Floral and reproductive biology ofDrosophyllum lusitanicum(L.) Link (Droseraceae)

Botanical Journal of the Linnean Society (1995), 118: 331–351. With 3 figures

Floral and reproductive biology of Drosophyllum lusitanicum (L.) Link (Droseraceae) ANA ORTEGA OLIVENCIA, JACINTO P. CARRASCO CLAVER AND JUAN A. DEVESA ALCARAZ Departamento de Biologıa y Produccio n Vegetal, Unidad de Bota nica, Facultad de Ciencias, Universidad de Extremadura, E-06071 Badajoz, Spain Received March 1995, accepted for publication June 1995

We studied the floral and reproductive biology of Drosophyllym lusitanicum (Droseraceae), a species endemic to the western Iberian Peninsula and northwest Morocco. Flowering lasted from March to August, peaking in April. The species is clearly homogamous, with pollen germination and stigma receptivity occurring even in preanthesis. This was reflected in the high rates of fruit set and seed set in bagged inflorescences. Fructification did not differ significantly between different pollination treatments, although we did find differences between some treatments in the numbers of viable seeds per flower and fruit. However, seed weight did not differ significantly. The importance of self-pollination over cross-pollination was also supported by the low percentages of fructification (24.67%) after emasculation and open pollination. Metameric deviations were seen in flowers, although this abnormality was not translated as any reproductive benefit or disadvantage (fructification, number or weight of seeds) in comparison with normal flowers. © 1995 The Linnean Society of London

ADDITIONAL KEY WORDS:*Drosophyllum – Droseraceae – metameric deviation – pollen : ovule ratio – self-pollination – abortion – fruit set – seed set. CONTENTS Introduction . . . . . . . . . Material and methods . . . . . . . Study area and plant characteristics . . Floral production . . . . . . . Flower characteristics . . . . . . Flowering and fructification phenology . Pollen and ovule production . . . . Self-pollen loads and pollen germination . Stigma receptivity . . . . . . . Breeding system . . . . . . . Pollinator limitation test . . . . . Results . . . . . . . . . . Floral production and flowering phenology Flower characteristics . . . . . . Pollen and ovule production . . . . Self-pollen loads and pollen germination . Stigma receptivity . . . . . . . Fructification . . . . . . . . 0024–4074/95/080331+21 $12.00/0

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INTRODUCTION

The family Droseraceae consists of four genera (Drosera, Dionaea, Aldrovanda and Drosophyllum), and nearly one hundred species (Moore, 1985; Cronquist, 1988). These plants are characterized by their ability to use special adaptations of their leaves to capture and absorb organic nitrogen from small insects (Boesewinkel, 1989). In the Iberian Peninsula, only the genera Drosera and Drosophyllum are represented, the latter being monospecific. The range of Drosophyllum lusitanicum (L.) Link is limited to the western Iberian Peninsula and Morocco (Fig. 1) (Franca, 1921; Quintanilha, 1926; Lloyd, 1942; Maire, 1976; Silvestre, 1987), where it can be considered rare (Slack, 1969), and where populations usually occupy an area no larger than

Figure 1. Range of Drosophyllum lusitanicum according to data from material deposited in the MA, MAF and UNEX herbaria, and published information.

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a few square metres (Quintanilha, 1926 and personal observation). In contrast with the other genera in this family, and with other members of the order Nepenthales, which usually inhabit damp soils (Cronquist, 1988) or even aquatic environments (Aldrovanda, sec.Moore, 1985), D. lusitanicum populations appear in dry, sunny areas on acid substrates (Harshberger, 1925; Lloyd, 1942; Slack, 1969; Lecoufle, 1989). These populations appear in clearings in pine, cork oak and oak forests or their successional shrublands, where the only water available during the dry period is supplied by condensation of atmospheric humidity (Lecoufle, 1989). These environments favour a great development of the root system (Franca, 1921). Like all Droseraceae, D. lusitanicum supplements its nitrogen (Quintanilha, 1926; Cronquist, 1988) and phosphorus supplies (Heslop-Harrison, 1978; Boesewinkel, 1989) by capturing small insects. The insects, attracted by the colour (Slack 1969; Joel, Juniper & Dafni, 1985) or by the odour of plant secretions (Meyer & Dewe`vre, 1894; Quintanilha, 1926; Slack, 1969), become trapped in the basal leaves (Franca, 1921; Quintanilha, 1926; Heslop-Harrison, 1978) and in other highly viscid aerial parts. Two types of glands are distinguishable in the leaves: sessile glands rich in digestive enzymes (Darwin, 1875; Franca, 1921; Quintanilha, 1926), and pedunculated glands that secrete a carbohydrate-rich mucilage that attracts insects. The latter glands are located on the abaxial surface and along the leaf margins (Lloyd, 1942). A number of features of this taxon related with its ‘insectivorous’ properties (Lloyd, 1942; Slack, 1969; Heslop-Harrison, 1978) are well known (Soland, 1870; Darwin, 1875; Penzig, 1877), especially the characteristics of the glands, their physiology, and the chemical bases of external digestion (Darwin, l.c.; Meyer & Dewe`vre, 1894; Dewe`vre, 1895; Franca, 1921, 1922 & 1925; Quintanilha, 1926; Lloyd, 1942; Schnepf, 1961). Patterns of light absorption of the glands and their secretions ( Joel et al., 1985), and growth requirements (Lecoufle, 1989) have also been described. However, little information on the reproductive biology of this species is available; the only publication to date appears to be Boesewinkel’s study (1989) of ovule and seed development. Some descriptive details of its flowers and fruits have appeared in basic texts on insectivorous plants (Slack, 1969). The present study was undertaken because of the scarcity of available information, and because most of the known populations of this species are located near areas subjected to intense pressure due to human activity, i.e. agriculture or construction, especially in coastal areas. The main objective of this work is to contribute to our understanding of the floral and reproductive biology of this species.

