Pollen Source Affects Female Reproductive Success and Early Offspring Traits in the Rare Endemic Plant Polemonium Vanbruntiae

Plant Species Biology (2011) 26, 244–253 doi: 10.1111/j.1442-1984.2011.00326.x

Pollen source affects female reproductive success and early offspring traits in the rare endemic plant

Polemonium vanbruntiae (Polemoniaceae)psbi_326 244..253

LAURA HILL BERMINGHAM* and ALISON K. BRODY† Departments of *Plant Biology and †Biology, University of Vermont, 63 Carrigan Dr., 111 Jeffords Hall, Burlington, VT 05405 USA

Abstract

Understanding the relative magnitudes of inbreeding and outbreeding depression in rare plant populations is increasingly important for effective management strategies. There may be positive and negative effects of crossing individuals in fragmented populations. Conservation strategies may include introducing new genetic material into rare plant populations, which may be beneficial or detrimental based on whether hybrid offspring are of increased or decreased quality. Thus, it is important to determine the effects of pollen source on offspring fitness in rare plants. We established pollen crosses (i.e. geitonogamous-self, autonomous-self, intrasite-outcross, intersite-outcross and open- pollinated controls) to determine the effects of pollen source on fitness (seeds/fruit and seed mass) and early offspring traits (probability of germination, number of leaves, leaf area and seedling height) in the rare plant Polemonium vanbruntiae. Open-pollinated, intrasite-outcross and geitonogamous-self treatments did not differ in fitness. However, plants receiving autonomous-self pollen had the lowest fitness and the lowest probability of seed germination. Intersite-outcross plants contained fewer seeds/fruit, but seeds germinated at higher frequencies and seedlings were more vigorous. We also detected heterosis at the seed germination stage. These data may imply that natural populations of P. vanbruntiae exhibit low genetic variation and little gene flow. Evidence suggests that deleterious alleles were not responsible for reduced germination; rather environmental factors, dichogamy, herkogamy and/or lack of competition among pollen grains may have caused low germinability in selfed offspring. Although self-pollination may provide some reproductive assurance in P. vanbruntiae, the result is a reduction in germination and size-related early traits for selfed offspring. Keywords: heterosis, inbreeding depression, outbreeding depression, rare plant, relative performance. Received 23 May 2010; revision received 7 September 2010; accepted 30 December 2010

Introduction depression may decrease if deleterious alleles are purged over generations of inbreeding (Ellstrand & Elam 1993). Rare plants existing within fragmented populations may However, plant populations that have been recently iso- be exposed to higher rates of self-fertilization or mating lated or reduced in size may not have sufficient time to with close relatives, which may result in a reduction in reduce the frequency of deleterious alleles by genetic plant fitness because of inbreeding depression (Buza et al. purging. If inbreeding depression leads to a reduction in 2000; Kéry et al. 2000). Inbreeding depression is main- plant fitness, populations may experience an increased tained in natural populations as generations of inbreeding risk of extinction, which may be particularly detrimental or mating with close relatives reveal deleterious recessive for threatened and endangered species (Barrett & Kohn alleles (Charlesworth & Charlesworth 1987). Inbreeding 1991; Ellstrand & Elam 1993; Frankham 2005). Correspondence: Laura Hill Bermingham The relative intensity of inbreeding depression depends Email: [email protected] on the level of gene flow among populations, which is

