Pollinators, mates and Allee effects : the importance of self-pollination for fecundity in an invasive lily

Pollinators, mates and Allee effects: the importance of self-pollination for fecundity in an invasive lily

James G. Rodger*

¹

, Mark van Kleunen

²

and Steven D. Johnson

¹

1

Centre for Invasion Biology, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg 3209, South Africa;

and

²

Ecology, Department of Biology, University of Konstanz, Universit€ atsstrasse 10, D-78457 Konstanz, Germany

Summary

1. Ability to self-fertilize is correlated with invasiveness in several introduced floras, and this has been attributed to its mitigating effect on fecundity when pollinator visitation and mate availability are inadequate. Cross-pollination opportunities are expected to be most limited in isolated individuals and small populations, both typical of the leading edge of an invasion.

Thus, self-pollination may promote invasion in part by mitigating pollen-limitation Allee effects.

2. We used emasculation and pollen supplementation experiments to test whether the impor- tance of self-pollination for fecundity increased as plant abundance decreased and isolation increased, in the hawkmoth-pollinated and autonomously self-pollinating invasive lily Lilium formosanum, in its introduced range in KwaZulu-Natal, South Africa. As inbreeding depres- sion is negligible in these populations, seed production through selfing is likely to be demo- graphically important.

3. In naturalized populations of L. formosanum, varying in size and degree of isolation, emasculation reduced seed production by two-thirds, indicating strong reliance on self-fertiliza- tion for fecundity due to inadequate pollinator visitation. However, this was not related to population size and was only greater for more isolated populations in one of the 3 years in which the experiment was carried out. Pollen supplementation experiments showed that pollen limitation was low – 12% on average – and significant in only one of 3 years, demonstrating that autonomous self-pollination was highly effective.

4. In artificial arrays, consisting of plants placed inside naturalized populations or in pairs iso- lated (3 – 702 m) from populations, the effect of emasculation on fecundity was greater in iso- lated plants than those inside the population in one of two populations. Isolation reduced fecundity when emasculated plants were placed next to a second emasculated plant, but not when emasculated plants were partnered with an intact plant, from which they could receive pollen.

5. We conclude that self-fertilization in L. formosanum compensates for inadequate pollinator visitation across all levels of population size and for a pollen-limitation Allee effect due to decreased mate availability in isolated plants, and may thus play an important role in invasion.

Key-words: abundance, aggregation, Baker’s Law, biological invasion, plant breeding systems, pollen limitation, reproductive assurance, Sphingidae

Introduction

Insufficient fecundity may prevent invasion entirely or reduce rate of spread of introduced species (Parker 1997), especially when it arises as an Allee effect (Veit & Lewis 1996; Leung, Drake & Lodge 2004; Taylor

et al.

2004). An Allee effect occurs when low abundance reduces fecundity

or any other aspect of performance, often resulting in pop ulation growth rate becoming negative (Stephens, Suther land & Freckleton 1999). Inadequate pollen receipt (pollen limitation) is a common cause of Allee effects in plants that cannot self fertilize (Knight

et al.

2005; Gascoigne

et al.

2009). One of the main reasons for this is that smal ler, sparser and more isolated patches of plants are less likely to be discovered by animal pollinators and, if discov ered, are less profitable for foraging (Sih & Baltus 1987;

*Correspondence author. E mail: rodgerjg@gmail.com

http://dx.doi.org/10.1111/1365-2435.12093

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-242496

Feinsinger, Tiebout & Young 1991; Agren 1996; Groom 1998). Moreover, when a plant species occurs at very low density, pollinators are likely to carry less or none of its pollen, even if visitation per plant is not reduced (Duncan

et al.

2004). Thus, ability to self pollinate may enhance invasiveness in plants by mitigating Allee effects.

Ability to self fertilize generally reduces or eliminates pollen limitation (Kalisz, Vogler & Hanley 2004; Knight

et al.

2005; Eckert, Samis & Dart 2006; Brys

et al.

2011).

Ability to self fertilize should provide an ecological (and evolutionary) advantage through increased reproduction, so long as the benefits of selfing in terms of fecundity (reproductive assurance benefits) outweigh the costs of inbreeding depression. Inbreeding depression occurs when progeny arising from selfing performs less well than those from outcrossing (Jain 1976). As self fertilization reduces the availability of ovules and pollen for outcrossing, less fit selfed progeny may be produced at the expense of fitter outcrossed progeny (gamete discounting; Lloyd 1992; Her lihy & Eckert 2002). As a result, ability to self fertilize is most likely to be advantageous when inbreeding depres sion and opportunities for outcrossing are both low.

Herbert Baker proposed that plants that can self fertilize should be better colonists than those that cannot, because selfing would allow single individuals, isolated from mates and pollinators by long distance dispersal, to found new populations (Baker 1955, 1967). This principle, known as Baker’s law or rule, has subsequently been expanded to include invasive species. As introduced plants have to reproduce successfully at low abundance along the leading edge of an invasion, reproductive assurance through self fertilization should increase their likelihood of becoming invasive. In other words, selfing may contribute to inva siveness by mitigating pollen limitation Allee effects (van Kleunen, Fischer & Johnson 2007; Ward, Johnson &

Zalucki 2012). This idea is consistent with evidence that ability to self fertilize is positively correlated with invasive status and size of invaded range among introduced species in several floras (van Kleunen & Johnson 2007; van Kleun en

et al.