MATERIAL AND METHODS

Study area and plant characteristics The present study was carried out between February and October 1990, June and July 1993, and March and July 1994. The population studied is located at 450 m above sea level, on a mesomediterranean level with a dry ombroclimate, in an area measuring approximately 350 m2 (population A). Because of the small size of the population, sample size was limited, and

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this may have biased some of our conclusions. This population is situated in the Sierra del Puerto del Centinela, 6 km from the village of Alburquerque (Badajoz, Spain), in a disturbed cork oak wood, and in a Pinus pinaster pine scrubland. Parts of this study were based on another population located in the Sierra Frı´a (population B) near Valencia de Alca´ntara (Ca´ceres, Spain; Fig. 1). The flowers of the species are hermaphroditic, actinomorphic and pentameric, with cymose, bracteate inflorescences of a characteristic pseudocorymbose appearance (Slack, 1969; Maire, 1976; Fig. 2). They have a dichlamydeous perianth consisting of five concrescent sepals at the base, and five large, free, yellow petals (Fig. 2). The stamens occur in two whorls of five each, and the gynoecia have five stigmas (Maire, 1976; Silvestre, 1987). Floral production To obtain information on the architecture of the inflorescence and the sequence of flower appearance and maturation, we traced the development of 200 inflorescences in 100 individuals. We recorded the total number of flowers produced per inflorescence (n  319 inflorescences), the total number of flowers open simultaneously per individual (n  200 plants), and the mean number of floral scapes (inflorescences) per individual (n  300 plants). Flower characteristics We marked 65 recently opened flowers, and with the help of a magnifying glass, recorded contact between the two sexual whorls. Contacts were also recorded in the laboratory under a dissecting microscope, from preanthesis until flower wilting. We also measured flower diameter at different stages (n  100 flowers). Because preliminary observations revealed notable variations in the number of elements of flower whorls (designated ‘abnormal’ in this study), 100 flowers were collected at random in each population studied (populations A and B) to estimate the frequency of elements. The numbers of sepals, petals, stamens and stigmas per flower were recorded. Flowering and fructification phenology In population A (spring 1990), 50 plants were examined at random to trace the evolution of flowering and fructification. The number of flowers and the state of the fruits, when present, were recorded at least once per week until dehiscence; losses due to abortion during flower development were also noted. To obtain information on the longevity of flowers, one flower on each of 50 different plants was marked and the number of hours each flower remained open was recorded. To determine whether there were significant differences in fructification between ‘normal’ and ‘abnormal’ flowers, 50 flowers of each type were examined in April 1994 and the presence (or absence) of fruits in each were

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Figure 2. Drosophyllum lusitanicum. Drawing by A. Cadete.

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recorded. We also examined the number of viable and aborted seeds, number of unfertilized ovules, and the weight of seeds in each fruit (in mg).

Pollen and ovule production The production of pollen and ovules was assessed in 75 randomly collected floral buds, which were kept in ethyl alcohol (70%) until laboratory examination. Pollen grains and ovules were counted using the procedure of Ortega Olivencia & Devesa (1993). Pollen grains in the stamens of the inner and outer whorl were counted separately to distinguish possible differences between the two. Furthermore, the number of stigmas per flower was recorded to check for possible relationships between this figure and the number of ovules in the ovary.

Self-pollen loads and pollen germination We examined 33 preanthetic and 56 anthetic (bagged) flowers, which were transported to the laboratory in jars of water with aspirin (acetylsalicylic acid). The stigmas were separated from the gynoecium and each was placed on a drop of lactophenol blue on a slide for observation with a light microscope. We counted the number of (self)-germinated pollen grains in each stigma. To determine pollen viability, we analysed the germination of pollen from flowers of different ages: (A) preanthetic, (B) anthetic, approx. one-half day of age, (C) anthetic, approx. 1.5 days, and (D) post-anthetic, aged, wilted, yellow-black flowers of approx. 3 days of age. We had previously found that percentage germination was highest in a culture medium composed of boric acid (100 ppm) and 20% sucrose. Germination was considered to have taken place when the pollen tube was equal in length or longer than the diameter of the pollen grain within 3 h after sowing.

