Population genetic structure and levels of inbreeding depression in the Mediterranean island endemicCyclamen creticum(Primulaceae)

Biological Journal of the Linnean Society (1997), 60: 527–549. With 10 figures

Population genetic structure and levels of inbreeding depression in the Mediterranean island endemic Cyclamen creticum (Primulaceae) LAURENCE AFFRE AND JOHN D. THOMPSON C.E.F.E.-C.N.R.S., Route de Mende, BP 5051, F-34033 Montpellier Ce´dex 1, France Received 22 March 1996, accepted for publication 17 October 1996

The variation and evolution of reproductive traits in island plants have received much attention from conservation and evolutionary biologists. However, plants on islands in the Mediterranean region have received very little attention. In the present study, we examine the floral biology and mating system of Cyclamen creticum, a diploid perennial herb endemic to Crete and Karpathos. Our purpose is to quantify (1) variation and covariation of floral traits related to the mating system, (2) the ability of the species to self in the absence of pollinators and its relative performance on selfing and outcrossing and (3) genetic diversity within and among populations. Pollen/ovule ratios were indicative of a xenogamous species. A controlled pollination experiment showed that the species is self-compatible but is unable to set seed in the absence of pollinators, probably due to stigma-anther separation. A multiplicative estimate of inbreeding depression based on fruit maturation, seed number and percentage seed germination gave d=0.38. Population genetic diversity was high, 54.76% polymorphic loci, a mean of 1.78 alleles per locus and a mean observed heterozygosity of 0.053. F-statistics nevertheless indicated high inbreeding rates (mean Fis=0.748) in natural populations, and low levels of population differentiation (mean Fst= 0.168). C. creticum thus appears to have a mixed-mating system with high levels of (pollinator) mediated inbreeding (either by facilitated selfing, geitonogamy or biparental inbreeding) in natural populations.  1997 The Linnean Society of London

ADDITIONAL KEY WORDS:—facilitated inbreeding – floral biology – genetic diversity – island biology – Mediterranean Basin – mixed mating-system – pollen to ovule ratio – reproductive assurance. CONTENTS Introduction . . . . . . . . . . . . Material and methods . . . . . . . . . Study species . . . . . . . . . . Populations studied . . . . . . . . Floral biology . . . . . . . . . . Temporal variation in stigma receptivity and Controlled pollinations . . . . . . . Germination trial . . . . . . . . . Enzyme electrophoresis . . . . . . . Results . . . . . . . . . . . . . . Floral trait variation and covariation . . Temporal variation in stigma receptivity and Self-fertility, self-compatibility and seed set . Seed germination and seedling vigour . . Inbreeding depression . . . . . . . Flower longevity . . . . . . . . . Amount and pattern of genetic variation . Discussion . . . . . . . . . . . . . 0024–4066/97/040527+23 $25.00/0/bj960119

. . . . . . . . . . pollen . . . . . . . . . . pollen . . . . . . . . . . . .

527

. . . . . . . . . . . . . . . . . . . . maturity . . . . . . . . . . . . . . . . . . . . maturity . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

528 529 529 529 529 532 532 533 534 535 535 536 538 538 539 540 540 543

 1997 The Linnean Society of London

528

L. AFFRE AND J.D. THOMPSON Genetic variation . . Floral biology . . . Inbreeding depression Acknowledgements . . . References . . . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

543 544 546 547 547

INTRODUCTION

Endemic species with restricted geographic distributions have become a central concern of biologists faced with the problem of preserving rare species endangered by habitat destruction and fragmentation and by the introduction of exotic species. Among the endemic species most prone to such effects are those endemic to islands where population numbers and sizes are often small. Island species have traditionally attracted the attention of biologists interested in documenting the effects of geographic isolation on patterns of species diversification (Carlquist, 1974; Warwick & Gottlieb, 1985; Witter & Carr, 1988; Westerbergh & Saura, 1994) and community structure (MacArthur & Wilson, 1967). There has however been much less work on the effects of habitat fragmentation on the population genetic structure of island species which are by definition isolated from source populations (Glover & Barrett, 1987; Husband & Barrett, 1991). The floras of several Mediterranean islands contain many endemic species that have evolved in isolation from ancestral taxa following changes in sea level and island formation. For example, endemic species make up 7% of the flora of the Balearic Islands (Cardona & Contandriopoulos, 1977), 8% on Corsica (Cardona & Contandriopoulos, 1977) and 8.6% on Crete (Greuter, 1979). Several of the species endemic to these islands have closely related congeners that are more widespread on the European continent (Greuter, 1979). Although there has been much interest in the cyto-geography and systematics of Mediterranean island endemics (Contandriopoulos & Favarger, 1974; Cardona & Contandriopoulos, 1977, 1979; Verlaque et al., 1991), there has been much less interest in the evolution of floral traits, reproductive systems and population genetic structure of such species. The only such study to our knowledge has shown the evolution towards selfing in Mediterranean island species of Nigella (Strid, 1969). The island of Crete provides the setting for such a study. Although the endemic flora of Crete has been relatively well described and its affinities have received much attention (Greuter, 1979; Turlan, Chilton & Press, 1993), there have been no studies of the reproductive biology and genetic diversity of Cretean endemic species. Cyclamen creticum is endemic to the Mediterranean islands of Crete and Karpathos. In this paper, we examine the reproductive biology and population genetic structure of C. creticum on Crete. Our six objectives are to quantify (1) floral traits that may be associated with the mating system in natural populations, (2) the temporal variation in stigma receptivity and pollen maturity, (3) the ability to set self-seed in the absence of pollinators (self-fertility), (4) the ability to set fruit on selfing relative to outcrossing (self-compatibility), (5) potential inbreeding depression affecting fruit maturation, seed set, seed germination and seedling performance, and (6) withinpopulation heterozygosity and genetic differentiation among populations using electrophoretic markers. Since C. creticum is closely related to C. balearicum, a species endemic to the Balearic Islands and southern France (Grey-Wilson, 1988; Anderberg, 1993), we discuss the

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

529

levels of genetic diversity and mating system attributes quantified here in relation to that observed previously in island populations of C. balearicum (see Affre, Thompson & Debussche, 1995, 1997).

MATERIAL AND METHODS

Study species Cyclamen creticum Hildebr. is a herbaceous, diploid long-lived perennial with a tuberous rootstock. The species has chromosome numbers of 2n=20 or 2n=22, the latter resulting from the multiplication of one chromosomal pair (Greilhuber, 1989) which does not influence its diploid meiotic behaviour. Leaves and flowers arise directly from the tip of a floral trunk which emerges from the upper side of the perennial tuber. The short-petiolate leaves which appear in late autumn show a greyish green lamina with grey or silver marbling on the upper surface and deeply dentate margins. The plant shows no evidence of vegetative reproduction. Flowering occurs in the spring (from the end of March to the end of May). The slightly scented flowers are solitary, pendant with reflexed plain white or pale-pink flushed petals. Flowers are hermaphroditic and do not produce nectar. The anthers are positioned within the corolla, attached to the base of the flower by very short filaments and held in a cone surrounding the simple style that protrudes from the tip of the anthers and the mouth of the corolla. The apical pore of the anthers (‘poricidal’ anthers) opens towards the centre of the flower. After fertilization, the erect pedicel coils from the apex downwards bringing the fruit to the soil surface where it gently releases the mature seeds.