© 2011 The Authors Journal compilation © 2011 The Society for the Study of Species Biology POLLEN SOURCE AFFECTS RARE PLANTS 245 influenced by both the plant’s mating system and dis- In contrast, geitonogamous selfing may cause pollen persal ability (Hamrick et al. 1979). If the genetic distance discounting and offers no reproductive assurance for among fragmented populations is large and dispersal populations experiencing pollinator scarcity (Goodwillie among populations is low, a decrease in fitness may occur et al. 2005). However, geitonogamous selfing may be an if distant populations are crossed (i.e. outbreeding inevitable consequence of plants producing large floral depression; Price & Waser 1979; Waser 1993). However, displays to attract pollinators (de Jong et al. 1993). Geito- if heterozygous individuals exhibit a fitness advantage nogamy may be particularly widespread in clonal plants, over homozygotes (‘overdominance’; Lande & Schemske such as P. vanbruntiae, which produce multiple flowering 1985), then hybridization between fragmented popula- ramets of the same genet (Harder & Barrett 1995). Sex tions may rescue a population from inbreeding depres- allocation theory posits that increased plant size and large sion and genetic drift by introducing new genetic material floral displays should increase male reproductive success (Lynch 1991; Hufford & Mazer 2003). Highly self- (i.e. pollen deposition) and that these fitness gains will compatible species with low interpopulation gene flow decrease with plant size (Charnov 1982). However, female are expected to exhibit higher frequencies of outbreeding reproductive success (i.e. seed production) may decrease depression when compared with self-incompatible, well- with plant size if geitonogamously produced offspring dispersed species (Dudash & Fenster 2000). However, are of lesser quality (de Jong et al. 1993). It is important to most species and populations fall somewhere in between discern the effects of geitonogamous selfing on offspring these two stable evolutionary endpoints depending on the fitness to predict patterns of selection on plant reproduc- relative performance of selfed versus outcrossed progeny tive and life-history traits, as well as determine whether (Eckert & Barrett 1994). the relative performance of offspring differs between In the present study, we examined whether seed pro- selfed and outcrossed progeny. duction and offspring performance differed between To determine whether the pollen source affects the rela- selfed and outcrossed progeny in the globally threatened tive performance of P. vanbruntiae offspring, we experi- perennial plant Polemonium vanbruntiae Britton (Eastern mentally applied five pollen crosses (autonomous-self, Jacob’s ladder, Polemoniaceae). Populations of P. vanbrun- geitonogamous-self, intrasite-outcross, intersite-outcross tiae may suffer from both inbreeding and outbreeding and open-pollinated controls) to treatment plants in depression because of its low dispersal ability and mixed- natural populations and to plants propagated in growth mating system, including the capacity for self-fertilization chambers. A series of plants were hand-pollinated in the and clonal reproduction (Hill et al. 2008). Clonal reproduc- laboratory in growth chambers because of concerns that tion occurs in P. vanbruntiae with vegetative shoots devel- intersite crosses would result in outbreeding depression oping from a thick underground rhizome. In natural and negatively impact natural populations. We compared populations, P. vanbruntiae clonal fragments contain an female reproductive fitness (seed production, seed average of two ramets/clonal fragments, although varia- mass and germination) among all pollen crosses. In addition is high (range: 1–22 ramets/clonal fragments; Hill tion, we compared offspring vigor (leaf number, leaf size Bermingham 2010). and seedling height) between intersite-outcross and Self-fertilization in P. vanbruntiae can result from both geitonogamous-self progeny. within-flower (autogamous) and between-flower (geito- We asked three main questions: (i) is there evidence for nogamous) pollen transfer (Herlihy & Eckert 2002). inbreeding depression in natural populations of the rare Within-flower selfing may be either spontaneous plant P. vanbruntiae (if inbreeding depression occurs, is it (‘autonomy’) or pollinator-facilitated (‘facilitated auto- evident in terms of a reduction in female reproductive gamy’), but geitonogamous pollination always requires a fitness, offspring vigor or both); (ii) does delivering out- pollen vector (Richards 1986; Schoen & Lloyd 1992). cross pollen from a distant population increase female When pollen is not limiting, autonomous selfing can reproductive success and/or offspring performance com- result in pollen discounting (i.e. the reduction of pollen pared with plants receiving outcross pollen from the same available for outcrossing, sensu Harder & Wilson 1998). population and plants receiving self-pollen; and (iii) do However, if populations are limited by pollen, only intersite-outcross offspring exhibit heterosis for early life- autonomous selfing provides reproductive assurance, history traits? thus allowing offspring to be produced even with little to no pollinator service (Baker 1955; Stebbins 1957; Charnov 1982; Schoen & Brown 1991; Lloyd 1992). The capacity for Materials and methods autonomous selfing may be adaptive for rare plant popu- Study species lations, such as P. vanbruntiae, which exist in small, fragmented populations that may receive stochastic pollinator Polemonium vanbruntiae Britton (Eastern Jacob’s ladder) is service (Kennedy & Elle 2008). a globally threatened clonal perennial plant in the Phlox