2008; Hao

et al.

2011; Py sek

et al.

2011).

As invasive plants are frequently visited by pollinators in the novel range (Richardson

et al.

2000; Memmott &

Waser 2002; Py sek

et al.

2011), it is not clear to what extent those that can self fertilize actually depend on this ability for their reproduction. Very few studies have assessed the benefits of selfing in the introduced range, par ticularly in relation to plant abundance (Knight

et al.

2005; Eckert, Samis & Dart 2006 although see van Kleun en, Fischer & Johnson 2007). It is therefore not generally known whether animal pollination of invasive plants is less reliable in small founder populations, such that plants in these populations rely more on selfing. While mate avail ability and pollinator visitation may decline at different rates as plant abundance decreases, their effects on cross pollen receipt are seldom distinguished (although see Kunin 1993; Duncan,

et al.

2004; Elam

et al.

2007). A functional approach incorporating both these processes

would allow us to understand why some plants are more vulnerable to pollen limitation Allee effects than others, and predict in which plants self pollination is most likely to be important for fecundity and, in the introduced range, invasiveness.

Lilium formosanum

Wallace (Fig. 1) is an autonomously self pollinating and hawkmoth pollinated geophyte that is invasive in South Africa. We explored the contributions of self fertilization and pollinators to fecundity of this species in its introduced range in South Africa, asking the follow ing specific questions: (i) What is the magnitude of repro ductive assurance benefits derived from self fertilization?

(ii) Are reproductive assurance benefits greater in smaller and more isolated populations of

L. formosanum? (iii) Are

reproductive assurance benefits higher in isolated plants than those in continuous patches? (iv) Do reproductive assurance benefits increase with distance from continuous patches? (v) Is any increase in reproductive assurance benefits with isolation attributable to reduced pollinator visitation or mate availability?

Materials and methods

S T U D Y S P E C I E S

Lilium formosanum(Fig. 1) is a bulbous perennial plant with erect, annual stems. Each stem terminates in an inflorescence of 1 8 white, nocturnally scented, trumpet shaped flowers (Rodger, van Kleunen & Johnson 2010). In South Africa, its principal pollinator is the native hawkmothAgrius convolvuli (Fig. 1b; Rodger, van Kleunen & Johnson 2010). Populations vary in self compatibility in its native range of Taiwan (Sakazonoet al.2012), and it is com pletely self compatible and autonomously self pollinating in its introduced range in Japan (Inagaki 2002) and South Africa (Ram buda & Johnson 2004). A molecular marker study in the native range showed that fixation indices (Fis) of populations range from 0032 to 0901, suggesting variation among populations in mating system (Hiramatsuet al.2001). As no inbreeding depression is evi dent in progeny up to 3 years of age in the introduced range (Rod ger, van Kleunen & Johnson 2010; Rodger 2012), the reproductive assurance benefits attained through selfing are likely to be impor tant for population growth and invasive spread.

P O P U L A T I O N S I Z E A N D I S O L A T I O N S T U D Y

Study region and populations

Experiments were conducted from January to March in 2005, 2006 and 2007 in naturalized populations in KwaZulu Natal, South Africa, 10 1700 m above sea level (Table A1, Supporting information). Observations of hawkmoth scales on stigmas (Fig. 1c) indicated that visitation of L. formosanum by these insects occurs throughout the study region (J.G. Rodger, unpub lished results). Populations were mainly in disturbed grassland adjacent to exotic tree plantations or on grassy road verges, with a few in exotic forests or in otherwise pristine natural grasslands and indigenous forests. Population size was taken as the number of flowering stems. We used 50 m as the minimum separation dis tance between populations, allowing us to span a large range of isolation from almost no separation to over 15 km from the near est population. An index of population isolation was calculated as the log10of the mean distance to the nearest three populations.

Data were obtained from 37 populations in 2005, 20 populations in 2006 and 22 populations in 2007 (Table A1, Supporting infor mation). Most populations were accessible or available in only one of the three study years, although eight populations were studied in 2 or 3 years (Table A1, Supporting information).

Reproductive assurance benefits

Emasculation experiments can be used to distinguish between the importance of self pollination vs. pollinator mediated cross polli nation (Lloyd 1992; Eckert, Samis & Dart 2006). The reduction in fecundity experienced by emasculated relative to intact flowers is a measure of reproductive assurance benefits in other words dependence on self pollination for fecundity (Schoen & Lloyd 1992; Kalisz & Vogler 2003). Lilium formosanum flowers were emasculated by opening buds and removing anthers with alcohol sterilized forceps. For naturally pollinated controls, buds were opened and forceps inserted. We considered it unlikely that emas culation would affect pollinator visitation to L. formosanum, as hawkmoths do not forage for pollen andA. convolvulireadily vis its emasculated flowers (J.G. Rodger, pers. obs). This was con firmed by data that showed that the presence of lepidopteran scales (Fig. 1c), a measure of pollinator visitation, did not differ between stigmas of emasculated and intact flowers in three popu lations in 2006 and in four populations in 2007 (7 17 flowers per treatment per population, J.G. Rodger, unpublished results).