Populations studied In this study, seven natural populations were sampled on Crete to encompass the full geographic and altitudinal distribution of the species on the island. The distribution of C. creticum comprises a wide variety of ecological conditions over an altitudinal gradient from 90 m to 1250 m. Specifically, the species ocurs in the forest understorey on hillsides, in gorges and on the banks of watercourses on northern to western exposures. The forest vegetation, which it inhabits, varies from oriental plane (Platanus orientalis L.) forests, through kermes oak (Quercus coccifera L.) and pine (Pinus brutia Ten.) forests, to cypress (Cupressus sempervirens L.) forests. The species shows no substrate specificity; it usually occurs on soil-free limestone crevices, but can also be found on varying soils from terra rossa, through clay-like, sandy, schisty or organic-rich soils, to pure leafmould (Aspland, 1994). The description of the populations sampled is shown in Figure 1 and Table 1.

Floral biology To quantify floral traits related to the mating system, we measured pollen and ovule production per flower, pollen volume, petal length and width, corolla diameter,

530

L. AFFRE AND J.D. THOMPSON

0

24°

50 km

25°

26° CRETE

PA TH

KE

LA

AS

35° TO

35° AN

24°

25°

26°

Figure 1. Distribution of the study populations of C. creticum on Crete. Abbreviations: TO: Topolia, PA: Papadiana, TH: Therisso, AS: Askifou, KE: Kera, LA: Lagou, and AN: Anatolia.

and style and stamen length on flowers collected, in spring 1993, from five or six natural populations on Crete (see Table 1). First, to quantify pollen and ovule numbers per flower, 25 floral buds (each flower sampled from a different individual) were randomly collected from each of five populations (see Table 1). The number of pollen grains and ovules per flower was counted following Affre et al. (1995). From the pollen and ovule production, pollenovule (P/O) ratios per flower were calculated. We also determined the mean pollen grain volume (v) by measuring the radius (r) of ten pollen grains per flower and then calculating v=(4/3)×p×(mean r)3 (lm3), this in order to quantify any potential relation between P/O ratio and pollen volume (e.g. Charnov, 1982). Second, to measure floral morphology, 25–40 mature flowers (at least four days old) were randomly collected from six populations (see Table 1) and conserved in F.A.A. (formalin–acetic acid–alcohol). Each mature flower was sampled from a different individual. Petal length and width, diameter of the corolla mouth, and style and stamen length were measured using digital calipers (to 0.01 mm). Stigmaanther separation was calculated by subtracting style length from stamen length. Differences among populations were examined for each floral trait in a one-way Analysis of Variance (ANOVA) (SAS, 1990). Due to the unbalanced nature of the data, the type III (missing values) partial sums of square computation was employed following Shaw & Mitchell-Olds (1993). Based on the pooled data for all populations, Pearson correlation coefficients between pollen and ovule production and among characters related to floral morphology (i.e. flower size, and style and stamen length) were calculated. Correlations between the two groups of floral traits were not possible since flowers on different individuals were used. It is impossible in the field to be sure that even closely adjacent flowers come from the same genotype without tracing flowers to tubers—which is often extremely difficult. Significance levels for the ANOVAs and Pearson correlations were adjusted using the sequential Bonferroni test for multiple comparisons (Rice, 1989).

4 km south of Papadiana

1.5 km west of Anatoli, edge of a dry stream bed 1 km north of Therisso

Papadiana (PA)

Anatolia (AN)

Lagou (LA)

Kera (KE)

Askifou (AS)

1 km south of Petres in forest above the road 1–2 km north of Kera in woodland on roadside Top of the pass between Kera and Lagou

3 km south of Topolia

Topolia (TO)

Therisso (TH)

Location

Populations

N

NW

NE

NW

W

NE

E

Exposure

1000 m

800 m

800 m

700 m

700 m

400 m

300 m

Altitude Woodland on schist (phylites) dominated by Quercus coccifera, Platanus orientalis, Arbutus unedo, with Sarcopoterium spinosum and Cistus sp. pl. Woodland on limestone dominated by Cupressus sempervirens, Olea europaea, Castanea sativa, with Erica sp. pl. Woodland and shrubland dominated by Pinus brutia and Cistus sp. pl. Woodland on schist (phylites) dominated by Platanus orientalis Woodland on limestone dominated by Cupressus sempervirens and Quercus coccifera Woodland on limestone dominated by Quercus coccifera and Olea europea Shrubland on limestone dominated by Quercus cocifera and Sarcopoterium spinosum

Vegetation

FB-FM-PO-GV

FB-FM-GV

FB-FM-GV

PO

FB-FM-PO-GV

FM-PO-GV

FM-FB-PO-GV

Experiments

T 1. Description of the seven populations of C. creticum on Crete. Experiments performed: FB: pollen and ovule traits in floral buds, FM: mature flower traits, PO: experimental pollination trials, and GV: genetic variation

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM 531

532

L. AFFRE AND J.D. THOMPSON

Temporal variation in stigma receptivity and pollen maturity During the spring 1993, tubers were collected from seven natural populations on Crete (see Table 1) and each tuber was transplanted to 13-cm individual plastic pot and kept in the CEFE–CNRS garden in Montpellier (France). The compost consisted of 1:3:2:1 (v/v) peat, coarse sand, thin sand and garden soil. After transplanting, liquid N-P-K fertilizer was applied to each individual pot. Individuals were then left to grow in the garden. In April 1995, temporal variation in stigma receptivity and pollen maturity were examined to determine whether C. creticum is dichogamous. Individual pots were placed in an insect-proof greenhouse. Insecticide ‘Cyfluthrine’ was then sprayed (0.5 cm3.l−1) over each pot to prevent any uncontrolled activity of small insects present in the compost. First, the onset and duration of stigma receptivity were examined for 108 flowers on 36 plants. As flowers opened (‘recipient’ flowers), they were bagged and emasculated by gentle removal all the anthers using tweezers to prevent self-fertilization. Using fresh pollen from a ‘donor’ flower that had been open for at least 3–4 days, flowers were cross-pollinated either 1, 2, 3, 4, 5, 6, 7, 11, or 15 days after ‘recipient’ flower opening and then left unmanipulated. Second, the onset and duration of pollen maturity were quantified in the greenhouse. Forty eight ‘recipient’ flowers on 18 plants were bagged and emasculated as they opened. When ‘recipient’ flowers were at least 4 days old, they were cross-pollinated with pollen from either 1, 3, 5, 7, 9, 13, 17, or 21 day old ‘donor’ flowers and then left unmanipulated. In both experiments, fruit initiation was recorded. Differences in fruit initiation on ‘recipient’ flowers of different age or on ‘recipient’ flowers pollinated with pollen from ‘donor’ flowers of different age were analysed using a chi-square test, with a Yates correction for small sample sizes (Sokal & Rohlf, 1981). Controlled pollinations In April and May 1994, controlled pollinations were carried out in the insectfree greenhouse on 58 flowering plants randomly placed on a bench and originating from five populations (see Table 1). Twenty floral buds, each on a different individual, were emasculated by gently removing anthers with tweezers and left unpollinated to check for extraneous pollination by unobserved insects. Hand pollinations were then conducted to quantify self-fertility, self-compatibility, and fruit and seed set following self- and cross-pollination. As floral buds opened, they were identified by placing a small coloured plastic ring around the base of the peduncle and then allocated to one of four pollination treatments (identified according to the colour of the ring): (1) 38 flowers were unmanipulated to quantify fruit set in the absence of pollinators, (2) 38 flowers were hand-pollinated using self-pollen from the same flower, (3) 35 floral buds were emasculated as they opened by gentle removal of the anthers using tweezers and flowers were hand-pollinated with self-pollen, and (4) 45 floral buds were emasculated as above and flowers were hand-pollinated using fresh pollen from another plant. In each treatment, all flowers were on different individuals (i.e. 38 individual plants for treatment one, etc.). All four treatments were carried out on the same genotype when possible, although sometimes only two or three ‘recipient’ flowers were produced by a given individual, hence, not all treatments

