Morph-specific differences in reproductive success in the distylous Primula veris in a context of habitat fragmentation

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a c t o e c

Original article

Morph-specific differences in reproductive success in the distylous Primula veris in a context of habitat fragmentation Fabienne Van Rossum*, Sara Campos De Sousa1, Ludwig Triest Plant Biology and Nature Management, Vrije Universiteit Brussel, Pleinlaan 2, BE-1050 Brussels, Belgium

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Article history: Received 22 April 2006 Accepted 20 June 2006 Available online 11 July 2006 Keywords: Distyly Floral morph Habitat fragmentation Pin reproductive advantage Pollination failure Population size Primula veris Seed abortion

Heterostylous self-incompatible plant species are particularly sensitive to habitat fragmentation and to disruption of pollination processes because of the need of intermorph cross-pollination for producing seeds. Heterostyly is characterized by sexual polymorphism through the occurrence of two (distyly) or three (tristyly) morph types that differ in floral traits (style length and anther position). We examined whether the long-styled (pin) and short-styled (thrum) morph types show differences in reproductive components and responses to habitat fragmentation in the distylous, self-incompatible perennial herb Primula veris. We documented reproductive components for pin and thrum individuals and their relationships with population size, plant density and morph ratio (pin frequency), in nine populations from Flanders (northern Belgium) located in fragmented habitats of the intensively used agricultural landscape. Seed abortion increased in small populations as a result of inbreeding depression. Fruit set increased with plant density. Seed set was positively related to pin proportion. Seed set was higher for pin than thrum in small populations, but lower in large populations. Two hypotheses can be considered to explain these morph-specific differences: a pollen transfer asymmetry, and a reproductive advantage for the partially self-compatible pin morph. Morph types appear to respond differently to habitat fragmentation constraints. A floral morph type showing partial selfcompatibility may be favored in populations under pollination failure, because it can increase reproductive success and mating opportunities through intramorph crosses. © 2006 Elsevier Masson SAS. All rights reserved.

1.

Introduction

Heterostylous self-incompatible plant species are characterized by sexual polymorphism, i.e. by two (distyly) or three

* Corresponding author. Present address: Department of Vascular Plants, National Botanic Garden of Belgium, Domein van Bouchout, B-1860 Meise, Belgium. Tel.: +32 2 269 3905; fax: +32 2 260 0945. E-mail address: [email protected] (F. Van Rossum). 1 Present address: Social, Genetic and Developmental Psychiatry Center (MRC), Institute of Psychiatry, PO 80, DeCrespigny Park, London SE5 8AF, UK.

(tristyly) genetically inherited floral morph types that vary in style length and anther position (herkogamy), and by genetic mechanisms that prevent self- and intramorph fertilization. These traits promote intermorph pollen transfer (disassortative mating), and therefore enforce an almost 100% outcrossing rate. Moreover, the resulting negative frequency-dependent selection contributes to a stable equilibrium state of equal morph frequencies (isoplethy) in populations (Barrett, 1992). Optimal compatible pollen availability and reproductive success are expected in populations with a 1:1(:1) morph ratio (Washitani, 2000; Kéry et al., 2003; Shibayama and Kadono, 2003).