A single bud was emasculated on each of three (2005 and 2007) or 10 (2006) plants per population, and the same number of flow ers was similarly allocated as controls, except in four populations for which we needed measures of within population variation for a separate study in 2007 (Table A1, Supporting information).

Control flowers were on separate plants to emasculated flowers in 2005 and on the same plants in other years. Because we emascu lated only a single flower per plant, pollinator mediated geitonog amy could have contributed to fecundity of emasculated flowers, making our estimates of reproductive assurance benefits conserva tive. However, this is unlikely to be important as preliminary anal

yses indicated that fecundity of emasculated flowers was not positively related to number of flowers per plant (J.G. Rodger, unpublished results).

Thirty four populations were used in 2005, 15 in 2006 and 22 in 2007. We chose low levels of replication within populations to avoid a bias in sampling effort against small populations, first because analysis of variance is less robust to unequal variance and non normality when data are unbalanced (Quinn & Keough 2002) and secondly because the effective replicate for a relationship between population attributes and plant performance is the popu lation, so statistical power is likely to be increased by maximizing the number of populations at the expense of sample size per popu lation (Quinn & Keough 2002). We calculated the overall repro ductive assurance benefit of selfing for each year as the proportional reduction in fecundity caused by emasculation:

RA 1009(1 emasculated/control) (Eckert, Samis & Dart 2006) with fecundity defined as seeds per flower (percentage fruit set9mean seeds per fruit). The fecundity values used were grand means of population means.

Pollen limitation

Pollen supplementation experiments were used to test for pollen limitation inL. formosanumas autonomous self pollination is not necessarily sufficient to fertilize all ovules (Rodger, van Kleunen &

Johnson 2010). The same populations and the same sample size regimes were used as for emasculations, but different plants (Table A1, Supporting information). Supplementation consisted of saturating the stigma with outcross pollen from a plant at least 5 m away in the same population. Plants sometimes allocate resources preferentially to flowers that have more fertilized ovules, which can lead to overestimation of pollen limitation in pollen supplementation experiments (Knight, Steets & Ashman 2006).

However, as pollen limitation was generally low in this study, any overestimation would be quite small.

We calculated pollen limitation from seed per flower as 1009(1 control/supplemented) (Larson and Barrett 2000).

(a) (b)

(c)

Fig. 1.Lilium formosanum growing in a disturbed habitat (a), being pollinated by the hawkmoth Agrius convolvuli (b) and stigma showing hawkmoth scales and pol len (c). Scale bars are 87 mm (a), 27 mm (b) and 13 mm (c).

Conducting emasculation and supplementation in the same popu lations also allowed us to assess how pollen limitedL. formosanum would have been, had it lacked the ability to self fertilize. This is termed pollinator failure and is calculated as 1009(1 emascu lated/supplemented) (cf Kalisz & Vogler 2003).

Fruit and seed scoring

Fruits were harvested for seed counting at maturity, 10 12 weeks after flowering. Seeds were counted if they contained an embryo that was at least half the length of the seed, excluding the wing.

For each fruit, we measured the mass of the entire contents and the mass and number of seeds in a random subsample containing approximately 50 seeds and used this information to calculate seeds per fruit. All seeds were counted in fruits containing fewer than 50 seeds. Seeds per flower data (fruit set9seeds per fruit) were zero inflated as there were many flowers that did not set fruit. Fruit set and seeds per fruit were therefore analysed separately.

Data analyses

Fruit set was analysed as a binomial response variable in general ized linear models incorporating a logit link function. Separate analyses of the effects of emasculation and pollen supplementation were carried out for each year, as most populations were used in only 1 year. Fruit set did not need to be analysed for the supple mentation experiment in 2007 as there was 100% fruit set in both treatments. Significance was assessed from quasiFstatistics in sequential analysis of deviance, analogous toFstatistics inANOVA

with type I sums of squares (Payne 2011). Models included floral manipulation (emasculation or supplementation) as a fixed factor, population as a random factor, log10 population size and log10

population isolation as covariates and population size by floral manipulation and population isolation by floral manipulation interactions. A type I approach was used because of the hierarchi cal structure of the data, with replicates occurring within popula tions and population size and isolation measured at the population level. Terms were entered in the same order as they appear in Tables 1 and 2. The order in which terms are entered may affect their significance in sequential analyses. Nevertheless,

reversing the order of population size and isolation, and the popu lation size9floral manipulation and population isolation9flo ral manipulation interactions gave very similar results to those presented here and did not affect the conclusions drawn from them (J.G. Rodger, unpublished results). Population size and iso lation were tested against population, and other terms were tested against the residual. Where models were not overdispersed (i.e.

where residual deviance residual d.f.) we assumed residual mean deviance 1 for the purposes of calculation of quasi F ratios, and when models were overdispersed we used the model calculated residual mean deviance (Payne 2011). Model validation consisted of checking plots of residuals against fitted values for patterns (Zuuret al.2009).