Plant Species Biology 26, 244–253 © 2011 The Authors Journal compilation © 2011 The Society for the Study of Species Biology 246 L. H. BERMINGHAM AND A. K. BRODY family (Polemoniaceae). This species has a global conser- pollen was again applied. This ensured that the majority vation rank of G3 (‘vulnerable to extirpation or extinction’; of flowers received a pollen treatment, as P. vanbruntiae NatureServe 2010), and fewer than 100 populations occur flowers remain receptive for an average of 3–5 days (Hill in eastern North America (Vermont Non-game and et al. 2008). Natural Heritage Program, Vermont Department of Fish For plants grown in the laboratory in growth chambers, and Wildlife 2001). Polemonium vanbruntiae is narrowly we carried out all of the aforementioned pollen crosses, endemic to cool, mountainous, wetland regions contain- with the exception of open-pollinated, but also included ing calcareous basal till soils with acidic to circumneutral intersite-outcross hand-pollinations. Intersite crosses pH in eastern North America (Deller 2002; Hill Berming- were only carried out in the laboratory because of stipu- ham 2010). lations outlined in the Vermont Threatened and Endan- Autonomous selfing in P. vanbruntiae may be limited gered Species permit. We carried out intersite crosses by because of both spatial separation of anthers and stigma collecting pollen on a paintbrush from all flowers dehisc- (herkogamy) and temporal separation of male and female ing pollen on a single donor plant from either site AP or function (dichogamy). Because P. vanbruntiae reproduces FR. We delivered the outcross pollen to the receptive clonally, geitonogamy may occur among ramets of a stigmas of a plant originating from the other site, resulting genet. Refer to Hill et al. (2008) for more detailed informa- in an AP ¥ FR cross. In both the laboratory and the field tion on the reproductive biology of P. vanbruntiae. crosses, our design was such that all pollination treatments were applied to each plant. We later collected fruits, scored them as expanded or not, counted all seeds per Pollination experiments fruit and weighed all seeds in the laboratory. We collected seeds from 70 plants in two Vermont populations, Forest Road 233 (FR) and Abbey Pond (AP), Effect of pollen source on early offspring traits located in the Green Mountain National Forest in central Vermont, USA, in September 2004. We collected ripened We planted seeds from each of the five pollination treat- fruits from 30 plants at site FR (1424 seeds) and from 40 ments in germination mix in 4.5 cm pots with six cells per plants at site AP (6019 seeds). We kept 10% of the seeds for pot. Each pot contained seeds from a single pollen treat- the pollination experiments and returned the remaining ment; the pots were randomly arranged in plastic trays in seeds to each respective population and passively dis- the growth chambers. We germinated all seeds from both persed the seeds around the maternal plant. The seeds for laboratory and field treatment plants after a 12-week the pollination experiments were randomly mixed and stratification period at 4°C in a growth chamber (Model germinated in the laboratory in December 2004. Eleven I-37LXX; Percival Scientific, Perry, IA USA) to examine if plants produced flowers in March 2006 and these were pollen source affected offspring quality. After the 12-week used for hand-pollination treatments in the laboratory. In stratification, we activated the 40 W fluorescent bulbs in addition, in June 2006, we conducted hand-pollinations in the chambers, which were programmed to run on an a natural population located in Lordsland Preserve (LP) in 18h/6h light/dark cycle. We humidified the chambers Otsego County, New York. Precise GPS coordinates for and kept the air temperature constant at 23°C. We checked the site locations are not provided for conservation the trays daily and watered the seedlings as needed so that reasons. the soil remained saturated. We randomized the position To examine the effects of pollen source on seed produc- of the trays in the chamber weekly throughout the plant tion and early offspring traits, we haphazardly selected trials. Seedling germination began to occur in March 2007 118 treatment plants throughout the entire LP population after 4 days under the lights. We assessed successful ger- in 2006. We randomly assigned individual flowers of a mination by emergence of the hypocotyl and calculated single plant to one of four pollination treatments: (i) the probability of germination (0/6–6/6). The seedlings geitonogamous-self-bagged, emasculated, self-pollinated began to open their first true leaves at 16 days, and all by hand with a mixture of within-plant self-pollen; (ii) seedlings had true leaves 40 days after germination. We autonomous-self-bagged, no emasculation, no hand- measured the following variables of all surviving seed- pollination; (iii) intrasite-outcross-bagged, emasculated, lings at 100 days after seedling germination: number of hand-pollinated with pollen from another flower on a true leaves, leaf area (mm2) and seedling height (cm). flowering stem at least 8 m away; and (iv) open-pollinated controls. All four pollination treatments were applied to Data analysis flowers within a plant. We carried out the pollination treatments twice per week on all open female-phase Differences in female reproductive fitness and offspring traits flowers throughout the blooming period. If stigmas of the We analyzed the data separately for treatment plants that treatment flowers were still receptive on the next visit, were pollinated in natural populations (‘field’) from those