1146-609X/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2006.06.005

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Beside herkogamy, the morph types can also differ in plant size and survivorship (McCall, 1996; Boyd et al., 1990), clonal growth (Mal and Lovett-Doust, 1997), pollen size and amount (e.g. Ornduff, 1980; Richards, 1997), reproductive success (Ågren and Ericson, 1996; Nishihiro et al., 2000), pollen transfer, and pollinator type and foraging behavior because of varying pollen accessibility and/or production (Wolfe and Barrett, 1987; Baker et al., 2000; Matsumura and Washitani, 2002). They can also differ in self- and intramorph compatibility levels (Barrett et al., 1989; Husband and Barrett, 1992a). These morph-specific differences can lead to assortative mating, skewed morph ratios (morph anisoplethy), spatial segregation of the morphs, morph gender specialization, morph niche differentiation and/or to differential responses of the morph types to less favorable ecological conditions (e.g. Levin, 1974; Barrett et al., 1989; Eckert and Barrett, 1995; McCall, 1996; Mal and Lovett-Doust, 1997; Larson and Barrett, 1998; Matsumura and Washitani, 2002; Kéry et al., 2003). Habitat fragmentation represents highly constraining conditions for heterostylous self-incompatible plant species, in particular when they are insect-pollinated (Wilcock and Neiland, 2002; Jacquemyn et al., 2003; Oostermeijer et al., 2003; Waites and Ågren, 2004). It consists of the subdivision of large continuous populations into small isolated remnants and in the disruption of pollination processes—and thus of gene flow—as a consequence of reduced pollinator abundance and changing patterns of foraging behavior. These can lead to pollen limitation and/or increased inbreeding, and consequently to reduced reproductive success and inbreeding depression (Kwak et al., 1998; Wilcock and Neiland, 2002; Oostermeijer et al., 2003). Moreover, small populations of heterostylous species also often show skewed morph ratios, as a result of demographic stochasticity and genetic drift (e.g. Eckert and Barrett, 1992; Husband and Barrett, 1992b; Endels et al., 2002; Kéry et al., 2003). The resulting insufficient availability of compatible mates can reinforce the negative effects of small population size and increase the extinction risk of small populations (Washitani, 1996; Shibayama and Kadono, 2003; Oostermeijer et al., 2003). Depending on their specificities, morph types can be expected to differently respond to habitat fragmentation. For instance, morph-specific differences in self- and intramorph compatibility can confer a reproductive advantage under poor pollination service for the partially self-fertile morph because of increased seed output through intramorph assortative mating (Barrett et al., 1989; Husband and Barrett, 1992a; Matsumura and Washitani, 2000). However it can also lead to higher inbreeding levels (Barrett and Husband, 1990; Eckert and Barrett, 1994) and inbreeding depression (Glémin et al., 2001; Mateu-Andrés and Segarra-Moragues, 2004). Only a few studies investigated reproductive responses to habitat fragmentation at the morph level (Matsumura and Washitani, 2000, 2002; Kéry et al., 2003; Waites and Ågren, 2004). Little is still known whether morph specificities can contribute to reduce or increase reproductive success in small populations from fragmented habitats. We studied reproductive success of Primula veris, a distylous self-incompatible perennial herb. In this species, the two flower morphs (pin or thrum) are coded as a “supergene”

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complex (Lewis and Jones, 1992). Pin plants are recessive homozygous (ss), and thrum plants are dominant heterozygous (Ss). Only legitimate crosses are expected to produce viable seeds (Richards, 1997). However, intrapin selfincompatibility is not totally strict in P. veris: under illegitimate hand-pollination, 14.5% and 0.6% of the ovules set viable seeds for intrapin and -thrum crosses, respectively, against 75.7% for intermorph crosses (Wedderburn and Richards, 1990). The offspring of a legitimate cross is expected to be half thrum Ss and half pin ss, whereas illegitimate pin × pin crosses and pin selfing only produce pin seedlings. This may contribute to a reproductive advantage of pins (more seeds produced) under legitimate pollination disruption. Over time, this may lead to a demographic advantage for the pin morph (increasing pin frequency), if the produced illegitimate offspring can establish, i.e. if they do not suffer from inbreeding depression (Barrett and Husband, 1990; Eckert and Barrett, 1995; Kéry et al., 2003; Van Rossum and Triest, 2006a). In Belgium, P. veris is common, but in Flanders (northern Belgium) it shows a scattered distribution and occurs in habitats that have been highly fragmented over the last decades, as a result of the intensification of agriculture and abandonment of the traditional management practices (Brys et al., 2003; Van Rossum et al., 2004). There, small, isolated populations suffer from genetic erosion (Van Rossum et al., 2004), reduced reproductive success due to pollination limitation, and morph anisoplethy (Brys et al., 2003). A positive relationship was also found between pin proportion and withinpopulation inbreeding coefficient, which suggests the existence of intrapin outcrosses in populations occurring in fragmented habitats (Van Rossum and Triest, 2006a). In this paper, we quantified reproductive success of pin and thrum morphs, in nine populations located in fragmented habitats of the intensively used agricultural landscape, and addressed the following questions: 1) Do pin and thrum morphs show differences in reproductive fitness components? 2) What are the relationships between morph fitness components and population demographic traits (population size, plant density, pin frequency)? We discuss the implications of our findings for conservation of heterostylous plant species in fragmented habitats.