Seeds per fruit was analysed in restricted maximum likelihood (REML) analysis of variance to accommodate differences in sam ple size between populations. REML analysis of variance used the same statistical design as the generalized linear model for fruit set except they also included the population by floral manipulation interaction as a random effect. Significance was evaluated using Wald Fstatistics for the fixed terms. For random terms, the change in deviance in the models when a term was dropped was compared with a chi squared distribution with one degree of freedom (Payne, Welham & Harding 2011). Residual plots were examined to check whether assumptions were met.

A R R A Y E X P E R I M E N T

Array layout

To test whether reproductive assurance was greater for plants iso lated from continuous patches and, if so, whether this was due to decreased visitation or mate availability, we created arrays of emasculated and intact plants transplanted either into central patches of L. formosanumor similar grassland habitat that was isolated from the patches. Plants used were sourced from the same populations. Two populations with discrete patches ofL. formosa numin open habitat (mainly natural grassland) were selected for experiments in February and March 2009. At Baynesfield (29 45162S, 30 21377E, Alt. 810 m), there was a population consist ing of a single large patch of 748 plants. In the Karkloof popula tion (29 20229, 30 17527, Alt. 1100 m), four patches of 67 610 Table 1.Significance levels from generalized linear models for fruit set and REML analysis for seeds per fruit in emasculation experiments across a range of populations differing in size and isolation. Full tables are in Appendix S1 (see Supporting information). Test statistics are QuasiFstatistics (ratios of mean changes in deviance) for fruit set analyses, WaldFstatistics for fixed effects in seed set analyses and change in deviance (tested against the chi squared distribution) for random effects (P, P9E) in the seed set analyses. Residual mean deviance shown in brackets for fruit set analyses

Effect

Fruit set Seeds per fruit

2005 2006 2007 2005 2006 2007

d.f.

Test

statistic d.f.

Test

statistic d.f.

Test

statistic d.f.

Test

statistic d.f.

Test

statistic d.f.

Test statistic

PS 1,30 003 1, 11 044 1, 18 424 1, 18 619 1, 10 06 1, 19 261

PI 1, 30 123 1, 11 421 1, 18 875** 1, 22 049 1, 10 001 1, 18 285

P^† 30, 30 160 11, 11 164 18, 17 067 1 314 1 2797*** 1 1579***

E 1, 30 1145** 1, 11 500* 1, 17 681* 1, 16 11313*** 1, 7 733* 1, 16 5426***

PS9E 1, 30 076 1, 11 003 1, 17 394 1, 16 831* 1, 10 029 1, 19 002

PI9E 1, 30 572* 1, 11 714* 1, 17 322 1, 20 429 1, 6 276 1, 17 002

P9E^† 1 018 1 194 1 517*

Residual 30 (120) 11 (043) 17 (21910 ⁵)

PS, population size; PI, population isolation; P, population; E, emasculation.

P<01;*P<005;**P<001;***P<0001.

†Random effects.

plants were used. The array experiments were conducted from 31 January to 14 February 2009 at Baynesfield and from 28 February to 10 March 2009 at Karkloof. We obtained data from 87 plants at Baynesfield and 59 at Karkloof.

Emasculated and intact plants were placed singly inside contin uous patches or in isolated pairs outside of these patches (Fig. C1, Supporting information). Isolated pairs consisted either of two emasculated plants or an emasculated plant and an intact plant, 1 m apart, to distinguish between effects of isolation on reproduc tive assurance benefits through reductions in pollinator visitation vs. mate availability. This approach is original to this study. Dis tances between successive pairs were chosen randomly from increasing intervals of the log2scale (2 4, 4 8, 8 16…), so that as distance away from the central patch increased, density decreased as well. Distance from central patches ranged from 3 to 702 m at Baynesfield and 3 to 561 m at Karkloof. After flowering, all trans planted individuals were re excavated and brought back to the University of KwaZulu Natal Pietermaritzburg campus and main tained in plant pots until fruits were mature.

Isolation

To test whether reproductive assurance compensated for reduced cross pollen receipt in isolated plants, we compared fecundity in emasculated and intact plants placed inside central patches and in isolated pairs. Statistical analyses of fruit set and seeds per fruit were again conducted separately. Reproductive assurance indices were calculated for plants inside patches. Fruit set was analysed in generalized linear models as before, but with number of flow ers (per plant) as the binomial total in an events/trials structure (Payne 2011). Using a type I analysis allowed us to test for the effect of distance from central patch after accounting for the effect of isolation (inside vs. outside patches). Terms in order of entry were isolation, distance from patch (log10 transformed), emasculation (intact vs. emasculated), emasculation by isolation, emasculation by distance. Distance was scored as zero for plants inside patches. For seeds per fruit, mean values were calculated for each plant, log10transformed to improve homogeneity of var iance and analysed in REML analysis of variance as sample sizes were unbalanced. The same model was used as described previ ously for fruit set. Terms were sequentially added to a model, and the significance of these terms was evaluated from Wald Fstatistics.