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

533

were performed on each plant used in the experiment. Treatments were randomly applied hence any phenological differences among flowers of the same plant were not confounded with treatment effects. Hand-pollinations were performed every two days from 08:00 to 15:00 hr using tooth picks (a different tooth pick was used for each pollination). The number of days between flower opening and corolla abscission (i.e. floral longevity) was recorded for all flowers. Fruit initiation, mean number of seeds per fruit and mean seed weight (i.e. the mean weight of individual seeds) were quantified on all flowers setting fruit. Two indices related to selfing were calculated for fruit initiation following Lloyd and Schoen (1992): (1) the self-fertility index as measured by the performance on spontaneous selfing (treatment 1)/performance on hand outcrossing (treatment 4), this quantifying the ability to set fruit/seed in the absence of pollinators, and (2) the self-compatibility index, i.e. the performance on hand selfing (treatment 3)/ performance on hand outcrossing (treatment 4). The number of flowers initiating and maturing a fruit, and the number of initiated fruits that aborted were compared among treatments using a two-way log-linear model. For all flowers maturing a fruit, differences in the mean number of seeds per fruit and the mean seed weight among treatments were analysed using a complete block Analysis of Variance design (Sokal & Rohlf, 1981) with individual plants as ‘blocks’ and pollination treatment as a fixed effect. Two such ANOVAs were carried out. First, to verify whether emasculation significantly affected seed production and seed biomass, the ANOVA was carried out for plants which were subject to both unemasculated and emasculated selfing. Second, we repeated the ANOVA using plants on which emasculated selfand cross-pollinations were performed. By only including in these analyses individuals on which both pollination treatments were carried out, we avoided bias due to unequal treatment sample sizes. Data were square root (mean seed number) and logarithm (mean seed weight) transformed to eliminate heteroscedasticity (Sokal & Rohlf, 1981). Pearson correlation coefficients, based on the pooled data for all individuals, were also calculated in each treatment for the mean seed number and seed weight. The relative performance (RP) of the emasculated self- and cross-pollination treatments was calculated (for each maternal plant) as RP=(wo–ws)/wmaximum where ws is the mean performance of selfed progeny and wo is the mean performance of outcrossed progeny. If the performance on outcrossing [ that on selfing then wmaximum=wo whereas if the performance on selfing > that on outcrossing then wmaximum=ws (following Agren & Schemske, 1993). For seed production and mass, inbreeding depression was calculated for the 23 plants which received both emasculated self- and cross-pollinations. Inbreeding effects being cumulative across the life cycle, we calculated a multiplicative estimate of inbreeding depression based on fruit maturation (1) and number of seeds per fruit (2). For each trait, we calculated the ratio self/outcross mean performance over all plants regardless of whether both treatments were carried out on the same plants. A multiplicative estimate of inbreeding depression was then calculated as 1 minus the product of the two above ratios i.e. 1-[(ws1/wo1)×(ws2/wo2)]. Germination trial Seeds were germinated in the greenhouse from September 1994 to June 1995. Twelve seeds from each of 19 emasculated hand-selfed maternal individuals and 26

534

L. AFFRE AND J.D. THOMPSON

emasculated hand-outcrossed maternal individuals were planted one cm deep in 13 cm plastic pots which were arranged in a fully randomized replicated block. Eight of these selfed and outcrossed families represent pairs originating from the same maternal parent. Pots contained a 2:4:1 (v/v) mixture of peat, thin sand and garden compost. To prevent fungal attack, Benomyl (0.3 g.l−1) was sprayed on individual pots at the beginning of the germination experiment and biweekly thereafter. All individual pots were covered with transparent plastic to maintain moisture, and cardboard to eliminate light. The cardboard and plastic were removed every 2 days to record seed germination and to water. The air temperature in the greenhouse was maintained at 15–18°C throughout the growing period. To reduce position and edge effects, individual pots were randomly rotated on a weekly basis. The number and date of germinated seed per individual pot were recorded every 2 days. Differences in germination between selfed and outcrossed progeny were examined using three different analyses. Percentage germination per maternal parent was analysed by a Kruskall Wallis non-parametric ANOVA. The number of maternal parents producing at least one germinating seed was analysed by a chi-square analysis. The mean number of germinating seeds per family in which at least one seed germinated was analysed using a one-way ANOVA (SAS, 1990). Cotyledon stalk length and cotyledon length and width were measured 30 and 120 days after the date of germination of each seed. Differences among the two treatments and dates for these three traits were examined using a multivariate Analysis of Variance with repeated measures (MANOVAR) (SAS, 1990). We used the Pillai’s trace statistic to quantify where significant differences occurred. Inbreeding depression on percentage seed germination, cotyledon stalk length, and cotyledon length and width was calculated as above for the eight plants which received both self- and cross-pollen. A multiplicative estimate of inbreeding depression was then calculated as above but incorporating percentage seed germination. Enzyme electrophoresis During the spring of 1993, two or three leaves on each of 22–31 individuals were sampled in six populations (see Table 1) which cover the east-west and altitudinal distribution of the species on Crete. The pedicel of each leaf was followed to its insertion on the tuber to be sure that even closely adjacent leaves were sampled from the same genotype. Enzyme extraction was carried out by grinding the leaf material with tris-HCl extraction buffer (Lumaret, 1981), and centrifuging the homogenate (20 min at 15 000×g). The supernatant was then immediately stored frozen in vials at −80°C prior to electrophoresis. Among 12 enzyme systems investigated, seven which encode nine enzyme loci, showed reliable results. The nine enzyme loci analysed were two glutamate oxalotransferases (GOT, E.C.2.6.1.1), one malate dehydrogenase (MDH, E.C.1.1.1.37), one leucine aminopeptidase (LAP, E.C.3.4.11.1), one phosphoglucose isomerase (PGI, E.C.5.3.1.9), two phosphoglucomutases (PGM, E.C.5.4.2.2), one a-este´rase (aEST, E.C.3.1.1.2), and one phosphogluconate dehydrogenase (PGD, E.C.1.1.1.44). Horizontal starch gel electrophoresis was conducted in a cold chamber at 4°C on crude protein extract of leaf tissue. Details concerning gel and electrode buffer systems, and the enzyme staining schedules on which the nine enzyme loci were resolved can be found in Affre et al. (1997).

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

535

The genetic basis of banding patterns for enzymes were inferred from the analysis of segregation patterns on offspring of controlled crosses (L. Affre, unpublished data). The number of independent banding zones as well as the banding patterns within each variable zone were largely consistent with expected enzyme sub-structure and compartmentalization for diploid inheritance and were treated as such in our analyses. Where more than one isozyme of a given enzyme was observed on a gel, they were considered to be the gene products of different loci and the loci were numbered sequentially with the most anodally migrating loci designated as 1. For each locus, the position of the most frequent allele in the gel was assigned the index value 1.00, and other indices were calculated as relative migration distances. Computation of allele frequencies, percentage of polymorphic loci (0.95 criterion, i.e. a locus is considered polymorphic if the frequency of the most common allele does not exceed 0.95), mean number of alleles per locus, mean unbiased expected panmictic heterozygosity and the fixation index (F) were calculated using BIOSYS1 (Swofford & Selander, 1981). A test for conformance to Hardy-Weinberg equilibrium was calculated for fixation indices using Levene’s (1949) correction for small sample size. Population structure was analysed using Wright’s (1965) F-statistics according to the protocol of Weir & Cockerham (1984). Using the computer program FSTAT (Goudet, 1995), we calculated three genetic parameters which are ‘capf’ (Fit), the overall fixation index, ‘smallf’ (Fis), the fixation index due to nonrandom mating within populations, and ‘theta’ (Fst), the fixation index due to population differentiation. Fst values were used to estimate inter-population number of immigrants per generation based on the relationship Fst=1/(4 Nm+1), where N is the local effective population size and m is the average rate of immigration (Wright, 1951; Slatkin, 1987). Genetic ¯ s and Gst) were calculated following Prentice & White (1988). diversity statistics (Ht, H We calculated the genetic distances among all populations following Nei (1972, 1973 and 1978) and also the chi-square method (Balakrishnan & Sanghvi, 1968) due to the presence of rare alleles.