2.

Materials and methods

2.1.

Study species

P. veris (Primulaceae) is a diploid, long-lived perennial rosette-forming herb. It occurs in dry to mesic calcareous grasslands and forest edges, but also in field and pasture boundaries. It is distributed from Spain to eastern Asia (Hegi, 1975). In April–May, it produces umbels with yellow flowers, which are primarily pollinated by Hymenoptera (mostly bumblebees), Lepidoptera and Diptera (Ornduff, 1980). The pin and thrum morphs differ in flower morphology: pin plants are long-styled and their anthers are attached within the corolla tube, whereas in thrum plants the style is

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short and located within the corolla tube and anthers are positioned above the style, at the mouth of the corolla. They also differ in pollen size and amount, with pin pollen grains smaller and about 2.4 times more numerous than thrum pollen (Ornduff, 1980). Vegetative propagation by rhizome branching appeared to be restricted to short distances (Van Rossum and Triest, 2006b), forming clusters of ramets easily identifiable on the field.

2.2.

Studied populations and demographic characteristics

The study was carried out in nine populations from the Westhoek region, in northwestern Flanders (51°05′N, 2°40′E; for more details, see Van Rossum et al., 2004). A population was defined as a group of individuals separated from the others by unsuitable habitat conditions for the species, such as intensive pastures and fields. In this region, an intensively used polder landscape, habitats are highly fragmented, populations of P. veris are scattered, with a density of 0.33 populations per km2 (Brys et al., 2003; Van Rossum et al., 2004). The geographical distance between two populations ranged from 0.32 to 12.58 km. Population size corresponded to the number of flowering individuals (genets) and ranged from 18 to 300 flowering individuals. Within-population plant density (= number of flowering plants m–2) was estimated from total population size and total population area measured on the field, and varied from 0.07 to 9.29 (Van Rossum et al., 2004). Morph type (thrum or pin) was noted for all flowering individuals (for a random sample ≥ 100 individuals in large populations) to calculate morph frequency. Only three individuals were found to be homostyle. The frequency of pin individuals in populations varied from 37.8% to 53.3%.

2.3.

Sampling procedure and measure of reproductive components

Depending on population size, reproductive traits were measured for nine to 27 individuals across the whole population area. During the flowering season, for each individual plant (total n = 134 individuals), we noted the morph type and counted the number of flowers. During the fruiting season, we counted the number of flowers developing fruits and collected up to five still closed fruits. For each fruit, the number of mature and aborted seeds was counted. Unfertilized ovules were also counted for eight populations (total N = 109), which allowed us to calculate the total number of ovules per flower as the sum of unfertilized ovules plus aborted and mature seeds. Fruit set and seed set were calcu-

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lated as the proportion of flowers developing fruits and as the proportion of ovules setting seeds, respectively.

2.4.

Data analysis

We used a mixed model two-way analysis of covariance (ANCOVA) to examine whether reproductive traits (fruit set, number of ovules per flower, seed set, number of mature and aborted seeds per fruit) differed between pin and thrum individuals and between populations, and whether the differences could be related to population demographic traits. Population and morph by population interaction (population × morph type) were entered as a random effect, and morph type as a fixed factor in the analysis. Population size, plant density and the frequency of pin individuals in the population (pin proportion) were used as covariates. Because several reproductive traits were influenced by floral display and/or by previous stages in the reproductive cycle (Table 1; see also Brys et al., 2003), the following components were used as covariates to distinguish these relationships from the tested effects: number of flowers per plant and/or fruit set for fruit and seed variables, and number of ovules per flower for seed set. The effects of population and individual covariates were tested against the variation among populations and against the residual (error) variation among plants, respectively. The degrees of freedom of the error terms were adjusted for statistical dependence using the Satterthwaite method. When covariate(s) had a significant effect, Pearson’s correlation coefficient, univariate and/or multiple regression analysis (with forward selection procedure) were calculated between the variable(s) and the tested component. When morph by population interaction (population × morph type) and/or pin proportion effect were found to be significant, we compared pin and thrum morphs by performing a test of homogeneity of slopes and mixed model regression analyses for the pin and thrum individuals separately, with population ID entered as a grouping factor and population size, plant density and pin proportion as independent variables. The variables were transformed (log- or angular transformation) to achieve homoscedasticity and normality when it was necessary. The normality was tested on the variables by a Kolmogorov–Smirnov test, and homoscedasticity was checked by a visual examination of the plots of the standardized residuals as a function of the regression standardized predicted value (Osborne and Waters, 2002). All analyses were performed with STATISTICA (version 7).