© 2011 The Authors Plant Species Biology 26, 244–253 Journal compilation © 2011 The Society for the Study of Species Biology POLLEN SOURCE AFFECTS RARE PLANTS 247 that were pollinated in the growth chambers (‘lab’). The assumptions of homoscedascity and normality: square- pollination treatments carried out in the field included root number of leaves and natural log of leaf area and autonomous-self, geitonogamous-self, intrasite-outcross offspring height. and open-pollinated controls, but lacked intersite- outcross. The pollination treatments carried out in the Heterosis and relative performance of offspring The ‘relative growth chamber included autonomous-self, performance of crosstypes’ (RP) estimate of intersite- geitonogamous-self, intrasite-outcross and intersite- outcross and geitonogamous-self offspring was modified outcross, but lacked open-pollinated controls. from the methods of Ågren and Schemske (1993) as Equa- To determine whether seeds/fruit and seed mass dif- tion 1: fered significantly among pollination crosses, we used []− = zzos separate linear mixed models. We designated ‘flower’ RPi (1) nested within ‘plant’, both as random factors, and parent zmax plant height (cm) as the covariate. We used the parent where zo is the mean phenotype of intersite-outcross height of the plants that were pollen receptors as the progeny, zs is the mean performance of geitonogamous- covariate to adjust for maternal effects (Becker et al. 2006). self progeny, and zmax = zo when zo > zs and zmax = zs when Pollination treatment was the independent variable and a zs > zo (Ågren & Schemske 1993; Johnston & Schoen 1994). fixed factor. We used a Bonferroni adjustment for pairwise ws We chose to not use the traditional ∂=1 − equation of comparisons among all pollen crosses. Because we could wo only assess seed production on a per-flower basis, our inbreeding depression because the relative performance analysis does not account for the potential of some flowers equation gives equal weight to inbreeding and outbreed- to produce variable numbers of seeds. In addition, the ing depression, whereas the traditional equation to results must be carefully interpreted as plants may shunt estimate ∂ does not (Ågren & Schemske 1993). We also did resources to flowers receiving higher quality pollen, and not use the traditional estimate of W for the relative per- those flowers may produce fruits containing higher- formance indices because we did not follow offspring to quality seeds (Mooney & McGraw 2007). To determine the flowering, and we therefore lack a complete estimate of relationship between female reproductive success (i.e. offspring fitness (W) of different crosstypes. The RP index seed production) and maternal plant size, we used a ranges from -1to+1, and positive values indicate inbreed- general linear model with plant height as the independent ing depression. variable and seeds/fruit as the dependent variable. Similarly, we used the relative performance index of We used a Poisson loglinear model to determine intrasite and intersite outcross laboratory pollen crosses whether the probability of germination (0/6–6/6 germi- as an estimate of outbreeding depression as Equation 2: nants) differed significantly among pollination crosses. A []− = zzintra inter Poisson distribution was chosen because it best fits the RPo (2) count data. We designated the number of germinants zmax as the dependent variable and pollination treatment as where zintra is the mean performance of intrasite progeny, the predictor variable. We analyzed the data separately zinter is the mean performance of intersite progeny, and for differences in germination in laboratory and field zmax = zintra when zintra > zinter and zmax = zinter when zinter > zintra. crosses. Positive values indicate outbreeding depression. A growth-chamber malfunction after seedling germina- We used the following equation to examine whether tion reduced the sample sizes of seedlings. Thus, we were offspring resulting from crosses between isolated popula- only able to compare offspring performance between tions exhibited heterosis (i.e. hybrid vigor): intersite-outcross and geitonogamous-self progeny. []− Although our offspring sample sizes were reduced, we zzinter intra H = (3) were still able to compare later-offspring traits (number of zintra leaves, leaf area and height) of the seedlings that survived.