RESULTS

Floral trait variation and covariation Mean pollen and ovule production and pollen volume for each population are shown in Figure 2. Over all populations, mean pollen production per flower (768 200±38 020) showed significant (F=6.72, df=4, 51, P=0.002) variation among populations whilst mean ovule production per flower (33.37±1.22) and mean pollen volume per flower (362.44±33.49) did not show significant among-population variation. For all three characters, the TO-population showed the lowest mean values. Mean values for the other floral traits are shown in Figure 3. Differences among populations were significant in mean corolla diameter (F=24.45, df=5, 164, P= 0.001), stamen length (F=19.08, df=5, 164, P=0.001), and stigma-anther separation (F=10.21, df=5, 164, P=0.001). Stigma-anther separation exceeded 1 mm in the AN-, TO-, PA- and KE-populations but was less than 1 mm (0.89 and 0.84 mm respectively) in the LA- and AS-populations.

L. AFFRE AND J.D. THOMPSON

10 8 6 4 2

m3)

a

0 750

Pollen volume (

60

A Ovule number

Pollen grain number ( 105)

536

b

b

ab

ab

TO AN AS KE LA

B

45 30 15 0

TO AN AS KE LA

C

600 450 300 150 0

TO AN AS KE LA

Figure 2. Mean (±SE) values for (a) pollen grain number, (b) ovule number, and (c) pollen volume in five populations of C. creticum on Crete. Symbols ‘a’ and ‘b’ represent significant differences (P<0.05) among populations following the Scheffe´ multiple range test. Abbreviations: TO: Topolia, AN: Anatolia, AS: Askifou, KE: Kera, LA: Lagou.

Correlations among characters were analysed by Pearson correlation coefficients and significance levels adjusted following Rice (1989). No negative correlation was found between pollen and ovule production, although a significant (r=0.94, n=56) positive correlation was found between ovule production and pollen volume. Petal length was positively correlated with petal width (r=0.24, n=170), corolla diameter (r=0.15, n=170), style length (r=0.28, n=170), and stamen length (r=0.32, n= 170). A positive correlation was also found between petal width and corolla diameter (r=0.26, n=170), between petal width and stamen length (r=0.24, n=170), between corolla diameter and stamen length (r=0.25, n=170), and between style and stamen length (r=0.33, n=170). Temporal variation in stigma receptivity and pollen maturity Forty-four percent of stigmas were receptive as early as the first day after flower opening. Maximum receptivity was however attained after seven days, when around 80% of pollinated flowers initiated fruit (Fig. 4). The duration of stigma receptivity was relatively long, even after 15 days, 71% of stigmas were still receptive. There were thus no significant differences (v2=1.51, df=8, P=0.992) among ‘recipient’ flower age classes in the number of flowers initiating fruit. Roughly 40% of flowers that were pollinated with pollen from newly opened flowers set fruit. Maximum fruit initiation was obtained with pollen from three to nine day old flowers (Fig. 4), although after pollination with pollen from 21 day-old flowers, 60% of pollinations still set fruit. There were thus no significant differences (v2=1.27, df=7, P=0.989) among ‘donor’ flower age classes in ability to set fruit.

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

10

4

2

0 6

bc ab ab

c

a

a

E

2 a

ab ab cd

d

bc cd

TO PA AN AS KE LA

TO PA AN AS KE LA D

4

2

0

TO PA AN AS KE LA

4

0

2

6

C

B

4

0

TO PA AN AS KE LA

Style length

6 Corolla diameter

Petal width

20

0

Stamen length

6

A

Stigma-anther separation

Petal length

30

537

2.0

TO PA AN AS KE LA F

1.5 1.0 0.5 0.0

bc

c

c

a

bc ab

TO PA AN AS KE LA

Figure 3. Mean (±SE) values (in mm) for (a) petal length, (b) petal width, (c) corolla diameter, (d) style length, (e) stamen length, and (f ) stigma-anther separation in six populations of C. creticum on Crete. Symbols ‘a’, ‘b’, ‘c’, and ‘d’ represent significant differences (P<0.05) among populations following the Scheffe´ multiple range test. Abbreviations: TO: Topolia, PA: Papadiana, AN: Anatolia, AS: Askifou, KE: Kera, LA: Lagou.

Percentage fruit set

100 80 60 40 20

0

2

4

6

8

10 12 14 16 18 20 22 Days

Figure 4. Percentage fruit set after pollination of stigmas on ‘recipient’ flowers of different ages (Β) and with pollen from ‘donor’ flowers of different ages (Χ). Age classes are in days after flower opening.

538

L. AFFRE AND J.D. THOMPSON

Percentage fruit initiation

100 80 60 40 20 0

1

2

3

4

Figure 5. Percentage fruit initiation on hand self-pollination without (1) and with (2) emasculation, and hand crosspollination (3), and spontaneous selfing (4).

Self-fertility, self-compatibility and seed set Emasculated but otherwise unmanipulated flowers set no fruit. Flowers that were unmanipulated had greatly reduced fruit initiation compared to all hand-pollinated flowers (Fig. 5). As a result, the self-fertility index calculated for fruit initiation was low (0.11). All three hand-pollination treatments showed more than 93% fruit initiation (Fig. 5). The self-compatibility index calculated for fruit initiation was thus high (1.04). Percentage fruit abortion was variable among the four treatments, (50.0% after spontaneous selfing, 19.4% after hand-selfing without emasculation, 38.2% after hand-selfing with emasculation, and 14.3% after hand-outcrossing). In an overall analysis, fruit initiation, abortion and maturation differed significantly over the four pollination treatments (v2=10.597, df=3, P=0.014). These differences are mainly due to (a) a lower fruit initiation and a greater fruit abortion in the spontaneous selfing treatment than the three other treatments, (b) a greater fruit abortion of selfed flowers than outcrossed flowers and (c) a greater fruit abortion in emasculated compared to non-emasculated selfed flowers. Mean seed number and mean seed weight in the three hand pollination treatments are shown in Figure 6. There was no significant effect of emasculation on seed number (F=3.22, df=1, 13, P=0.096) or seed weight (F=0.95, df=1, 13, P= 0.348). Likewise, there was no significant difference in seed number (F=1.92, df= 1, 22, P=0.179) and seed weight (F=1.27, df=1, 22, P=0.272) between selfed and outcrossed flowers. After outcrossing, mean seed number was negatively correlated (r=−0.38, n=29, P=0.039) with mean seed weight.

Seed germination and seedling vigour No significant differences between selfed and outcrossed seeds were observed for percentage seed germination (Kruskall Wallis non-parametric ANOVA gave a v2= 1.69, df=1, P=0.193), the number of maternal parents producing at least one germinating seed (v2=0.036, df=1, P=0.849) and the number of germinated seeds per maternal parent (F=0.42, df=1, 16, P=0.523). Mean cotyledon stalk length, cotyledon length and width in hand-selfed and

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

Seed number

30

A

20 10 0 8

Seed weight (mg)

539

1

2

3

1

2

3

B

6 4 2 0

Figure 6. Mean (±SE) seed number (a) and mean individual seed weight (b) following hand self-pollination without (1) and with (2) emasculation, and hand cross-pollination (3).