Stigma receptivity We determined stigma receptivity as the capacity of pollen grains on the stigma to germinate after hand pollination. To record the beginning and end of stigma receptivity, preanthetic flowers were marked and brought to the laboratory, where they were emasculated, bagged and distributed into four age groups as defined in the previous section. All flowers were pollinated by hand with pollen from donors of the same population, and examined 3 h later. Stigmas were removed individually, and pollen grains were counted as described above. Hand pollination of all flowers was done by the same author, who gently dragged the stamens across each stigma of the gynoecium. The mean number of pollen grains transferred to each stigma was 98.2240.4 (n  85 stigmas); this figure was not significantly different (one-way ANOVA, F  3.34, P  0.070) from natural pollen loads (x  115.7260.3 grains; n  35 stigmas).

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Breeding system Data from 1990 showed that there were no significant differences between fructification and seed set between bagged plants and plants that remained exposed to open pollination. This indicated not only the existence of selfcompatibility, but also varying degrees of selfing in the population studied. To estimate the selfing rate we used four treatments: (A) hand crosspollination, (B) hand self-pollination, (C) open pollination (control), and (D) spontaneous self-pollination. In treatment A, flowers were pollinated with pollen from several donors growing approx. 100 m away. In treatment B, pollen was obtained from another, older flower on the same plant (geitonogamous cross). In each treatment we counted the number of fruits set, the number of viable seeds per flower and per fruit, the number of aborted seeds, the number of unfertilized ovules, the total number of ovules, and weight of the seeds (in mg). When the data did not show a normal distribution, we used nonparametric tests to analyse the means (Sokal & Rohlf, 1981). Data from these treatments were used to calculate the selfing ratio according to Charlesworth & Charlesworth (1987). At the end of June 1993 and at the beginning of April 1994 we marked 30 preanthetic flowers, which were emasculated and bagged. Several weeks later we recorded the fruit and seed set in order to check the agamospermy presence. Pollinator limitation test To determine whether pollinators were a limiting factor in pollination, we used two treatments: emasculation + open pollination (1), and emasculation + hand cross-pollination (2). In treatment 2 we used pollen from several donors growing at least 100 m away, and the flowers were bagged until fructification. On April 17, April 30, May 2 and May 8 1994, we recorded all visits to D. lusitanicum flowers by potential pollinators between 8.00 and 12.00 h GMT (period of maximum activity). Total observation time was 7 h in periods of 15 min at 2-h intervals. In all, 464 flowers were examined for the presence of potential pollinators. We recorded the number of visits, the duration of each visit, and the visitor’s behaviour. RESULTS

Floral production and flowering phenology In the populations of D. lusitanicum studied, individuals began to produce inflorescences in March, with one floral scape per foliate rosette. The number of inflorescences produced depended on the number of rosettes per plant, which was indirectly related with the age of the individual (the older the individual, the greater the number of rosettes). The mean number of inflorescences per plant was 2.6922.17 (n  300 plants; data from 1990 and 1994, population A). The flowers are arranged in inflorescences of pseudocorymbose appearance,

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Early flowering

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Figure 3. Flowering and fructification phenology in D. lusitanicum. Number of flowers throughout the flowering period (*); mean temperature in population A (· · ·); number of fruits produced throughout the fruiting period (– – –). The top part of the graph shows average flower abortion per plant at three flowering periods. ***, P  0.001; a, P × 0.05. Temperature data from the Presa del Aguila Meteorological Station (Villar del Rey, Badajoz).

and have a centripetal pattern of development. The mean number of flowers per inflorescence was 5.0522.16 (n  319 inflorescences), and the most frequent numbers of flowers per inflorescence were 4 or 3. Nevertheless, at any given moment the number of open flowers per inflorescence was 1.720.95; this variable was significantly correlated (r  0.624, P ³ 0.001, n  200 plants) with the number of inflorescences per plant. The flowering period lasted from March to August (population A) (data from 1990; Fig. 3). The first flowers appeared on 24 March and the last on 19 August. However, flowering was maximal between 7 and 21 April, coinciding with mean temperatures between 14°C and 16°C (Fig. 3). Flowering was inversely correlated with mean temperature (r  −0.73, P  0.000). At