Mate availability

To distinguish between effects of reduced mate availability vs. pol linator visitation on reproductive assurance benefits in isolated plants, we compared fecundity of emasculated plants inside popu lations, isolated and paired with another emasculated plant or iso lated and paired with an intact plant as a test for the effect of mate availability. Fruit set and seeds per fruit were analysed using gen eralized linear models and REML analysis of variance as described above. Analyses included mate presence as a fixed factor, distance as a continuous variable and the mate presence by isolation dis tance interaction. As appreciable heterogeneity of variance remained for seeds per fruit, even after transformation, we con ducted pairwise comparisons between groups with Mann Whitney Utests. Although corrections for multiple comparisons are some times applied for pairwise comparisons, we have not done so because in this case each comparison tests a different hypothesis, so the multiple comparisons do not inflate type 1 error.

Scale and pollen deposition

We also addressed the question of whether isolated plants experi enced decreased pollinator visitation or mate availability by scoring emasculated flowers for the presence of lepidopteran scales, an indica tion of visitation, and presence of pollen on stigmas, an indication of successful pollination, using a 20X hand lens. Each plant was scored once, 3 4 days after transplanting, for all flowers that had been open for at least one night. Scale and pollen deposition were analysed in general linear models for binomial data with a logit link function including isolation as a fixed factor and distance as a covariate.

All statistical analyses were performed in Genstat 12.1 (VSN International, Hemel Hempstead, UK).

Results

P O P U L A T I O N S I Z E A N D I S O L A T I O N S T U D Y

Reproductive assurance benefits

Emasculation significantly reduced fruit set and number of seeds per fruit in naturalized populations in all 3 years,

Table 2. Significance levels from generalized linear models for fruit set and REML analysis for seeds per fruit in pollen supplementation experiments across a range of populations differing in size and isolation. Full tables are in Appendix S1 (see Supporting information). Test statistics are QuasiFstatistics (mean change in deviance) for fruit set analyses, WaldFstatistics for fixed effects in seed set analyses and change in deviance (tested against the chi squared distribution for random effects in the seed set analyses. Residual mean deviance shown in brackets for fruit set analyses. 2007 data were not analysed for fruit set as all replicates of both treatments set fruit in this experiment

Effect

Fruit set Seeds per fruit

2005 2006 2005 2006 2007

d.f.

Test

statistic d.f.

Test

statistic d.f.

Test

statistic d.f.

Test

statistic d.f.

Test statistic

PS 1, 32 419* 1, 12 137 1, 25 077 1, 12 121 1, 14 021

PI 1, 32 024 1, 12 311 1, 27 201 1, 10 054 1, 15 287

P† 32, 32 237** 12, 12 078 1 770** 1 1918*** 1 2809***

S 1, 32 003 1, 12 002 1, 74 586* 1, 138 171 1, 134 026

PS9S 1, 32 433* 1, 12 077 1, 75 204 1, 139 213 1, 134 003

PI9S 1, 32 121 1, 12 236 1, 76 002 1, 138 048 1, 134 098

P9S† 1 000 1 000 1 000

Residual 32 (2029) 12 (37910 ⁴)

PS, population size; PI, population isolation; P, population;S, supplementation.

*P<005;**P<001;***P<0001; †, random effects.

with a mean reduction in total fecundity (reproductive assurance benefits) of 67%: 90% in 2005, 45% in 2006 and 66% in 2007 (Table 1; Fig. B1, Supporting informa tion). The effect of emasculation was not greater in smaller or more isolated populations except that there was a greater effect of emasculation in more isolated populations for fruit set in 2005 (Table 1; Figs 2 and 3).

In other cases where there were significant population size by emasculation and isolation by emasculation inter actions, these were not attributable to fruit set or seeds per fruit declining more for emasculated than control flowers as population size decreased or isolation increased (Table 1; Figs 2 and 3).

Pollen limitation

Pollen supplementation increased fecundity (indicating pollen limitation) by an average of 12% (16% in 2005, 11% in 2006 and 10% in 2007), but this was only signifi cant in 2005 (Table 2; Fig. B1, Supporting information).

A significant supplementation by population size interac tion in this year showed that supplementation increased fruit set only in smaller populations (Table 2; Fig. B2, Supporting information). There was no evidence for any

effect of population isolation on pollen limitation as the interaction between population isolation and pollen sup plementation was never significant (Table 2; Fig. B3, Sup porting information). Pollinator failure was estimated as 92% in 2005, 48% in 2006 and 72% in 2007.