35 30

35

A

30

25

25

20

20

15

15

10

10

5

5

0

SL

CL

CW

0

B

SL

CL

CW

Figure 7. Mean (±SE) values (in mm) of cotyledon stalk length (SL), cotyledon length (CL) and cotyledon width (CW) for seedlings resulting from hand-selfed (open bars) and hand-outcrossed (closed bars) treatments at 30 days (a) and 120 days (b) after seed germination.

hand-outcrossed offspring on two dates are shown in Figure 7. The MANOVAR showed that outcrossed seedlings were significantly (F=5.60, df=3, 53, P=0.002) bigger than selfed seedlings, predominantly as a result of differences in cotyledon stalk length (Fig. 7). Seedlings were significantly (F=166.25, df=3, 53, P<0.001) larger at the second date of measurement. The interaction between treatment and date was not significant (F=0.68, df=3, 53, P=0.568).

Inbreeding depression Inbreeding depression calculated for each plant which received both emasculated self- and cross-pollinations was low for mean seed number (d=0.15) and nonexistent for mean seed weight (d=−0.08). When based on mean values after selfing and outcrossing calculated over all plants regardless of whether both treatments

540

L. AFFRE AND J.D. THOMPSON

Flower longevity (days)

40

30

20

10

0

1

2

3

4

Figure 8. Mean (±SE) flower longevity on hand self-pollination without (1) and with (2) emasculation, hand crosspollination (3), and spontaneous selfing.

were carried out on the same plants, the higher levels of inbreeding depression on fruit maturation (d=0.28) and seed production (d=0.21) gave however a relatively high multiplicative value of d=0.43. No inbreeding depression on seed germination (d=−0.19) was detected. The multiplicative estimate of inbreeding depression, incorporing fruit maturation, seed number per fruit and percentage seed germination then decreased to 0.38. Finally, inbreeding depression was generally low or non-existent for each seedling trait at both dates (d=0.07 and 0.08 for cotyledon stalk length, d=0.14 and −0.008 for cotyledon length and d=0.05 and −0.12 for cotyledon width after 30 and 120 days respectively).

Flower longevity The mean longevity of flowers (Fig. 8) showed significant (F=262.11, df=3, 121, P<0.001) variation among the four pollination treatments due to a significantly longer flowering life for unpollinated flowers in the spontaneous selfing treatment.

Amount and pattern of genetic variation At the species level, four loci (Got-1, Lap-1, Pgi-1, and Pgm-1) of the nine enzyme loci investigated were monomorphic. Got-2 showed banding patterns which are consistent with a dimeric enzyme, but the instability of the banding intensity in different gels prevented us from interpreting many individuals, hence the locus was excluded from analyses. No bands were observed at the Pgm-2 locus that was been found to be polymorphic in the related C. balearicum (Affre et al., 1997). Allele

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

A

24°

0 50 km 25°

B

26°

24°

541

0 50 km 25°

26°

CRETE

35°

35°

24°

25°

CRETE

35°

26°

35°

24°

25°

Mdh-1 1.00 C

24°

0.76

26°

Pgm-3 1.29

0 50 km 25°

1.00 D

26°

24°

1.12

0 50 km 25°

26°

CRETE

35°

35°

24°

25°

26°

CRETE

35°

35°

24°

25°

Est-1 1.00

0.95

0.98

26°

Pgd-1 1.06

1.09

1.00

0.94

Figure 9. Allele frequencies in the six sampled populations of C. creticum on Crete.

T 2. Summary of genetic variability for the six sampled populations.The values shown are means (±SE) Mean heterozygosity Populations TO PA AN AS KE LA Mean

Percentage of polymorphic locia 42.86 57.14 57.14 57.14 57.14 57.14 54.76

Mean no. of alleles per locus 1.57 2.00 1.86 1.86 1.86 1.57 1.78

(0.30) (0.44) (0.34) (0.34) (0.34) (0.20) (0.07)

Direct-count 0.000 0.051 0.060 0.078 0.058 0.070 0.053

(0.000) (0.036) (0.036) (0.054) (0.037) (0.045) (0.011)

HdyWbg expectedb 0.163 0.253 0.272 0.159 0.256 0.230 0.222

(0.085) (0.102) (0.104) (0.064) (0.095) (0.083) (0.020)

a

A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.95. b Unbiased estimate (see Nei, 1978).

frequencies at the remaining four polymorphic loci (Mdh-1, Pgm-3, aEst-1, and Pgd1) are shown in Figure 9. Among populations, the percentage of polymorphic loci ranged from 42.86 to 57.14%, the mean number of alleles per locus from 1.57 to 2.00, and the mean observed heterozygosity from 0.051 to 0.078 (Table 2). No heterozygotes were found in the TO-population which thus showed complete fixation at the three polymorphic

542

L. AFFRE AND J.D. THOMPSON

T 3. (a) Fixation indices (F) and (b) summary of F-statistics for the four polymorphic loci (A) Locus

TO

PA

AN

AS

KE

LA

Mdh-1 Pgm-3 aEst-1 Pgd-1

1.000∗∗∗ —a 1.000∗∗∗ 1.000∗∗∗

0.868∗∗∗ 0.326 0.948∗∗∗ 1.000∗∗∗

0.892∗∗∗ 0.326 0.833∗∗∗ 1.000∗∗∗

0.513∗∗∗ −0.216 1.000∗∗∗ 1.000∗∗∗

0.932∗∗∗ 0.354∗ 0.782∗∗∗ 1.000∗∗∗

0.887∗∗∗ 0.292 0.636∗∗ 1.000∗∗∗

Locus is monomorphic; ∗, P<0.05, ∗∗, P<0.01, ∗∗∗, P<0.001 indicate significant deviation from the expected heterozygosity under Hardy-Weinberg equilibrium. (B) a

Locus

Fis

Fit

Fst

¯s H

Ht

Gst

Mdh-1 Pgm-3 aEst-1 Pgd-1 Mean ±SE

0.862 0.251 0.880 1.000 0.748∗∗∗ 0.068

0.902 0.296 0.916 1.000 0.778∗∗∗ 0.057

0.292 0.060 0.301 0.018 0.168∗∗∗ 0.028

0.483 0.318 0.484 0.259 0.386 0.057

0.657 0.343 0.669 0.277 0.486 0.103

0.265 0.073 0.277 0.067 0.170 0.058

∗∗∗ F-statistics significantly (P<0.001) different from zero using the computer software FSTAT (Goudet, 1995).

T 4. Fst values calculated among all pairs of populations Populations TO PA AN AS KE LA

TO

PA

AN

AS

KE

LA

— 0.144 0.051 0.276 0.313 0.405

— 0.109 0.292 0.223 0.322

— 0.192 0.153 0.219

— 0.244 0.260

— 0.206

—

loci (Table 3A). This population also showed the lowest percentage of polymorphic loci and number of alleles per locus (Table 2) and was the only population monomorphic at the Pgm-3 locus. A significant heterozygote deficiency (Tables 2 and 3A) was found for the Mdh-1, aEst-1 and Pgd-1 loci in all six populations and only at the Pgm-3 locus in the KE-population (Table 3A). The fixation indice is relatively high with Fis equal to 0.748±0.068 (Table 3B). All Fst values were significantly different from zero (P<0.001). Mean Fst and Gst values were respectively 0.168±0.028 and 0.170±0.058 (Table 3B), which suggests that differentiation among populations is relatively low. Fst values, calculated for all possible combinations of two populations (Table 4), indicate lower population differentiation among the eastern KE-, AN- and LA-populations (mean Fst=0.192) which are closer together than the three western AS-, PA- and TO-populations (mean Fst=0.235). The slightly greater genetic differentiation among the three western populations is probably due to the allelic fixation observed in the TOpopulation at the Pgm-3 locus. Genetic differentiation among western and eastern populations was intermediate in extent (mean Fst=0.229). The correlation between geographic distance and number of immigrants (Nm) among populations was significant (r=−0.52, n=15, P=0.044).