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the end of April, flower production decreased markedly, with only slight peaks around 19 May, 18 June and 2 July. Mean longevity of the flowers was 55.2420.98 h (approx. 2.3 days; n  50 flowers; data from 1990), after which time the petals become tightly furled and turned blackish, remaining on the plant until emergence of the fruit. Nevertheless, this longevity was strongly temperature-dependent, and became shorter as air temperature increased (personal observation). Flower opening was maximal between approximately 9.30 and 11.30 h GMT (air temperature 20–26°C), although that period varied depending on the temperature. Flower characteristics The attractive yellow or sulphur-yellow flowers have a mean diameter of 2.720.3 cm in anthesis (n  100 flowers). In anthesis, the corolla is bellshaped, but in the previous stage it is tubular or cylindrical, with a mean diameter of 0.9–1.2 cm. The flowers are photonastic, and became smaller (0.3–0.6 cm) and slightly twisted in the evening, in cloudy weather, in deep shade, and as they wilted. In the field and in the laboratory, we found that the sexual whorls clearly made contact, even in preanthesis. In 63.1% of the flowers, we observed fusion between the two stamen whorls: usually, the outer (longer) whorl was fused throughout the lower half of the anther, whereas the inner (shorter) whorl made contact along the upper half. In 39.9% of the flowers, we observed fusion of the inner whorl, but not the outer one. The stamens of the inner whorl are located at the same level as the stigmas, and thus made direct contact, whereas the outer whorl (located slightly higher than the stigmas) probably facilitates self-pollination by falling pollen grains. Selfpollination is also favoured by the insect visits, and by the furling of the corolla as a result of photonasty or wilting. Although D. lusitanicum flowers are usually pentameric, in both populations A and B we found many deviations from this type. In 100 randomly sampled flowers in population A, 59% showed metameric deviation; the corresponding figure in population B was 87%. At both sites, variations were noted in the numbers of petals and sepals, as well as in the number of stamens and stigmas. These variations most frequently affected only the perianth whorls, but also occurred in the sexual whorls, affecting all of them in only 6% and 4% of the flowers in populations A and B respectively. Pollen and ovule production Mean pollen grain production per flower was 14002.7, with differences between whorls, the inner whorl producing fewer (6237.3322579.9 grains) than the outer one (7685.322813.3 grains). The difference was statistically significant (One-way ANOVA, F  10.79, P  0.001). Although mean ovule production was 20.4, the difference between gynoecia with five stigmas (19.723.5) and gynoecia with six or seven stigmas (22.522.7) was also significant (one-way ANOVA, F  9.42, P  0.003). These two parameters were used to calculate the P:O ratio, 687.4, closer to that obtained by

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TABLE 1. Percentage germination of pollen in vitro (% Gv), and percentage pollen germination per stigma (stigma receptivity, % Ge) after hand cross-pollination of flowers of different ages. SD in parentheses –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Mean number Flower %Gv pollen:stigma state n  10 flowers n  10 flowers %Ge –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — A 85.53 68.95 17.05 (preanthetic) (14.1) (35.65) (11.12) n  20 stigmas B 82.9 97.52 55.28 (anthetic) (23.4) (49.47) (30.04) n  29 stigmas C 8.68 101.43 47.04 (anthetic) (6.3) (39.04) (20.8) n  28 stigmas D 3.57 95.68 35.86 (postanthetic) (2.2) (30.83) (12.33) n  28 stigmas –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– —

Cruden (1977) for facultative xenogamy (796.6287.7) than to the ratio for facultative autogamy (168.5222.1). Self-pollen loads and pollen germination In preanthetic flowers the mean number of self-pollen grains per stigma was 65.7262.8 (n  112 stigmas), of which 13.4% had germinated. Flowers in anthesis (approx. one-half day old) held a self-pollen load of approx. 83.0259.6 grains (n  66 stigmas), of which 19.35% had germinated. In only four stigmas in preanthetic flowers no pollen grains were found and in only one stigma in anthesis. Pollen was viable from the preanthetic stage (stage A) of the flower almost until wilting (stage D). However, viability was greatest in stages A and B (Table 1), and decreased rather sharply from stage C onward (approx, 1.5 days). Stigma receptivity The stigma was receptive from preanthesis (stage A, with 17.1% germination) until the corolla wilted (stage D, with 35.9% germination). However, stigma receptivity was greatest in stage B (approx. one-half day) and stage C (approx. 1.5 days) (Table 1). Consequently, Drosophyllum flowers were potentially able to self-pollinate from preanthesis until postanthesis, as pollen was viable and stigmas were receptive throughout this period. Fructification After open pollination, capsules matured to a shiny brown colour after a mean time of approximately 17.524.29 days (n  49 fruits). After ripening, a mean period of 46.9226.75 days was required for all seeds to be released,

