spot. T. repens occurs together with T. occidentale. Type: Falaise. Sample: TR 332.

17. On the NE part of Ile Mole`ne, 400}500 m from the coast. Type: Dune. Sample: TRO 333.

18. On the NE point of Ile Mole`ne, 1.5 m from the cli! edge. Type: Dune. Sample: TR 334. Bretagne:

19. Plage de Porsliogan. S of le Conquet, near the parking lot. On shallow loam on the falaise. T. occidentale, and nearby, on deeper soil, T. repens. Type: Falaise. Sample: TRO 305.

20. Presqu'm( le de Kermorvan. Along the path that runs on the N side of the Pointe de Kermorvan. Populations of T. repens and T. occidentale adjacent, but not mixed. Type: Falaise. Samples: TRO 197, TRO 198, TO 202, TRO 306.

21. Presqu'm( le de Kermorvan. 200 m NW of the Phare, very exposed on shallow soil. Only T. occidentale. Type: Falaise. Sample: TO 307.

22. Presqu'm( le de Kermorvan. On the edge of a cultivated "eld in the middle of the penunsula. Only T. repens. Type: Inland. Sample: TR 308.

23. TreH mazan. On the south side of the bay, on a grazed meadow, 100 m from the sea. Only T. repens. Type: Dune. Sample: TR 309.

24. TreH mazan. On the falaise 10 m below 23. Mixed populations of T. repens and T. occidentale. Type: Falaise. Sample: TRO 310. P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 647

25. Presqu'm(le de St. Laurent. On the peninsula, N of Porspoder. On the dry top, grazed by rabbits. Only T. repens. Type: Dune. Sample: TR 311. 26. Presqu'm( le de St. Laurent. The same area as 25, but amongferns. Type: Dune. Sample: TR 313. 27. Lochrist. On a meadow S of the graveyard. T. repens mixed with T. rubens. Type: Inland. Sample: TR 314. 28. Pointe du Grand Minou. Somewhat inland from the very exposed cli!. Along the track that runs beside the water reservoir. Type: Dune. Sample: Tr 315. 29. Lampoul Plouarzel. W of the Port de Porspaul. 2 m from the high watermark onwards. Type: Falaise. Sample: TRO 316. 30. Lampoul Plouarzel. The same location as 29, but more inland. Type: Dune. Sample: TRO 317. 31. Anse des Blanc Sablons. On the dunes that surround the bay. Type: Dune. Sample: TR 318. 32. Pointe du Corsen. On the falaise that lies over the beach. On dry soil, with many small snails. T. occidentale is abundant on the more exposed parts. T. repens is found appr. 10 m away. Type: Falaise. Sample: TRO 319. 33. Port de Lilia. NW of the harbour, on an abandoned "eld. Type: Falaise. Sample: TRO 335. 34. Plage de Pors Meur. On a meadow on rocks, between cultivated "elds. Type: Dune. Sample: TR 336. 35. St. Mathieu On the falaise S of the church. T. repens and T. occidentale occur near to each other, T. occidentale on the more exposed ridges. Type: Falaise. Sample: TO 203. 648 P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649

36. Presqu'm( le de St. Marguerite. In the dunes N of the road to the beach. Dry dunes, with Calystegia soldanella. Many snails. Only T. occidentale. Type: Dune. Sample: TO 194, TO 200. 37. Ile Tariec. Small island W of Presqu'm( le de St. Marguerite, that can be reached by foot during low water. T. occidentale grows near the high watermark, both on sand and in crevices on the rocks. Many snails, among others Helix aspersa. Type: Dune/Falaise. Sample: TO 195, TO 201. 38. St. Marguerite. Along the road to the beach, 1 km from the high water mark. Only T. repens. Type: Inland. Sample: TR 196. Normandie: 39. Gatteville. Along the road to the light house. Shallow soil on granite. T. repens and T. occidentale both present, but not mixed. Type: Falaise. Samples: TO 404, TR405, TO 414. 40. Point Levy. On the north coast of the Peninsula, E of the lighthouse. Like 39, but with deeper soil. Only T. repens. Type: Falaise. Samples: TR406, TR 415. 41. Cap de Flamandville. On the south side of the peninsula, along the footpath, on steep rocky exposed slopes. Type: Falaise. Samples: TRO 407, TRO 416. 42. Ecalgrain. On a rocky promontory, between the Baie Ecalgrain and the Anse du Cul Rono. Exposed cli! vegetation. Many snails. Type: Falaise. Samples: TO 408, TO 417. Cornwall: 43. Mullion Cli!. S of Mullion Cove, on the cli!s. Type: Falaise. Sample: TRO 419. 44. Carthillian Cli!. On the Crane ledges, W. of the village of Lizard. This is the type locality of T. occidentale, described by Coombe (1961). T. occidentale occurs on the more exposed points, T. repens in less exposed situations with deeper soil. The minimum distance between the species is 5}10 m. Type: Falaise. Samples: TRO 410, TRO 418. P. Kakes, A.N. Chardonnens / Biochemical Systematics and Ecology 28 (2000) 633}649 649

45. St Ives. On the W and NW facing slopes of St. Ives Head. The soil is deeper and the vegetation more luxurious than 44, but the species composiition is the same. T. repens is rare in this locality. Type: Falaise. Samples: TRO 411, TRO 421. 46. Sennen Cli!. Between Sennen Cove and Lands End. (Mayon Cli!). W facing slope. T. occidentale and T. repens occur together. Type: Falaise. Samples: TRO 412, TRO 420.

References

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