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

% 100

A

80

60

60

40

40

20

20

0

0

KERA LAGOU TOPOLIA ANATOLIA PAPADIANA ASKIFOU

80

B

KERA LAGOU TOPOLIA ANATOLIA ASKIFOU PAPADIANA

% 100

543

Figure 10. Genetic distance relationships among the six sampled populations as summarized by cluster analysis using (a) Nei’s and (b) chi-square genetic distance coefficients (given as percentage).

Based on 15 pairwise comparisons, the mean Nei and chi-square genetic distances were 0.155±0.021 and 0.195±0.010 respectively (Fig. 10). The western and eastern populations were not clearly separated into two clusters since the AN-population was classed with the western populations. The two closest and highest altitude populations (KE and LA) are however clearly connected and isolated from the other populations.

DISCUSSION

Genetic variation Stebbins (1942) suggested that endemic species should have lower levels of genetic variation than more widespread species. Hamrick, Linhart & Mitton (1979), Loveless & Hamrick (1984), Karron (1987, 1991) and Hamrick & Godt (1989) have since documented the relationship between life history traits, mating system, geographic distribution and genetic diversity in a wide variety of plant species. According to these reviews, geographic range and mating system are important determinants of genetic diversity and population genetic structure. In this study, we have found that the percentage of polymorphic loci (54.76%), but not the number of alleles per locus (1.78±0.07) in the endemic C. creticum is higher than that found for other endemic (40% and 1.80±0.08 respectively) species (see Hamrick & Godt, 1989). These levels of genetic variation are also markedly higher than that found in a previous study of the closely related C. balearicum for which the percentage of polymorphic loci and the mean number of alleles are 24.20% and 1.30±0.04 respectively on the Balearic Islands (Affre et al., 1997). The

544

L. AFFRE AND J.D. THOMPSON

proportion of total diversity among populations of C. creticum (Gst=0.170±0.058) is low compared to other endemic (Gst=0.248±0.037) species (Loveless & Hamrick, 1984; Hamrick & Godt, 1989). It is also markedly less than that observed among populations of C. balearicum on the Balearic Islands (Gst=0.299±0.058). Genetic ¯ s=0.386±0.057) is however higher than mean values diversity within populations (H ¯ =0.163±0.016) species (Hamrick & Godt, 1989), and that found of other endemic (H ¯ =0.231±0.078). Overall, genetic diversity for C. balearicum on the Balearic Islands (H is relatively high in C. creticum, despite its restricted geographic distribution. The genetic similarity of the KE- and LA-populations (Fig. 10) suggests a common evolutionary history for these two populations which are found on the same massif on the eastern part of the island. A possible explanation for this pattern of genetic diversity lies in the history of Crete, which is a remnant of an Aegean landmass that initially emerged from the sea during the Oligocene (i.e. −37.5 Myr). At this point in time, Crete was part of a chain of mountains that connected the Peloponnese in southern Greece with the Taurus mountains in south-west Turkey. Due to sea level changes in the Pliocene (i.e. −5 Myr), the chain of mountains split into a series of islands, the contemporary massifs of Lefka Ori (2452 m) to the west, Oros Idi (2456 m) in the centre, and Oros Dikti (2148 m) and Afendis Kavousi (1476 m) in the east. These joined to form a single island, the present day Crete, during the Pleistocene (i.e. less than −1.8 Myr) (Turlan et al., 1993). The genetic distance data suggest that the KE- and LA-populations have been together for a longer period of time, i.e. they were on the same ‘island’ in the Pliocene. If C. creticum was present on the initial Aegean landmass, it is likely that the populations of C. creticum, studied here, have experienced the isolation and subsequent joining of the islands. The populations of C. creticum may thus not have experienced severe genetic bottlenecks that would have occurred during colonization of Crete from mainland populations, hence the high levels of within-population genetic diversity observed. Another important factor determining the pattern and levels of genetic diversity is the mating system. We found significant fixation indices (mean Fis=0.748±0.068) due to heterozygote deficiencies at most loci in each population. This is indicative of high levels of historical inbreeding (either by autogamy, geitonogamy or biparental inbreeding) in these populations of C. creticum. The fixation index is slightly less than that observed for C. balearicum which has a mean Fis=0.919±0.048 on the Balearic Islands (Affre et al., 1997). The TO-population, which showed the lowest amount of genetic variation (see Table 2), also shows the lowest pollen production and the smallest stamens (see Figs 2 and 3) of all the study populations. Inbreeding may thus be greater in this population. Floral biology The mean P/O ratio of 25 700±2050 and the subsequent mean logarithm P/O ratio of 4.35±0.03 obtained in our study of C. creticum suggest that the species is xenogamous (Cruden, 1977). A previous study (Affre et al., 1995) of natural populations of the closely related C. balearicum showed a similar albeit lower value (log P/O ratio=4.09±0.01). However, as just discussed, populations of both species contain significant heterozygote deficiencies at most studied loci, this suggesting high degrees of inbreeding in both species. The P/O ratio may not be a good predictor of the mating system in this species for several reasons.

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

545

First, the lack of nectar production means that pollen is an important component of pollinator attraction, hence P/O ratios remain high, as is predicted to occur when inbreeding is facilitated (Lloyd, 1988). Second, inbreeding may be relatively recent, and P/O ratios may not have yet evolved towards ratios observed in other inbred species (Cruden, 1977). An important feature of the floral biology of C. creticum is the stigma-anther separation which, as we found in our pollination trial, may prevent autonomous self-pollination (self-fertility index=0.11; see Fig. 5) and provide little reproductive assurance in the absence of pollinators. Hence, the high levels of inbreeding in C. creticum appear to be due to facilitated inbreeding, which is likely competing (i.e. self-pollinations occurring at the same time as cross-pollinations, sensu Lloyd & Schoen, 1992) due to the lack of any temporal separation in female and male functions (see below). In contrast, the high levels of inbreeding found in C. balearicum are probably due to autonomous selfing due to the close proximity (<1 mm) of the stigma and anthers in this species (Affre et al., 1995). Despite the inability of C. creticum to set fruit in the absence of pollinators, no pollinators were observed during four hours of observation in two populations nor on 100 flowers coated with clear unscented glue. Many flowers were however observed to be ‘visited’ by ants, small hymenoptera and spiders ( J.D. Thompson, pers. observ.), which may cause passive selfing (e.g. Svensson, 1986; Peakall & Beattie, 1989; Peakall & James, 1989; Gomez & Zamora, 1992). However, in the absence of more detailed observations, we are unable to conclude on the pollination biology of this species. Individual flowers showed very little temporal separation of pollen maturity and stigma receptivity. The slight variation which occurred was due to the three-nine day pollen age classes showing higher fruit initiation than the respective stigma age classes (Fig. 4). However as Figure 4 shows, whereas maximum fruit initiation for the different pollen age classes reached 100%, that for the stigma receptivity classes only reached 75%. Hence, the difference in the early classes between pollen and stigma fertilities may be a spurious effect. If all the stigma receptivity classes are compared to the maximum value of 75%, and an index of pollen fertility at different ages calculated in the same way (as is automatically done in the case of the pollen maturity classes since a maximum of 100% was attained) then the difference between the stigma receptivity and pollen maturity schedules becomes negligible. We can thus suggest that this species shows very little or no evidence for any temporal separation in male and female function. Stigmas remained receptive for at least 15 days and pollen retained in the anthers remains mature for at least 21 days. This long longevity of stigma receptivity and pollen maturity, combined with a long mean flower longevity in unpollinated flowers of 36.50±1.49 days, suggests that pollinator uncertainty may be an important component of the reproductive biology of this species (Primack, 1985). Such long floral longevity is likely to be favoured by the fact that flowers do not produce nectar and thus have low daily resource demands, hence the cost of maintaining flowers once they have opened is low, and a long floral longevity may ensue (see Primack, 1985; Ashman & Schoen, 1994; Schoen & Ashman, 1995). However, Primack (1985) pointed out that flower longevity in natural conditions is usually lower than that observed in flowers protected from pollinators.