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TABLE 2. Fructification, mean viable and unviable seed production, unfertilized ovules and weight of seeds (in mg) in normal and ‘abnormal’ flowers. SD in parentheses. The same small superscript within a column indicates no significant difference ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Fruits number Viable Aborted Unfertilized Mass percent seeds:fruit seeds:fruit ovules:fruit seed ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — a b c d 8.93 0.61 6.20 3.44e Normal 46 flowers 94% (4.78) (1.33) (5.38) (0.41) (n  49) n  41 seeds a b c d Abnormal 47 10.13 0.52 6.98 3.52e flowers 96% (4.74) (1.25) (4.59) (0.37) (n  49) n  43 seeds ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — a One way ANOVA, F  0.207, P  0.65 b Mann Whitney test, Z  1.05, P  0.29 c Mann Whitney test, Z  0.49, P  0.62 d Mann Whitney test, Z  1.12, P  0.26 e Mann Whitney test, Z  1.42, P  0.15

as the process was mediated by the progressive separation of the valves (normally five or six valves; x  5.120.30). Seeds were released passively, either simply by the force of gravity, or assisted by incidental movements of the inflorescence by external agents. In any case, the seeds fell close to the mother plant. In the 50 plants used for phenological studies, percentage fructification was 84.42% and percentage fruit abortion during development was 15.58% (data from 1990). At the individual plant level, fruit abortion occurred in 2.7623.20 flowers (n  50 plants). Although the incidence of abortion was not correlated with mean temperature in the study area (r  −0.246, P  0.325), there were significantly more abortions among flowers formed at the end of the flowering period of the taxon, in comparison with earlier flowers and flowers formed in mid-period (see Fig. 3; one-way ANOVA, F  7.55, P  0.001). Nevertheless, there was no significant difference (Fisher’s multiple comparison test) between mid-period flowers and flowers formed at the end of the flowering period. Open pollination had no affect on fructification by ‘abnormal’ (see above) flowers (Table 2), nor were there significant differences in the number of viable and aborted seeds, number of unfertilized ovules or weight of seeds in comparison with normal flowers. Breeding system Data from 1990 showed no significant differences between bagged and unbagged plants (Table 3) in number of viable seeds per fruit or per flower, or in number of aborted seeds per fruit. The results from 1994 were similar (Table 4). In that year the sample size was larger and we discriminated between viable and abortive seeds and unfertilized ovules in each fruit. In addition, we recorded seed weight. In all four pollination treatments tested in 1994, fructification was greater than 91%, with no significant difference between treatments. The viable seeds set per flower and per fruit were greatest after open pollination; the differences

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TABLE 3. Fruit and seed production in bagged and unbagged inflorescences in 1990. SD in parentheses. The same small superscript within a column indicates no significant difference ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Fruits Flowers number Viable Viable Aborted number percent seeds:flower seeds:fruit seeds:fruit ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — 10.13b 12.02c 1.03d Inflorescences 72 59a bagged 81.94% (6.15) (4.71) (1.66) (n  15 plants) Inflorescences 74 53a 9.82b 13.16c 0.98d unbagged 71.62% (7.01) (4.67) (1.64) (n  14 plants) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — a One way ANOVA, F  1.89, P × 0.05 b Mann Whitney test, Z  0.11, P  0.91 c Mann Whitney test, Z  1.22, P  0.22 d Mann Whitney test, Z  0.08, P  0.94

were significant in comparison with the two hand-pollination treatments (cross- and geitonogamic), but not when compared with spontaneous selfpollination (Table 4). The number of aborted seeds was greatest after hand geitonogamous pollination, although the difference was significant only in comparison with open pollination; also, the differences in this variable were significant between hand cross-pollination and open pollination. The number of unfertilized ovules per flower did not differ significantly between treatments, nor did seed weight. However, the total number of ovules per flower showed significant differences (Table 4), and may therefore have influenced other variables. Total ovule number showed a significant direct correlation with the number of seeds per fruit (P ³ 0.001) in all treatments, a relationship that may account for the smaller numbers of seeds formed after hand pollination. However, the inverse correlation between total ovule number and number of aborted seeds was significant only after open pollination (P ³ 0.01) and spontaneous self-pollination (P ³ 0.001), but not after either of the hand pollination treatments (P × 0.05). These results suggest that manipulation may have caused alterations in flowers. Because the values for reproductive parameters were higher after open pollination in comparison with the other three treatments, we could not estimate the selfing ratio (S) (Charlesworth & Charlesworth, 1987), as the values of S in our sample were negative. When reproductive efficiency for each treatment was calculated as the product of the quotients of Fr:F1 and S:O according to Wiens et al. (1987), the resulting values were likewise negative. The poor success of cross-pollination can be seen from the data in Table 5 (emasculation + open pollination). This treatment led to 24.7% fructification and 7.19 viable seed set per fruit, which yielded a reproductive efficiency of 11.5%. This result was clear evidence of the importance of self-pollination over cross-pollination. Neither flowers marked at the end of June 1993 nor those marked at the beginning of April 1994 produced fruits or seeds after they were emasculated and bagged. This result apparently rules out agamospermy in this species.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Flowers Fruits Viable Ovules Pollination number number Fr:F1 Viable seeds:fruit Aborted Unfertilized per fruit Seed Fr:F1×S:O treatment F1 Fr percent seeds:flower S seeds ovules:fruit O mass S:O percent ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — 11.15e,f 1.07i 3.65 15.76j,k 3.5 0.71 66.60 Hand cross103 97 94.17 10.5a,b pollination (4.49) (3.77) (1.99) (3.3) (3.1) (0.5) 9.8e,g,h 1.14o 4.21 15.14l,m 3.53 0.65 61.27 Hand geito113 107 94.69 9.28a,c,d nogamic (4.53) (4.07) (1.82) (3.11) (2.66) (0.36) 12.49f,g 0.39i,o 3.97 16.86j,l 3.48 0.74 68.91 Open polli128 119 92.97 11.61b,c nation (5.43) (4.56) (0.78) (3.44) (3.29) (0.3) d h k,m 11.72 0.78 4.37 16.87 3.49 0.70 63.52 Spontaneous 139 127 91.37 10.72 self-polli(5.58) (4.7) (1.55) (4.51) (2.96) (0.25) nation ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — h Z  3.28, P  0.0011 Mann Whitney test a Z  1.99, P  0.46 b i Z  2.49, P  0.013 Z  2.23, P  0.026 c j Z  4.07, P  0.000 Z  2.32, P  0.02 d k Z  2.49, P  0.013 Z  2.26, P  0.024 e l Z  2.24, P  0.025 Z  4.09, P  0.000 f m Z  2.93, P  0.003 Z  4.10, P  0.000 g n Z  4.84, P  0.000 Z  2.70, P  0.007