All offspring examined for later-offspring performance The value zintra represents the mean performance of traits were the progeny of laboratory-raised plants. intrasite progeny and zinter represents the mean perfor- Because offspring performance estimates may be corre- mance of intersite progeny. Positive values indicate het- lated, we initially ran a manova. We assigned treatment as erosis (Busch 2006). a ﬁxed factor and offspring height (cm), number of leaves We estimated RPi,RPo and H for all early offspring and leaf area (mm2) as response variables. We also carried traits, including seeds/fruit, seed mass, probability of ger- out univariate tests for all response variables following a mination and the cumulative-offspring measure (seed signiﬁcant manova. We carried out the following transfor- mass ¥ probability of germination). Because all intersite- mations for offspring performance data to meet anova outcross pollinations were carried out in the laboratory,

Plant Species Biology 26, 244–253 © 2011 The Authors Journal compilation © 2011 The Society for the Study of Species Biology 248 L. H. BERMINGHAM AND A. K. BRODY we only used geitonogamous-self and intrasite-outcross (a) treatments carried out in the laboratory for the relative performance indices. We generated 95% conﬁdence intervals to test whether the RPi,RPo and H indices deviated signiﬁcantly from zero. Our statistical analyses were carried out using the JMP version 7.0 (SAS 2000) and SPSS version 16.0 statistical software programs (SPSS 2001).

Results Differences in early offspring traits Pollen source had a significant effect on early offspring traits for all treatment plants, including those that were hand-pollinated in the field and in the growth chambers. (b) The number of seeds per fruit was significantly different among pollination treatments for plants pollinated in the field (F4,156 = 4.99, P = 0.0008) and in the growth chambers (F4,52 = 3.24, P = 0.02). In the field, flowers from the geitonogamous-self, intrasite-outcross and open- pollinated controls set equivalent seeds/fruit, and autonomous-self flowers set the fewest seeds/fruit (Fig. 1a). The same trend was observed for the pollen crosses carried out in the laboratory as autonomously- selfed flowers set the fewest seeds. However, there was no statistical difference between autonomous-self, intersite- outcross or intrasite-outcross flowers in the laboratory crosses; the only significant difference was between geitonogamous-self and autonomous-self flowers (Fig. 1b). There were no significant differences between the number of seeds/fruit set by plants in natural populations compared with those raised in a growth chamber Fig. 1 Differences in female reproductive success of Polemonium

(F1,299 = 0.02, P = 0.88). Parent height in the ancova models vanbruntiae among pollination treatments in (a) field-pollinated was not significant in any of the analyses and therefore the plants and (b) laboratory-pollinated plants. The numbers under results are presented without the covariate. Seed mass was the x-axis pollination treatments refer to the sample size (number of flowers) for each pollination treatment. Error bars represent not different among hand-pollination treatments the standard error of the mean. Pollination treatments with the F = P = ( 4,161 0.64, 0.64). Larger plants, based on plant height, same lowercase letter do not differ significantly. produced significantly more seeds/fruit than their smaller counterparts (F1,92 = 11.19, P = 0.001; Fig. 2). Only the offspring resulting from field-raised parents Nonetheless, we did detect significant differences in later exhibited significant differences in probability of germi- offspring traits between progeny resulting from outcross nation among the pollination treatments (field: c2 = 8.1, and self pollination. Intersite-outcross seedlings per- P = 0.04, d.f. = 3; Fig. 3a; laboratory: c2 = 4.8, P = 0.19, formed significantly better than the geitonogamous-self d.f. = 3; Fig. 3b). Seeds from intersite pollen crosses tended seedlings in the number of leaves produced (F = 17.03, to have the highest probability of germination, but inter- 1,7 P = 0.006; Fig. 4a), leaf area (F = 18.83, P = 0.005; Fig. 4b) site germination was statistically equivalent to open- 1,7 and seedling height (F = 29.82, P = 0.002; Fig. 4c). pollinated, intrasite and geitonogamous seedlings. 1,7 Autonomously selfed seeds had the lowest probability of germination. Heterosis and relative performance of offspring Owing to the growth chamber malfunction reducing the sample sizes of seedlings, we were only able to compare Overall, intersite-outcross offspring were of higher offspring performance between the laboratory pollinated quality than geitonogamous-self offspring, indicated by intersite-outcross and geitonogamous-self seedlings. positive RPi values for seed mass, probability of germina-