A R R A Y E X P E R I M E N T

Isolation

Fruit set and seeds per fruit were significantly lower in emasculated than in intact plants for both the Baynesfield and Karkloof sites (Table 3; Fig. 4). Indices of reproduc tive assurance were 75% for plants inside continuous patches and 96% for isolated plants at Baynesfield; 80%

inside patches and 84% for isolated plants at Karkloof. At Baynesfield, emasculation reduced seeds per fruit more strongly in isolated plants than those in populations [sig nificant isolation by emasculation interaction with a nonsignificant trend in the same direction for fruit set (Table 3; Fig. 4a,b)]. However, at Karkloof, the effect of emasculation on fruit set and seeds per fruit was not related to isolation (Table 3; Fig. 4c,d). The effect of emas culation on fruit set and seeds per fruit did not increase

0·0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0

0·0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0 0·0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0 0·0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0 0·0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0 0·0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 4·0

Proporton frut set

–0·2 0·0 0·2 0·4 0·6 0·8 1·0 1·2

(a)

2005

PS^ns E**

PS . E^ns

–0·2 0·0 0·2 0·4 0·6 0·8 1·0 1·2

Emasculated Control

(b)

2006

PS^ns E* PS . E^ns

(c)

2007 PS^† E*

PS.E^†

(d)

PS*

E***

PS . E*

(e)

PS^ns E*

PS . E^ns –0·2

0·0 0·2 0·4 0·6 0·8 1·0 1·2

(f) PS^ns

E***

PS . E^ns

Seeds per frut

0 200 400 600 800 1000 1200 1400 1600

Log₁₀ population size

0 200 400 600 800 1000 1200 1400 1600

0 200 400 600 800 1000 1200 1400 1600

Fig. 2.Fruit set (model adjusted, a c) and seeds per fruit (d f) of emasculated and intact, naturally pollinated plants in relation to popula tion size for 3 years. PS, population size, E, emasculation; ns, nonsignificant;^†P<01;*P<005;**P<001;***P<0001. Circles repre sent predicted values for populations for fruit set (adjusted for population isolation) and mean populations values for seeds per fruit.

Regression lines shown for seeds per fruit.

with distance of isolation in either population (Table 3;

Fig. C2, Supporting information). Although the distance by emasculation interaction was significant for seeds per fruit at Baynesfield, the effect of emasculation actually decreased with distance due to an outlier (Fig. C2, Supporting information).

Mate availability

Mate availability had a significant effect on fruit set at Baynesfield (Table 3): isolated emasculated plants with no mates available (i.e. with an emasculated partner) had sig nificantly lower fruit set than those inside populations (two tailed

t

tests;

t

296, d.f. 48,

P

0009) or iso lated with a potential mate (intact partner,

t

207, d.f. 48,

P

0044). Isolated plants with intact partners did not differ significantly from those in continuous popu lations (t 1 08, d.f. 48,

P

0 286). In isolated plants at Baynesfield, seeds per fruit was not affected by mate availability although it did increase with isolation distance (Table 3; Fig. 5), contrary to the expectation of decreased pollen transfer in more isolated plants (Table 3; Fig. C3, Supporting information). At Karkloof, mate availability had a significant effect on seeds per fruit (Table 3;

Fig. 5d): emasculated plants with no mates available

(emasculated partner) had fewer seeds per fruit than those inside populations (Mann Whitney

U

test

u 05, P 0019, n 4, 6) or isolated with a potential mate (u 00,P 0016,n 4, 5). Isolated plants with intact part ners did not differ significantly from those in continuous populations (u 13,P 0792,n 6, 5).

Scale and pollen deposition

Scale deposition was not related to isolation or distance at either Baynesfield or Karkloof (Table 3). Isolated plants had significantly lower pollen receipt than those in the main patch at Baynesfield (Table 3), but not at Karkloof (Table 3).

Discussion

These results show that

L. formosanum

relies heavily on self fertilization for fecundity even though it has an effective hawkmoth pollinator in its invasive range (Rodger, van Kleunen & Johnson 2010). On average, reproductive assur ance benefits from self pollination, as assessed by floral emasculations, accounted for 67% of the total fecundity of naturally occurring plants, but this did not vary according to population size, contrary to expectations from other

2·5 3·0 3·5 4·0 4·5

2·5 3·0 3·5 4·0 4·5 2·5 3·0 3·5 4·0 4·5 2·5 3·0 3·5 4·0 4·5

2·5 3·0 3·5 4·0 4·5 2·5 3·0 3·5 4·0 4·5

Proporton frut set

–0·2 0·0 0·2 0·4 0·6 0·8 1·0 1·2 1·4

2005

PI^ns E**

PI . E*

–0·2 0·0 0·2 0·4 0·6 0·8 1·0 1·2 1·4

2006

PI E*

PI . E*

2007

PI**

E*

PI.E

PI^ns E***

PI . E

PI^ns E*

PI . E^ns –0·2

0·0 0·2 0·4 0·6 0·8 1·0 1·2 1·4

Emasculated Control

PI^ns E***

PI . E^ns

Seeds per frut

0 200 400 600 800 1000 1200 1400 1600

Log₁₀ population isolation

0 200 400 600 800 1000 1200 1400 1600

0 200 400 600 800 1000 1200 1400 1600

(a) (b) (c)