TABLE 4. Level of fructification, seed set and reproductive success (Fr:F1×S:O) after four different pollination treatments in Drosophyllum lusitanicum. Number of weighed seeds for each treatment was n  89 (hand cross-pollination), n  103 (hand geitonogamic pollination), n  115 (open pollination) and n  125 (spontaneous self-pollination). SD in parentheses. The same small superscript within a column indicates significant difference

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––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Flowers Fruits Viable Ovules Pollination number number Fr:F1 Viable seeds:fruit Aborted Unfertilized per fruit Seed Fr:F1×S:O treatment F1 Fr percent seeds:flower S seeds ovules:fruit O mass S:O percent ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — 1.77b 7.19c 1.19 7.05 15.43d 3.45 0.47 11.51 Emasculation 150 37 24.7a + open-pollination (3.77) (4.3) (2.02) (4.71) (2.39) (0.64) n  31 9.64b 9.91c 1.21 5.75 16.87d 3.53 0.59 57.1 Hand cross-pollination 73 71 97.3a (4.78) (4.56) (1.76) (3.66) (3.11) (0.59) n  64 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — a One way ANOVA, F  198.7, P × 0.001 b Mann Whitney test, Z  9.78, P  0.000 c Mann Whitney test, Z  2.85, P  0.004 d One way ANOVA, F  5.97, P  0.016

TABLE 5. Level of fructification, seed set and reproductive success (Fr:F1×S:O) after two different pollination treatments in Drosophyllum lusitanicum. SD in parentheses. The same small superscript within a column indicates significant difference

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Pollinator limitation test Table 5 shows that fructification after emasculation + open pollination (24.7%) was significantly lower than after hand cross-pollination (97.3%). The number of seeds set per flower and per fruit were also significantly lower after the former treatment. No significant differences were found between treatments for the number of aborted seeds, the number of unfertilized ovules, or seed weight. These results suggest that pollinators are limiting factors in cross-reproduction in D. lusitanicum. Although pollinators are active between approx. 7.30 and 15.00 h GMT, maximum activity was seen between 8.00 and 12.00 h GMT. We observed a total of 198 visits to 464 flowers; the mean number of visits per flower was thus 0.43. The most frequent visitor was Panurgus sp (Hymenoptera, Apoideae), which accounted for 46.5% of all visits, followed by Homaloplia ruricola (Coleoptera) (33.3%) and Eristalis tenax (Diptera, Syrphidae) (5%). Other species accounted for the remaining 14% of the visits. Visits by Panurgus sp lasted a minimum of 10–30 sec; visits by H. ruricola lasted a minimum of 2 min; and those by E. tenax, a minimum of 5–15 sec. Panurgus sp spends long periods (at times the entire afternoon or night) resting on the flower parts, and the behaviour of H. ruricola was similar, although the presence of several individuals simultaneously in a single flower suggested that this species occasionally mated in flowers. The best pollinator, however, was probably E. tenax: although less frequent, visits were briefer than those of the other two species, which showed greater preference for other flowering plants in the area, such as Cistus ladanifer and some species of the Asteraceae.