Table 1 – Pearson’s correlation coefficients between reproductive components (see text and Table 2 for details and sample sizes, respectively) Variable

Flowers per plant

Fruit set Ovules per flower Seed set Mature seeds per fruit Aborted seeds per fruit * P < 0.05, ** P < 0.01, *** P < 0.001.

0.019 0.356*** 0.109 0.275** –0.021

Fruit set

0.142 0.187 0.341*** 0.034

Ovules per flower 0.314*** 0.707*** 0.080

Seed set

0.822*** 0.001

Mature seeds per fruit

–0.205*

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3.

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429

Results

Several reproductive components were positively related to former stages of the reproductive cycle (Table 1). Amongst others, seed set was positively correlated with the number of ovules per flower (Pearson’s correlation coefficient r = 0.314, P = 0.001). Based on a sampling of nine populations, no significant (P > 0.05) relationship was found between population size, plant density and pin proportion (r ranging from –0.498 to 0.409). The two-way ANCOVA (Table 2) revealed significant differences among populations (P < 0.05) for the number of mature seeds per fruit. There were significant relationships between several reproductive components and population variables: the number of aborted seeds was negatively related to population size (F1,7 = 6.57, P = 0.039) (Fig. 1A). Seed set was positively related to pin proportion (F1,6 = 44.22, P < 0.001; partial regression coefficient β = 0.826, P = 0.016) (Fig. 1B). There was also a positive relationship between plant density and fruit set (F1,8 = 7.05; r = –0.700, P = 0.036) (Fig. 1C). No significant difference (P > 0.05) was found between pin and thrum morphs for the studied reproductive traits when based on a sampling of nine populations (results not shown). The ANCOVA (Table 2) also indicated a (marginally) significant interaction between population and morph for the total number of ovules per flower and for seed set (F6,67 = 2.63, P = 0.024; F6,66 = 2.21, P = 0.053, respectively). The homogeneity of slopes test revealed that seed set in pins and thrums responded differently to changes in population size (F1,98 = 4.78, P = 0.031), but this was not the case (P > 0.05) for the number of ovules. No significant (P > 0.10) regression was found between population size and pin and thrum seed set (r = –0.318 and 0.563, respectively), but when analyzing the within-population difference in seed set between morphs in

Table 2 – Mixed model two-way ANCOVA of the effects of population and morph type on reproductive success, with population size, plant density, pin proportion and reproductive components (see text for details) used as covariates. Effect: direction (positive or negative) of the effect for the covariates. Only effects with P < 0.10 are shown Variable

Source

Effect

df

F

Fruit set (n = 134) Ovules per flower (n = 109)

Plant density Flowers per plant Population × morph type Pin proportion Ovules per flower Population × morph type Flowers per plant Fruit set Population Population size

Positive Positive

1, 8 1, 25

7.05* 7.75**

6, 67

2.63*

1, 6 1, 43

44.22*** 7.18**

6, 66

2.21(*)

Positive

1, 56

10.70**

Positive

1, 40 5, 8 1, 7

7.95** 13.33*** 6.57*

Seed set (n = 109)

Mature seeds per fruit (n = 131)

Positive Positive

Aborted seeds Negative per fruit (n = 131) n sample size, df degrees of freedom, (*) 0.05 < P < 0.10, * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 1 – Relationship between (A) population size (logtransformed) and number of aborted seeds per fruit ± S.E.; (B) pin proportion and seed set (%, ± S.E.); (C) between plant density (log-transformed) and fruit set (percentage of flowers setting fruits, ± S.E.) in nine and eight populations of P. veris (r = Pearson’s correlation coefficient, R = quadratic coefficient of determination of the univariate regression model, β = standardized partial regression coefficient of a multiple regression, with population size and rosettes per plant as selected predictor variables with pin proportion and number of ovules per plant as selected predictor variables).

relation to population size, the relationship was significantly negative (r = –0.905, P = 0.002), the seed set values tending to be higher for pin than thrum individuals in small populations (flowering population size N < 60), whereas the inverse trend was observed in large populations (N > 100) (Fig. 2).