(d) (e) (f)

Fig. 3.Fruit set (model adjusted, a c) and seeds per fruit (d f) of emasculated and intact naturally pollinated plants in relation to popula tion isolation for 3 years. PI, population isolation [mean distance (m) to nearest three populations], E, emasculation; ns, nonsignificant;

†P<01;*P<005;**P<001;***P<0001. Circles represent predicted values for populations for fruit set (adjusted for population size) and mean populations values for seeds per fruit. Curves for fruit set were fit in generalized linear models using logit transformed data and back transformed.

studies that show component Allee effects via a decrease in animal mediated pollination in small populations ( Agren 1996; Groom 1998; Brys

et al.

2011). The 71% average esti mate of pollinator failure shows that

L. formosanum

would be highly pollen limited if it was self incompatible.

Although we have previously documented variation in the ability of

L. formosanum

to self pollinate autonomously in the study region, the fact that pollen supplementation

increased fecundity by only 12% on average indicates that most populations have high levels of autofertility.

There was no evidence for an effect of population size on reproductive assurance benefits in the survey of natural pop ulations, indicating that population size did not affect polli nator visitation (Table 1; Fig. 2). However, reproductive assurance mitigated a detectable Allee effect for isolated plants lacking nearby mates in the array experiment

Fruit set

0 0 0 2 0 4 0 6 0 8 1 0

Fruit set

0 0 0 2 0 4 0 6 0 8 1 0

8

9 19

9 12

8

12 16

Inside patch Isolated Inside patch Isolated

Mean seeds per fruit

0 200 400 600 800

Intact Emasculated

8

17

7

6

Mean seeds per fruit

0 200 400 600 800 1000 1200

9

11

6 10

Inside patch Isolated

Inside patch Isolated Isol *

Dist * Emas ***

E × I ns E × D ns

Isol ns Dist ns Emas ***

E × I ns E × D ns

Isol ns Dist ns Emas ***

E × I * E × D *

Isol ns Dist ns Emas ***

E × I ns E × D ns

(a) (b)

(c) (d)

Fig. 4.Fruit set and seeds per fruit for emasculated and intact plants in array experiment at Baynesfield (a, b) and Kark loof (c, d). For fruit set (a, c), bars repre sent means of fruit set values for individual plants. For seeds per fruit (b, d), back transformed means and error bars are plot ted. Numbers above bars are numbers of plants.

Table 3.Significance levels from generalized linear models for fruit set, scale deposition and pollen deposition and REML analyses for seeds from array experiments onLilium formosanum. Test statistics are QuasiFstatistics (mean change in deviance) for fruit set, pollen and scale analyses, WaldFstatistics for fixed effects in seed set analyses and change in deviance (tested against the chi squared distribu tion) for random effects in the seed set analyses. Residual mean deviance shown in brackets for fruit set, pollen and scale analyses

Effect

Fruit set Seeds per fruit

Baynesfield Karkloof Baynesfield Karkloof

d.f.

Test

statistic d.f.

Test

statistic d.f.

Test

statistic d.f.

Test statistic Emasculation analyses

Isolation 1, 46 449* 1, 35 023 1, 32 037 1, 30 002

Distance 1, 46 493* 1, 35 024 1, 32 03 1, 30 134

Emasculation 1, 46 8041*** 1, 35 1637*** 1, 32 3107*** 1, 30 2332***

E9I 1, 46 136 1, 35 004 1, 32 731* 1, 30 015

E9D 1, 46 002 1, 35 0305 1, 32 654* 1, 30 001

Residual 46 (102) 35 (11)

Mate availability analyses

Distance 2, 46 528** 2, 30 084 2, 17 038 2, 10 709*

Donor presence 1, 46 006 1, 30 058 1, 17 567* 1, 10 003

DP9D 1, 46 000 1, 30 060 1, 17 003 1, 10 017

Residual 46 (172) 30 (152)

Pollen and scale analyses

Scales Pollen

Isolation 1, 26 003 1, 22 108 1, 26 2588*** 1, 22 001

Distance 1, 26 051 1, 22 002 1, 26 000 1, 22 131

Residual 26 (267) 22 (147) 26 (065) 22 (177)

*P<005;**P<001;***P<0001

(Fig. 5). Reproductive assurance benefits were greater for isolated plants than those placed in continuous populations at only one of the two sites (Baynesfield, Fig. 4a,b). How ever, in both populations, emasculated plants that were iso lated with no potential mate (i.e. placed next to another emasculated plant) had lower fecundity than those isolated with a single intact plant nearby, or placed in continuous populations. This shows that the greater reproductive assur ance benefits for isolated plants at Baynesfield were due to decreased mate availability, not reduced pollinator visita tion (Table 3, Fig. 5a,d). Findings of lower stigmatic pollen deposition on isolated than on nonisolated plants at Baynes field and the lack of effect of isolation on lepidopteran scale deposition on stigmas (Table 3) support this conclusion.