(a)

Fig. 2 Female reproductive success as a function of plant size (r2 = 0.11).

(b)

(c)

Fig. 3 Probability of germination of Polemonium vanbruntiae among pollination treatments in (a) ﬁeld-pollinated plants and (b) laboratory-pollinated plants. Error bars represent the stan- Fig. 4 Comparison of early offspring traits of Polemonium van- dard error of the mean. Pollination treatments with the same bruntiae seedlings resulting from intersite-outcross and lowercase letter do not differ signiﬁcantly. geitonogamous-self pollination treatments. (a) Number of true leaves, (b) leaf area and (c) seedling height. Error bars represent the standard error of the mean. tion and the cumulative measure of offspring quality Intrasite-outcross plants produced more and larger off-

(Fig. 5a). Yet, RPi was signiﬁcantly greater than zero for spring than intersite-outcross treatment plants, as indi- germination (RPi = 0.11) and cumulative offspring quality cated by the signiﬁcant RPo index for seeds/fruit and seed

(RPi = 0.11), but was not significant for seed mass (i.e. 95% mass (seeds/fruit: RPo = 0.21; seed mass: RPo = 0.13; confidence intervals overlapped zero). Fig. 5b). The RPo index was significantly less than zero for

germination (RPo = –0.16), implying that intersite offspring germinated at a higher rate than intrasite-outcross offspring. This trend is again observed as significant heterosis for intersite-outcross offspring germination (Fig. 5c). The cumulative measure of offspring quality for heterosis was not significantly different from zero (H = 0.07); thus, heterosis is only significant at the seedling germination stage (H = 0.20).

Discussion We found that pollen source has a significant effect on reproductive success and early offspring traits in P. vanbruntiae. Open-pollinated, intrasite-outcross and geitonogamous-self treatments did not differ in reproductive success, which may indicate that natural populations of P. vanbruntiae exhibit low genetic variation because of either a lack of gene flow, past population bottlenecks or both (Charlesworth & Charlesworth 1987). The combina- tion of simultaneously open flowers on a single plant and clonal reproduction in P. vanbruntiae increases the probability of geitonogamy as pollinators regularly visit multiple flowers on the same genetic individual, thus increasing the selfing rate (de Jong et al. 1992; Eckert 2000). Plants receiving only autonomous-self pollen con- sistently performed most poorly in terms of the number of seeds/fruit and germination. Intersite-outcross seedlings germinated at higher frequencies, had more, larger leaves, and were taller than offspring produced from geitonogamous-self pollen. Although self-pollination may provide some reproductive assurance of offspring production in P. vanbruntiae, the result is a reduction in germination and size-related early offspring traits for geitonogamously selfed offspring. Self-fertilization is expected to expose and purge deleterious alleles from populations, therefore reducing levels of inbreeding depression, particularly at loci affect- ing early life-history traits (Lande & Schemske 1985; Char- lesworth & Charlesworth 1987; Husband & Schemske 1996; Byers & Waller 1999). Yet, in selfing populations, the Fig. 5 Relative performance (RP) and heterosis (H) estimates of mutation rate may be high enough to overcome the effects early offspring traits in the rare plant Polemonium vanbruntiae. of purging and maintain inbreeding depression (Ågren &