DISCUSSION

Drosophyllum lusitanicum is a suffruticose plant with a flowering period that extends from March to August, and peak floral production in April. This phenological behaviour is similar to that described for most Mediterranean species (Pacini & Franchi, 1984; Dafni & O’Toole, 1994), although many other factors besides temperature may condition the pattern of flowering (Sedgley & Griffin, 1989). Each inflorescence produced a mean of five flowers during the flowering period; the mean number of flowers per plant depended on plant age. Population A produced an average of 2.7 inflorescences per plant. Aside from the total number of flowers per individual, a more informative parameter was the number of flowers that open simultaneously (approx. 1.7), as this number may reflect the likelihood of geitonogamy. Because of the strong correlation between the number of flowers open simultaneously and the number of inflorescences per individual, geitonogamy would be expected to increase with plant age, as older plants bear more inflorescences. On the other hand, although flower production was mainly concentrated in the first 2 months of the flowering period, it covered a 5-month period; this species therefore displayed an extended flowering period, a well known characteristic in many taxa (Bawa, 1983). This extended flowering, and the fact that very few (approx. 1.7) flowers were open at any given moment,

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may represent attempts to control the resources available for flowering and fruiting (Bawa, 1983), an important strategy aimed at ensuring reproductive success in the face of environmental fluctuations. The metameric deviations observed in floral whorls with respect to the base number for typically pentameric flowers has not been reported in previous studies except in the androecium (10 to 30 stamens; Maire, 1976; Lecoufle, 1989). This phenomenon, which may be relatively frequent in some angiosperms (Heslop-Harrison, 1952; Astie´, 1962; Meyer, 1966; Huether, 1969; Briggs & Walters, 1984; Wilder, 1991), may be a consequence of this species’s type of reproduction (see below). The results (Table 2), which showed no differences between normal and ‘abnormal’ flowers in fruit or seed set, can be interpreted in two ways: (1) metameric deviations may represent adaptations aimed at making flowers more attractive to pollinators, although this has not yet to be reflected in the amount (seed set) or quality (seed weight) of the progeny; (2) the deviations may be a consequence of inbreeding in the population we studied. As in other plants, not all flowers produced become fruits (Stephenson, 1981; Bawa & Webb, 1984; Nakamura, 1986; Sutherland, 1986; Mikesell, 1988; Bawa & Buckley, 1989; Charlesworth, 1989a; Ehrle´n, 1991; Karoly, 1992; Guitia´n, 1993). Under our study conditions, floral abortion was not correlated with mean temperature, although we did note that abortion rates fluctuated throughout the flowering and fruiting periods (Fig. 3; see Lloyd, 1980; Stephenson, 1981; Bawa & Webb, 1984; Marshall, Levin & Fowler, 1985; Nakamura, 1986), with maximum values appearing from 2 July onward. This observation may be interpreted as a consequence of limitations in available resources, as the middle and the end of the flowering period coincide with fruit maturation and seed release from the earliest flowers formed. Studies in other species have yielded many hypotheses to explain seed abortion and unfertilized ovules. Limitations in pollinators or resources (Lloyd, 1980; Stephenson, 1981; Lee & Bazzaz, 1982; Bawa & Webb, 1984; Marshall et al., 1985; Nakamura, 1986) and genetic loads (Wiens et al., 1987; Charlesworth, 1989a,b, but see also Bawa, Hedge & Uma Shaanker, 1989) have been suggested, although none of these hypotheses was tested in our study. However, the pollinator limitation test (Table 5) failed to reveal significant differences between the two treatments in number of aborted seeds or unfertilized ovules, so the pollinator limitation hypothesis cannot account for seed abortion. Limitation in resources appears an unlikely explanation, as treatments were done in April, during a time when the plants devote more energy to flowering than to the formation of the as yet scarce fruits. There is clear evidence that D. lusitanicum flowers use self-pollination from very early moments of flower formation. In preanthetic flowers, the stigmas bear large numbers of self-pollen grains, and the pollen itself shows germinative capacity even before the corolla unfolds (anthesis). Self-pollen reaches the stigmas of a given flower not only as a result of direct contact between the two sexual whorls in anthesis, but also as a result of the tube-like shape of the corolla during preanthesis. Moreover, as in other plants, self-pollination in D. lusitanicum also results from the repeated opening and closing of the corolla (Faegri & Van der Pijl, 1979) due to photonasty. Pollen grain viability