As pollen limitation of self incompatible plants is gener ally higher in the introduced than in the native range (Burns

et al.

2011), it can be expected that invasive plants obtain substantial reproductive assurance benefits from selfing. However, no reproductive assurance benefits were found in hummingbird pollinated

Nicotiana glauca

plants invasive in North America (Schueller 2004), while large reproductive assurance benefits were found in hawkmoth pollinated

Lilium formosanum

(RA 67%, this study) and

Datura stramonium

(RA 83%, van Kleunen, Fischer &

Johnson 2007). Clearly, more studies spanning a range of pollination systems, geographic areas and life forms are needed before it will be possible to assess the importance of selfing for fecundity of introduced plants generally.

Mitigation of increased mate limitation by selfing in iso lated plants, as demonstrated in the array experiment (Fig. 5), could be especially important for invasion of

L.

formosanum, given that reproduction by isolated individu

als should have a dramatic impact on the invasion process (Kot, Lewis & Driessche 1996; Clark, Lewis & Horvath 2001). We are not aware of any previously published stud

ies showing that selfing mitigates mate limitation Allee effects in invasive species, and only one for a native species (Brys

et al.

2011). Indirect evidence from some studies of the effect of plant abundance on fecundity suggests that mate limitation may generally be more important than reduced pollinator visitation in reducing cross pollen receipt of isolated individuals (Kunin 1993; Duncan,

et al.

2004; Elam

et al.

2007). This is one of the first studies to distinguish between mate availability and pollinator visita tion components of Allee effects, yet this approach is essential for deriving the functional understanding that would allow us to predict which plants should be most vul nerable to Allee effects, and to allow more refined predic tions about the effects of reproductive assurance and pollen limitation on invasiveness.

The absence of a detectable effect of plant abundance on hawkmoth visitation to

L. formosanum

is consistent with some other studies of hawkmoth pollinated plants (e.g.

Johnson, Torninger & Agren 2009) and contrasts with results for plants with other pollinators ( Agren 1996;

Groom 1998; Brys

et al.

2011). This could be because hawkmoths are more nomadic in their movements and opportunistic in their foraging than other pollinators (as suggested by Johnson, Torninger & A 2009) or because foraging primarily by olfactory rather than visual cues ren ders them less capable of assessing population size prior to arrival in populations.

Conclusions

We have used a functional approach to assess the relation ship between plant abundance and reproductive assurance benefits in

L. formosanum, distinguishing between effects

of pollinator visitation and mate availability as well as between isolation and population size. Although we found

Fruit set

0·0 0·2 0·4 0·6 0·8 1·0

Fruit set

0·0 0·2 0·4 0·6 0·8 1·0

19

15

9

9

Single None

Seeds per fruit

0 100 200 300 400 500

7

3

Seeds per fruit

0 50 100 150

200 5

4

Single None

Mate **

Dist ns D × M ns

Mate ns Dist ns D × M ns

Mate ns Dist * D × M ns

12

6 9

8

Many Many Single None

Single None Many Many

Mate * Dist ns D × M ns

Mate availability

(a) (b)

(c) (d)

Fig. 5.Fruit set (a, c) and seeds per fruit (b, d) of emasculated plants at different lev els of mate availability in array experiment at Baynesfield (a, b) and Karkloof (c, d).

For fruit set, bars represent means of fruit set values for individual plants. For seeds per fruit, back transformed means and standard errors of plant means on log2

scale shown. Numbers above bars are num bers of plants. For seeds per fruit, all fruits had at least one seed.

no evidence that pollinator visitation was related to abun dance, our finding that selfing mitigated decreased mate availability in isolated plants is, to the best of our knowl edge, the first evidence that selfing may contribute to inva siveness by mitigating an Allee effect. Because of this finding, because reproductive assurance benefits are high even in the absence of Allee effects, and because progeny trials have revealed almost no evidence for inbreeding depression in

L. formosanum

in South Africa (Rodger, van Kleunen & Johnson 2010; Rodger 2012), reproductive assurance benefits may well translate into a demographic advantage. This makes it likely that ability to self pollinate contributes to the invasiveness of

L. formosanum. Demo

graphic analysis will be required to assess the effect of sel fing on invasiveness, and the relative importance of its compensating for generally inadequate pollinator visitation vs. mate limitation in isolated individuals.

Acknowledgements

Thanks to Dalton Nyawo for assistance with seed counting, Wade Shrives for assistance with floral manipulations and Ben Khumalo for help setting up the arrays. We are grateful to Craig Morris, Mike Ramsey and Law- rence Harder for statistical advice and to Karl Duffy, Chris Eckert, Eliza- beth Elle, Taina Witt and Lorne Wolfe for comments on previous drafts of this manuscript. We also thank Baynesfield Estates, the Engelbrechts and the Shaws for permission to work on their properties and UKZN Botanical Garden for space to maintain plants. This study was supported by the DST-NRF Centre of Excellence for Invasion Biology (CIB).

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