Following the tradition of Ågren and Schemske (1993), RPi is an Schemske 1993). In fact, deleterious alleles having a small estimate for inbreeding depression (a), RPo is an estimate for effect may also become fixed as a result of genetic drift in outbreeding depression (b) and H is an estimate of heterosis (c). small populations. Based on our detection of heterosis at The cumulative function is the product of seed mass and prob- the germination stage as a result of outcrossing from a ability of germination. The RP and H estimates are significant if distant site, small, isolated populations of P. vanbruntiae the 95% confidence intervals do not overlap zero and if the RP and H values are greater than zero. Significant values are denoted may contain a substantial mutational load caused by drift by an asterisk. (Crow 1948; Levin 1984; Ouborg & van Treuren 1994; Paland & Schmid 2003; Busch 2006). It is also plausible that the clonal nature of P. vanbruntiae has limited the purging of deleterious alleles that act on early seed traits (Hill et al. 2007).

Sex allocation theory (Charnov 1982) speculates that in germination; autonomous-self seeds germinated at a increased plant size and large floral displays should significantly lower rate than geitonogamous-self seeds. If increase male reproductive success (i.e. pollen deposi- deleterious alleles were responsible for decreased ger- tion), but female reproductive success (i.e. seed produc- minability, then both types of selfing should have low tion) may decrease with plant size if geitonogamous-self germination. Instead, the low germination rate for the offspring are of lesser quality (de Jong et al. 1993). We did autonomously self cross may result from older pollen not find significant differences in female reproductive because P. vanbruntiae exhibits temporal separation of success between geitonogamous-self and outcross male and female function (dichogamy) where the anthers progeny, but did find that geitonogamously produced off- dehisce pollen prior to the stigma becoming receptive. spring are of lesser quality when compared with intersite- When the stigma becomes receptive and can receive outcross offspring. In addition, smaller plants produce pollen, within-flower pollen is no longer freshly dehisced. fewer seeds/fruit; thus, size affects female reproductive In addition, P. vanbruntiae has spatial separation of success in P. vanbruntiae. Taken together, these results anthers and stigma (herkogamy) where the stigma is may indicate selection against increased plant size and exserted beyond the anthers and corolla (Hill et al. 2008). large floral displays in P. vanbruntiae in order to avoid For this reason, few pollen grains may be reaching the inbreeding depression and the production of inferior gei- receptive stigma without pollinator assistance. Finally, tonogamously selfed offspring. autonomous pollen was from a single donor, whereas in We expect self-fertilization to be maintained in popula- the four other crosses pollen was collected from multiple tions of P. vanbruntiae because natural selection will favor flowers or donor plants. Young and Stanton (1990) decreases in selfing if the inbreeding depression index is showed that when pollen is received from multiple >0.5 (Byers & Waller 1999). Our estimates of inbreeding donors and there is competition among pollen grains, the depression fall far below this threshold. Husband and pollen of superior quality fertilizes the ovules. In general, Schemske (1996) found average inbreeding depression the quality of offspring from multiple-donor pollinations levels of 0.23 for self-fertilizing species and 0.53 in pre- has been shown to be higher than that resulting from dominantly outcrossing angiosperms. Polemonium van- single-donor crosses (Schemske & Paulter 1984; Young & bruntiae is fully self-compatible (Hill et al. 2008), so low Stanton 1990), as was shown here with the single-donor levels of inbreeding depression as a result of the purging autonomous-self pollen cross. of deleterious alleles is to be expected in this rare plant We detected outbreeding depression for female repro- (Charlesworth & Charlesworth 1987) because the genetic ductive success in plants receiving intersite-outcross load has been exposed to selective pressures (Eckert & pollen. However, these effects may be negligible as plants Barrett 1994). Inbreeding depression was most prevalent receiving outcross pollen from a distant site had a higher at the germination stage, which is an important stage in probability of germinating, thus exhibiting heterosis for the life cycle for establishment in natural populations. In the germination stage. Offspring that have a higher germi- addition, previous studies of P. vanbruntiae and other nation rate and grow significantly faster are more vigorous clonal species have found that seedling survival and and may outcompete neighboring plants for resources growth contribute significantly to future population under natural conditions, thus giving them a selective growth, highlighting the importance of offspring recruit- advantage. Previous studies in other plant species have ment, establishment and survival in order for rare plant also found increased performance of first generation populations to persist over time (de Kroon et al. 1987; hybrids relative to parents; however, hybrid fitness can Kéry et al. 2000; Hill Bermingham 2010). decrease in later generations owing to the breakup of Although we detected inbreeding depression in the co-adapted gene complexes (Hufford & Mazer 2003). relative performance index RPi for germination, we must Our findings corroborate studies examining the off- be cautious in our interpretation of these results. First, we spring quality of long-lived perennial plants with mixed assessed offspring quality and performance under green- mating systems existing within small populations. For house conditions, whereas effects of inbreeding depres- example, Mooney and McGraw (2007) examined off- sion are often magnified under field conditions spring quality in self and cross-pollinated populations of (Goodwillie et al. 2005). However, field experiments are Panax quinquefolius and found a significant reduction in inherently risky because of stochastic events that may leaf area and height in selfed offspring when compared obscure differences in offspring performance and under- with outcross offspring. Michaels et al. (2008) detected estimate inbreeding depression (Byers & Waller 1999). substantial inbreeding depression in populations of the Ultimately, though, sowing seeds in a natural environ- perennial plant Lupinus perennis because self-pollination ment simulates the actual environment in which the reduced seed production, seedling emergence and seed- plants will mature (Schemske, 1983). Second, seeds from ling growth. As in these studies, our experiment did not the two self-pollen crosses showed significant differences follow the offspring to reproduction because of the mul-