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et al., 2003). When restricting the range of population size to 18–300 as done in the present study, values of fruit set (~50–80%) and seed number (~20–40) reported by Brys et al. (2003) are similar to what we found, and the relationship with population size is also no longer significant. This indicates that the reproductive failure due to pollination limitation observed in P. veris in fragmented habitats (Brys et al., 2003; see also Kéry et al., 2000) mostly occurs at extremely small populations sizes. Nevertheless we found that fruit set tended to increase with plant density. Plant density influences pollination patterns and pollinator behavior. Visitation rates can be lower at low than at high plant densities, and result in fewer flights between neighbors, lower visitation rates of plants, and therefore in reproductive failure (Richards, 1997; Wilcock and Neiland, 2002). In fragmented habitats, small populations often tend to have lowered plant densities (Kwak et al., 1998). Even if no significant relationship was found between population size and plant density (r = 0.344, P > 0.10) in the Westhoek region (Van Rossum et al., 2004), we can expect a higher risk of reproductive failure for small populations when they are also sparse.

Fig. 2 – Relationship between population size (logtransformed) and (A) seed set (%, ± S.E.) of pin (○) and thrum (●) morphs and (B) the difference in seed set (%) between pin and thrum (P–T) in eight populations of P. veris. (A) r = –0.318 and 0.563, respectively, P > 0.10; (B) r = –0.905, P = 0.002.

4.

Discussion

As previously found for several heterostylous plant species (e.g. Washitani et al., 1994; Ågren, 1996; Van Rossum et al., 2002; Brys et al., 2004), the present study revealed that the variation of population demographic traits related to habitat fragmentation processes significantly influenced reproductive fitness components: the number of aborted seeds decreased with population size. Seed abortion can result from a limitation in the maternal resources available for fruit maturation and/or from inbreeding depression in the seeds produced by selfing or biparental inbreeding (Charlesworth, 1989; Eckert and Barrett, 1994; Glémin et al., 2001). Inbreeding depression was reported at early stages of plant development in small populations of P. veris (Kéry et al., 2000). In our study, no significant relationship was found between population size and fruit set or seed production. However, when considering a larger number of populations of P. veris from Westhoek (n = 26), including also the extremely small and larger ones (N ≥ 3 up to 1470 flowering individuals), these relationships were significantly positive (Brys

More surprising are the findings of the positive relationship between seed set and pin frequency and the morphspecific response of seed set to population size. Two hypotheses may be considered to explain these results. First, as a result of reciprocal herkogamy, the pin stigmas and thrum anthers are more accessible to pollinators than the pin anthers and thrum stigmas (Husband and Barrett, 1992a; Nishihiro et al., 2000). Pin plants may be more efficient pollen recipients than thrums, whereas due to their anther position thrums may rather function as pollen donors. Higher seed set can therefore be expected for the pin morph (Casper, 1992). Contrary to expectations, the present results showed a trend towards higher seed set in large populations for the thrum morph, suggesting that the thrum morph may be more female-biased and the pin morph may be rather a pollen donor. However, in small populations, seed set is lower for thrums than for pins, suggesting that pollen transfer asymmetry may shift when there is a change in pollination frequency, the pin becoming more female-biased under pollination limitation (Beach and Bawa, 1980; González et al., 2005). Second, morph-specific differences in partial selfcompatibility can confer a reproductive advantage for the most self-fertile morph under disrupted pollination: the reproductive output of the most self-fertile morph can be maintained through illegitimate (intramorph) fertilization, whereas reproductive failure can be expected for the strongly self-incompatible morph (Barrett, 1992; Nishihiro et al., 2000). Intramorph crosses can lead to higher selfing and biparental inbreeding levels, and therefore to inbreeding depression, but can also produce outcrossed progeny when individuals are unrelated (Barrett et al., 1989; Matsumura and Washitani, 2000; Glémin et al., 2001; Mateu-Andrés and SegarraMoragues, 2004). Previous studies on genetic variation of a larger set of P. veris populations in fragmented habitats from Flanders (Van Rossum and Triest, 2006a, b) showed a negative relationship between pin proportion and population size (r = –0.546, P = 0.043), lower Wright inbreeding coefficient (FIS)