546

L. AFFRE AND J.D. THOMPSON

Inbreeding depression The role of inbreeding depression in the evolution and maintainance of selfing versus outcrossing is a fundamental component of mating system evolution (Fisher, 1941; Lande & Schemske, 1985; Charlesworth & Charlesworth, 1987; Uyenoyama, Holsinger & Waller, 1993). Inbreeding depression evolves in relation to the breeding history of a population (Husband & Schemske, 1995). Inbreeding depression will be high in populations that are historically outbred and heterozygous if it is the result of lethal or sub-lethal deleterious recessive alleles as is commonly believed. However, in inbred populations, inbreeding depression is likely to be low, since recessive alleles will be continually exposed in the homozygote state and thus purged by selection. Inbreeding depression may also differ markedly among life cycle stages (Wolfe, 1993; Agren & Schemske, 1993; Husband & Schemske, 1995; Carr & Dudash, 1995). This is due to the fact that the levels of inbreeding depression are determined partly by its genetic basis (Husband & Schemske, 1995) and because genes causing inbreeding depression may be to some extent independent across life-history stages (Husband & Schemske, 1996). The review of these authors illustrates how inbreeding depression is often during seed development and seed set in outcrossed species and later in the life cycle, at the growth and reproduction stages, in inbred species. This may be due to the fact that lethal recessive alleles cause most early-acting inbreeding depression and this genetic load can be rapidly purged from a population by even a small amount of inbreeding. In contrast, much of the late-acting inbreeding depression may be due to weakly deleterious mutations or to polygenic mutations and this genetic load is difficult to purge, even under extreme inbreeding (Lande & Schemske, 1985; Charlesworth & Charlesworth, 1987; Charlesworth, Morgan & Charlesworth, 1990). We found no evidence of inbreeding depression on seed weight, percentage seed germination and seedling traits. Inbreeding depression was however important during fruit maturation. When based on mean values calculated over all plants regardless of whether both treatments were carried out on the same plants, we found high inbreeding depression on fruit maturation (d=0.28) and seed production (d=0.21) and no inbreeding depression on seed germination (d= −0.19) which give a multiplicative value of d=0.38. This greater inbreeding depression on seed set than on seed germination fits the trend described above for outcrossing species (Husband & Schemske, 1996) and may result from the same reasons they suggest. The overall level of inbreeding depression may also be more severe in natural situations than in controlled conditions (e.g. Schemske, 1983; Dudash, 1990). Inbreeding depression may be high in C. creticum, despite high levels of inbreeding due to high rates of mutation to mildly deleterious alleles with partial dominance (Charlesworth et al., 1990). Other inbred species maintain high levels of inbreeding depression (Husband & Schemske, 1996), hence such mutations with partial dominance may be important. In conclusion, C. creticum appears to have a mixed-mating system. Given the high Fis values reported here, plus the lack of reproductive assurance and intermediate values of inbreeding depression, it would now be most interesting to examine whether such a mating system may be maintained as a result of factors linked to population structure (Uyenoyama et al., 1993; Ronfort & Couvet,

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

547

1995) or to loss of outcrossed siring success due to pollen discounting (Holsinger, Feldman & Christiansen, 1984; Harder & Barrett, 1995).

ACKNOWLEDGEMENTS

The authors thank Franc¸ois Bretagnolle and Virginie Quesnay for help with data analysis, Christian Collin and the greenhouse staff at the CEFE-CNRS for help with the cultivation of plant material, France Di Giusto, Lamjed Toumi, Michele Tarayre and Roselyne Lumaret for help and advice on electrophoresis procedures and genetic data interpretation, and Max Debussche and Susan Mazer for helpful discussion. John D. Thompson is particularly grateful to Hristo Stankov for his help in the field and the staff and students at the MAICH on Crete for their hospitality and help. The Ministe`re de la Recherche et de la Technologie, the Ministe`re de l’e´ducation nationale de l’enseignement supe´rieur, de la recherche et de l’insertion professionnelle (contract ACC SV3 N°9503025), and the CNRS provided financial support for the work.

REFERENCES Affre L, Thompson JD, Debussche M. 1995. The reproductive biology of the Mediterranean endemic Cyclamen balearicum Willk. (Primulaceae). Botanical Journal of the Linnean Society 118: 309–330. Affre L, Thompson JD, Debussche M. 1997. Genetic structure of continental and island populations of the Mediterranean endemic Cyclamen balearicum (Primulaceae) American Journal of Botany (in press). Agren J, Schemske DW. 1993. Outcrossing rate and inbreeding depression in two annual monoecious herbs Begonia hirsuta and B. semiovata. Evolution 47: 125–135. Anderberg AA. 1993. Phylogeny and subgeneric classification of Cyclamen L. (Primulaceae). Kew Bulletin 49: 455–467. Ashman TL, Schoen DJ. 1994. How long should flowers live? Nature 371: 788–791. Aspland V. 1994. Expedition to Crete 1994. The Journal of the Cyclamen Society 18: 42–44. Balakrishnan V, Sanghvi LD. 1968. Distance between populations on the basis of attribute data. Biometrics 24: 859–865. Cardona MA, Contandriopoulos J. 1977. L’ende´misme dans les flores insulaires Me´diterrane´ennes. Mediterranea 2: 49–77. Cardona MA, Contandriopoulos J. 1979. Endemism and evolution in the islands of the western Mediterranean. In: Bramwell D, ed. Plants and islands. London: Academic Press, 133–169. Carlquist S. 1974. Island biology. New York: Columbia University Press. Carr DE, Dudash MR. 1995. Inbreeding depression under a competitive regime in Mimulus guttatus: consequences for potential male and female function. Heredity 75: 437–445. Charlesworth D, Charlesworth B. 1987. Inbreeding depression and its evolutionary consequences. Annual Review of Ecology and Systematics 18: 237–268. Charlesworth D, Morgan MT, Charlesworth B. 1990. Inbreeding depression, genetic load, and the evolution of outcrossing rates in a multilocus system with no linkage. Evolution 44: 1469–1489. Contandriopoulos J, Favarger C. 1974. Proble`mes pose´s par l’ende´misme en Me´diterrane´e. La flore du bassin Me´diterrane´en. Essai de syste´matique synthe´tique 175–194. Cruden RW. 1977. Pollen-ovule ratios: a conservative indicator of breeding systems in flowering plants. Evolution 31: 32–46. Dudash MR. 1990. Relative fitness of selfed and outcrossed progeny in a self-compatible, protandrous species, Sabatia angularis L. (Gentianaceae): a comparison in three environments. Evolution 44: 1129–1139. Fisher RA. 1941. Average excess and average effect of a gene substitution. Annals Eugenics 11: 53–63. Glover DE, Barrett SCH. 1987. Genetic variation in continental and island populations of Eichhornia paniculata (Pontederiaceae). Heredity 59: 7–17. Gomez JM, Zamora R. 1992. Pollination by ants: consequences of the quantitative effects on a mutualistic system. Oecologia 91: 410–418. Goudet J. 1995. Fstat version 1.2: a computer program to calculate F-statistics. Journal of Heredity 86: 485–488.