Plant Species Biology 26, 244–253 © 2011 The Authors Journal compilation © 2011 The Society for the Study of Species Biology 252 L. H. BERMINGHAM AND A. K. BRODY tiple years it takes for long-lived perennial plants to Hypochoeris radicata: experiments with plants from three Euro- flower. This limits our ability to measure the cumulative pean regions. Biological Conservation 132: 109–118. effects of inbreeding depression on offspring survival and Busch J. W. (2006) Heterosis in an isolated, effectively small, and self-fertilizing population of the flowering plant Leavenwor- fitness to determine whether inbreeding depression poses thia alabamica. Evolution 60: 184–191. significant fitness consequences for P. vanbruntiae. Buza L., Young A. & Thrall P. (2000) Genetic erosion, inbreeding However, if selfed offspring suffer from a reduction in and reduced fitness in fragmented populations of the endan- survival to reproductive maturity, there could be selection gered tetraploid pea Swainsona recta. Biological Conservation against self-fertilization in P. vanbruntiae populations 93: 177–186. (Eckert & Allen 1997; Morgan et al. 1997). Byers D. L. & Waller D. M. (1999) Do plant population purge their For plants like P. vanbruntiae with a mixed mating genetic load? Effects of population size and mating history on inbreeding depression. Annual Review of Ecology and System- system, self-pollination can provide reproductive assur- atics 30: 479–513. ance when pollen is scarce. However, even self-fertile Charlesworth D. & Charlesworth B. (1987) Inbreeding depres- species can have inbreeding depression at various life- sion and its evolutionary consequences. Annual Review of history stages, as we observed in P. vanbruntiae, which can Ecology and Systematics 18: 237–268. result in reduced offspring quality of selfed progeny. Charnov E. L. (1982) The Theory of Sex Allocation. Princeton Uni- Genetically mixing plant populations has been shown to versity Press, Princeton, NJ. lead to substantial levels of heterosis in the survival and Crow J. F. (1948) Alternative hypotheses of hybrid vigor. Genetics 33: 477–487. reproduction of interpopulation hybrid offspring (Levin Deller M. B. (2002) Polemonium vanbruntiae Britton (Appalachian 1984; van Treuren et al. 1993; Heschel & Paige 1995; Paland Jacob’s ladder) New England Plant Conservation Program & Schmid 2003) and may be an effective conservation Conservation and Research Plan for U.S. Forest Service management strategy for P. vanbruntiae populations if Region 9. New England Wild Flower Society, Framingham, hybrid fitness does not decrease in later generations. 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