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and stigma receptivity coincide with flowering (approx. 2.3 days), beginning in preanthesis and ending when the corolla wilts. Further evidence of the importance of self-pollination in this population was reflected in the high percentages of fructification and seed set in inflorescences bagged in 1990 and 1994 (Tables 4 and 5). The data suggest that these plants are not dependent on pollinators. The high S:O and Fr:F1 ratios (see Table 4, open pollination) are more characteristic of inbreeders than outbreeders (Wiens, 1984; Molau, 1993), as is the product of the two ratios or pre-emergent reproductive success (Wiens et al., 1987). We found no significant differences between the four pollination treatments in seed weight, although the greatest numbers of viable seeds and lowest numbers of aborted seed were obtained after open and spontaneous selfpollination. These results may have been influenced by the fact that these flowers had more ovules than those subjected to either of the hand pollination treatments. The higher rate of seed abortion after hand treatments (crossand geinotogamic) may have resulted from manipulation of the flowers. In hand cross-pollination, the distance between the crossed plants (approx. 100 m) may also have affected the results, as crossing distance is correlated with genetic similarity between the parents (Waser & Price, 1993). The geitonogamous cross may also have been affected by pollen transfer, as the donor pollen was from older flowers, and so with less viable pollen grain. The data we obtained from our pollination treatments did not allow us to calculate the selfing ratio with the formula proposed by Charlesworth & Charlesworth (1987). However, emasculation + open pollination in 150 flowers (Table 5) led to low fruit and seed production, which was translated indirectly as a considerable level of selfing. The P:O ratio of 687.4 places this species in an intermediate position between facultative autogamy and facultative xenogamy (Cruden, 1977). However, the best estimate of the level of selfing in this species must be obtained by analysis of isoenzymes in the study population as well as in additional populations, given the likelihood of interpopulation variations in plant species that use mixed reproduction (Schoen, 1982; Ayre, Whelan & Reid, 1994). Two arguments support the predominance of self-pollination over crosspollination in D. lusitanicum. Firstly, at times when (presumably effective) pollinators are most active, Drosophyllum flowers contain large amounts of self-pollen, part of which has already germinated. The potential cross-pollen would thus be unlikely to supersede self-pollen in amount or germination time. In other self-compatible plants, no evidence has been found that selfpollen is less successful than cross-pollen in fertilization (Walsh & Charlesworth, 1992) and even Johnson & Mulcahy (1978) reported that in maize, the fertilization capacity of self-pollen increased with the number of selfing generation. Moreover, the main vectors in Drosophyllum also help increase the amount of self-pollen on the stigmas during their visits and movements within the flower. Secondly, pollinators were limiting factors, as shown by the low rates of cross-pollination in this population (Table 5). This limitation was probably conditioned by the number and efficiency of available pollinators. Insects that spend a long time visiting each flower or inflorescence are probably more important for selfing, since they visit fewer plants (McMullen & Naranjo,

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1994). In addition, both H. ruricola and Panurgus sp tended to prefer species such as C. ladanifer with more rewards than Drosophyllum (Talavera, Gibbs & Herrera, 1993), and some species of Asteraceae that flowered at the same time as Drosophyllum. Interspecific competition, together with the low number of visits per flower and the prolonged duration of some visits (lasting the entire afternoon or overnight, especially in windy, rainy or cloudy weather) reflected the nonspecific nature and relative unimportance of insects as pollinators of this species. In the population we studied, E. tenax, a pollenivorous species ( Jordano, 1993; Guitian, 1993) that makes short visits (5–15 sec) was generally rare in our study area, but was especially abundant in a subplot that contained many old plants (with more flowers open simultaneously); thus possible instances of cross-pollination in this subplot probably also comprised some level of geitonogamy. It is nonetheless difficult to explain the clear evidence for the importance of self-pollination in the light of the pattern of extended flowering with few flowers per inflorescence, which may represent an adaptation to reduce geitonogamy and increase xenogamy. If autogamic reproduction is a derived condition (Stebbins, 1957), this phenologic behaviour may be evidence (or a vestige) of a cross-pollination more important in the past. Autogamy is common in many other members of the Droseraceae (Mu¨ller, 1883; East, 1940; Fryxell, 1957), regardless of the importance other asexual mechanisms of reproduction may have (Ridder & Dhondt, 1992 a, b). The production of large, attractive flowers may likewise be a vestige of cross-pollination. The different metameric deviations found in populations A and B are probably a consequence of inbreeding depression. Levin (1984) noted that the effects of inbreeding depression were often pronounced during seed development, and although we had significant differences in seed set after geitonogamic and xenogamic treatment, there were no differences in the numbers of aborted seeds or unfertilized ovules. In similar studies, Karron (1989; Astragalus spp) and Schoen (1983; Gilia achilleifolia) found no significant differences in abortion rates. Because the absence of inbreeding depression in early stages of the life cycle does not necessarily mean the absence in later stages (Waller, 1984; Karron, 1989), further studies will need to examine the life cycle of Drosophyllum in detail. Moreover, inbreeding depression is not necessarily absent in species with high levels of autogamy (Charlesworth & Charlesworth, 1987; Karron, 1989; Molina-Freaner & Jain, 1993). The type of seed dispersal may help explain the usually small area occupied by populations of Drosophyllum. The presence of this species on both sides of the Straits of Gibraltar can only be explained with reference to the natural history of the western Mediterranean region (see Messinian Model; Bocquet, Widler & Kiefer, 1978). Many plant species show a distribution similar to that of Drosophyllum. Because we found no evidence of asexual reproduction, and because the agamospermy tests were negative, most of our evidence suggests that in the population of Drosophyllum lusitanicum we studied, selfing is the most important mechanism of sexual reproduction. This species currently displays facultative autogamic behaviour. Although xenogamy cannot be ruled out, it is probably of little importance given to nonspecific nature of the pollinators and the scarcity of their visits.

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ACKNOWLEDGEMENTS

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