548

L. AFFRE AND J.D. THOMPSON

Greilhuber J. 1989. Karyotype structure and evolution in Cyclamen L. subgen. Psilanthum Schwz. (Primulaceae). Flora 183: 103–113. Greuter W. 1979. The origins and evolution of islands floras as exemplified by the Aegean archipelago. In. Bramwell, D, ed. Plants and islands. London: Academic Press, 87–105. Grey-Wilson C. 1988. The genus Cyclamen. Portland, Oregon: A Kew Magazine Monograph. Hamrick JL, Godt MJW. 1989. Allozyme diversity in plant species. In: Brown AHD, Clegg MT, Kahler AL, Weir BS, eds. Plant population genetics, breeding and genetic resources. Sunderland, MA: Sinauer, 43–63. Hamrick JL, Linhart YB, Mitton JB. 1979. Relationships between life history characteristics and electrophoretically detectable genetic variation in plants. Annual Review of Ecology and Systematics 10: 173–200. Harder LD, Barrett SCH. 1995. Mating cost of large floral displays in hermaphrodite plants. Nature 373: 512–515. Holsinger KE, Feldman MW, Christiansen FB. 1984. The evolution of self-fertilization in plants: a population genetic model. The American Naturalist 124: 446–453. Husband BC, Barrett SCH. 1991. Colonization history and population genetic structure of Eichhornia paniculata in Jamaica. Heredity 66: 287–296. Husband BC, Schemske DW. 1995. Magnitude and timing of inbreeding depression in a diploid population of Epilobium angustifolium (Onagraceae). Heredity 75: 206–215. Husband BC, Schemske DW. 1996. Evolution of the magnitude and timing of inbreeding depression in plants. Evolution 50: 54–70. Karron JD. 1987. A comparison of levels of polymorphism and self-compatibility in geographically restricted and widespread plant congeners. Evolutionary Ecology 1: 47–58. Karron JD. 1991. Patterns of genetic variation and breeding systems in rare plants species. In. Falk D, Holsinger K, eds. Genetics and conservation of rare plants. Oxford: Oxford University Press, 87–98. Lande R, Schemske DW. 1985. The evolution of self-fertilization and inbreeding depression in plants. I. Genetic models. Evolution 39: 24–40. Levene H. 1949. On a matching problem arising in genetics. Annals of Mathematics and Statistics 20: 91–94. Lloyd DG. 1988. Benefits and costs of biparental and uniparental reproduction in plants. In. Michod RE, Levin BR, eds. The evolution of sex. Sunderland, Massachusetts: Sinauer Associates Inc., 233–252. Lloyd DG, Schoen DJ. 1992. Self- and cross-fertilization in plants. I. Functional dimensions. International Journal of Plant Sciences 153: 358–369. Loveless MD, Hamrick JL. 1984. Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics 15: 65–95. Lumaret R. 1981. Structure ge´ne´tique d’un complexe polyploide: Dactylis glomerata L. Unpublished D. Phil. Thesis, Universite´ des sciences et techniques du languedoc, France. MacArthur RH, Wilson EO. 1967. The theory of island biogeography. Princeton, NJ: Princeton University Press. Nei M. 1972. Genetic distance between populations. The American Naturalist 106: 283–292. Nei M. 1973. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences, USA 70: 3321–3323. Nei M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: 583–590. Peakall R, Beattie AJ. 1989. Pollination of the orchid Microtis parviflora R. Br. by flightless worker ants. Functional Ecology 3: 515–522. Peakall R, James SH. 1989. Outcrossing in an ant pollinated clonal orchid. Heredity 62: 161–167. Prentice HC, White RJ. 1988. Variability, population size and isolation: the structuring of diversity in ¨ land Gypsophila fastigiata. Acta Oecologica/Oecologia Plantarum 9: 19–29. O Primack RB. 1985. Longevity of individual flowers. Annual Review of Ecology and Systematics 16: 15–37. Rice WR. 1989. Analysing tables of statistical tests. Evolution 43: 223–225. Ronfort J, Couvet D. 1995. A stochastic model of selection on selfing rates in structured populations. Genetic Research, Cambridge 65: 209–222. SAS Institute Inc. 1990. SAS/STAT User’s guide. version 6, vol. 2, Cary, North Carolina. Schemske DW. 1983. Breeding system and habitat effects on fitness components in three neotropical Costus (Zingiberaceae). Evolution 37: 523–539. Schoen DJ, Ashman TL. 1995. The evolution of floral longevity: resource allocation to maintenance versus construction of repeated parts in modular organisms. Evolution 49: 131–139. Shaw RG, Mitchell-Olds T. 1993. ANOVA for unbalanced data: an overview. Ecology 74: 1638–1645. Slatkin M. 1987. Gene flow and the geographic structure of natural populations. Science 236: 787–792. Sokal RR, Rohlf FJ. 1981. Biometry. 2nd ed. New York, USA: W.H. Freeman. Stebbins GL. 1942. The genetic approach to problems of rare and endemic species. Madron˜o 6: 241–258. Strid A. 1969. Evolutionary trends in the breeding system of Nigella (Ranunculaceae). Botaniska Notiser 122: 380–397. Svensson L. 1986. Secondary pollen carryover by ants in a natural population of Scleranthus perennis (Caryophyllaceae). Oecologia (Berl.) 70: 631–632. Swofford DL, Selander RS. 1981. BIOSYS-1. A computer program for the analysis of allelic variation in genetics. User’s manual. Urbana, IL: University of Illinois.

REPRODUCTIVE BIOLOGY OF CYCLAMEN CRETICUM

549

Turlan NJ, Chilton L, Press JR. 1993. Flora of the Cretan Area. Annotated checklist Atlas. The Natural History Museum, London, HMSO. Uyenoyama MK, Holsinger KE, Waller DM. 1993. Ecological and genetic factors directing the evolution of self-fertilization. In: Futuyma D, Antonovics J, eds. Oxford surveys in evolutionary biology. Oxford: Oxford University Press, 327–381. Verlaque R, Aboucaya A, Cardona MA, Contandriopoulos J. 1991. Quelques exemples de spe´ciation insulaire en Me´dite´rrane´e occidentale. Botanika Chronika 10: 137–154. Warwick SI, Gottlieb LD. 1985. Genetic divergence and geographic speciation in Layia (Compositae). Evolution 39: 1236–1241. Weir BS, Cockerham CC. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370. Westerbergh A, Saura A. 1994. Genetic differentiation in endemic Silene (Caryophyllaceae) on the Hawaiian Islands. American Journal of Botany 81: 1487–1493. Witter MS, Carr GD. 1988. Adaptive radiation and genetic differentiation in the Hawaiian silversword alliance (Compositae: Madiinae). Evolution 42: 1278–1287. Wolfe LM. 1993. Inbreeding depression in Hydrophyllum appendiculatum: role of maternal effects, crowding, and parental mating history. Evolution 47: 374–386. Wright S. 1951. The genetical structure of populations. Annals of Eugenics 15: 323–354. Wright S. 1965. The interpretation of population structure by F-statistics with special regard to mating systems. Evolution 19: 358–420.