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values with increased pin proportion, and a higher fine-scale spatial genetic structure for pin than thrum morphs, suggesting the existence of intramorph crosses for the pin but not for the thrum morph. Our present results on P. veris indicate that seed set is higher for pin than thrum in small populations, and that it increases with pin-biased morph ratio. Moreover, inbreeding depression has been observed in selfed pin offspring of P. veris (Darwin, 1876), which suggests that their recruitment and survival may be restricted (Eckert and Barrett, 1994), and that only intrapin crosses might produce viable seeds and seedlings. We may therefore hypothesize a pin reproductive advantage in P. veris populations under pollination limitation in fragmented habitats. Investigating additional populations with extreme morph bias would contribute to strengthen our findings. The observed fitness patterns might also reflect a combination of interacting factors rather than a differential response to a single factor, as suggested by the nonlinear relationship between seed set and pin proportion (Fig. 1b), by the variation of seed set among populations, and by the population by morph interaction for the number of ovules per fruit that could not be related to the studied demographic factors. Stochastic factors, such as demographic stochasticity, genetic drift and temporal environmental stochasticity, may randomly modify morph frequencies, population flowering patterns and plant–pollinator interactions, and therefore affect reproductive success, especially in extremely small populations (Matsumura and Washitani, 2000; Washitani, 2000; Kéry et al., 2003; Oostermeijer et al., 2003). Moreover, morph-specific selection can be highly localized and variable among populations (Eckert and Barrett, 1995; Mal and Lovett-Doust, 1997). Indeed, there may be among-site variability of a broad range of abiotic (e.g. soil moisture and nutrients, climate) and biotic factors (e.g. vegetation, abundance of reward-producing flowering species, pollinator abundance, predation) (Kwak et al., 1998; Larson and Barrett, 1998; Waites and Ågren, 2004). Individual plant traits may also influence plant reproduction: vegetative plant size, on which depends the availability of nutrient resources; floral display, which represents the attractive signal to pollinators; and genetic factors, such as levels of genetic variability, inbreeding, and/or self-compatibility (e.g. Casper, 1992; Kwak et al., 1998; Oostermeijer et al., 2003; Brys et al., 2004). In conclusion, habitat fragmentation, through small population size and disruption of the pollination processes, seems to have created an opportunity for morph-specific fitness differences to be expressed, the morph types responding differently to these constraints. Moreover, being partially self-compatible may represent a selective advantage for a morph in a context of habitat fragmentation, because it can increase reproductive success and mating opportunities through intramorph crosses (Barrett, 1989; Husband and Barrett, 1992a). However, depending on demographic features (population size, plant density), there may also be a risk of inbreeding depression through higher levels of illegitimate biparental inbreeding (Glémin et al., 2001; Mateu-Andrés and Segarra-Moragues, 2004; Van Rossum and Triest, 2006b). On the long term, morph types might attain frequencies that would reflect their relative fitness (Eckert and Barrett, 1995),

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provided that populations, in particular in the small ones, do not incur high disturbance due to intensive agricultural practices (e.g. spread of herbicides and partial destruction of the verges where the populations occur). Indeed, this may lead to a stochastic loss of individuals (demographic stochasticity), and therefore to a random change of morph frequencies in the populations (Husband and Barrett, 1992b; Kéry et al., 2003). Our study highlights the need for further thorough investigation on morph-specific reproductive and mating processes in heterostylous species in a context of habitat fragmentation, and their consequences on population dynamics and viability, for developing appropriate conservation strategies.

Acknowledgements We thank P. Endels for population location on the field, and two anonymous referees for comments on the manuscript. This work was funded by the Ministry of the Flemish Community (contract VLINA 98/03), the Vrije Universiteit Brussel (OZR Funding) and the European Programme Erasmus (S.C.D.S.).

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