Southeastern Naturalist 405 M.A. Burt and L.A. Brudvig 22001199 SOUTHEASTERN NATURALIST 1V8o(3l.) :1480,5 N–4o1. 83 Pollen Limitation and Self-Compatibility in Three Pine Savanna Herbs Melissa A. Burt1,* and Lars A. Brudvig2 Abstract - There is substantial interest in the restoration of the Pinus palustris (Longleaf Pine) savannas in the southeastern US Coastal Plain, one of the most endangered ecosystems in the world and also home to a diverse plant assemblage. Better understanding of the pollination ecology of these plants is necessary for their successful conservation and restoration. In this study, we assessed the rates of self-compatibility and pollen limitation in Carphephorus bellidifolius (Sandywoods Chaffhead), Liatris squarrulosa (Appalachian Blazing Star), and Aristida beyrichiana (Wiregrass)—3 perennial understory herbaceous species of conservation interest. We measured self-compatibility with a pollination-exclusion– bag experiment and pollen limitation with a pollen-supplementation experiment within South Carolina populations. We found no evidence of pollen limitation in any of these 3 species. This result is surprising given the high incidence of pollen limitation typically found in other study systems. Our pollination-exclusion bag experiment showed that Sandywoods Chaffhead likely requires out-cross pollen for successful pollination, whereas both Appalachian Blazing Star and Wiregrass appear to exhibit at least low levels of selfcompatibility. Taken together, our results may indicate that the active management of the Longleaf Pine ecosystem with prescribed fire and overstory tree removal has supported sufficient pollination in these plant populations. Introduction Currently reduced to less than 3% of their original range, Pinus palustris Mill. (Longleaf Pine) ecosystems are among the most endangered habitats in North America (Frost 2006; Noss 1988, 2013; Outcalt 2000; Outcalt and Sheffield 1996). Longleaf Pine ecosystems are part of the southeastern US Coastal Plain biodiversity hotspot and support species-rich plant assemblages, including at least 191 endemic species (Hardin and White 1989, Noss 2013, Noss et al. 2014, Peet and Allard 1993). Most of this diversity is contained in the shrubby and herbaceous ground layer, which can support >50 plant species per square meter (Walker and Peete 1984). As a result, there is substantial interest in the conservation and restoration of Longleaf Pine ecosystems (Brudvig et al. 2014, Noss 2013, Noss et al. 2014, Walker and Silletti 2005). Given the interest in re-introducing these populations (Frost 2006, Walker and Silletti 2005), and the fragmented nature of Longleaf Pine landscapes (Frost 2006), an understanding of the pollination biology of ground layer species will be critical to assist these conservation and restoration efforts. 1Kellogg Biological Station, Michigan State University, Hickory Corners, MI 49060. 2Department of Plant Biology and Program in Ecology, Evolutionary Biology, and Behavior, Michigan State University, East Lansing, MI 48824. *Corresponding author - melissa.ann.burt@gmail.com. Manuscript Editor: Justin Hart Southeastern Naturalist M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 406 An understanding of pollination ecology is essential for the successful conservation and restoration of plant diversity (Schemske et al. 1994). Reductions in plant population size and increased isolation among populations from habitat loss, fragmentation, and the creation of small founder populations during reintroduction can affect pollen transfer among individuals (e.g., Aguilar et al. 2006, Rusterholz and Baur 2010, Winfree et al. 2009). As a result, pollen limitation may develop when receipt of pollen is insufficient for adequate seed or fruit production, which, in turn, may affect population growth or persistence. Such effects will be most pronounced in obligate out-crossing species (i.e., species that require pollen from other individuals for successful fertilization) and less or not evident in species that are capable of self-pollination (Burd 1994, Larson and Barrett 2000, Schemske et al. 1994). Yet, the details of the pollination ecology of many plant species remain unknown (e.g., self-compatibility and evidence for pollen limitation), and further studies are needed to assess the extent to which pollen limitation may be an important consideration during conservation and restoration activities. Pollen limitation is a well-documented, widespread phenomenon in flowering plants. In fact, reviews and syntheses on the topic have reported that pollen limitation occurs in a majority of published cases, with an estimated 62–73% of plant species exhibiting pollen limitation at some time or place (Ashman et al. 2004, Burd 1994, Knight et al. 2005, Larson and Barrett 2000). As a result of lower seed production, species subject to pollen limitation may subsequently undergo a variety of demographic consequences due to low levels of subsequent recruitment, potentially leading to a reduction in population size. Pollen limitation over long periods of time may even result in evolutionary changes, such as the ability to self-fertilize (Vallejo-Marín and Uyenoyama 2004). We experimentally assessed the degrees of pollen limitation and self-compatibility in 3 understory herbs associated with the Longleaf Pine ecosystem: Carphephorus bellidifolius (Michx.) Torr. and A. Gray, (Asteraceae; Sandywoods Chaffhead), Liatris squarrulosa (Green) K. Schum (= L. earlei [Greene] K. Schum.) (Asteraceae; Appalachian Blazing Star), and Aristida beyrichiana Trin. and Rupr. (Poaceae; Wiregrass); taxonomy according to Radford et al. (1968). To our knowledge, pollen limitation and self-compatibility have not been investigated in these species. We focused on these species because they are indicative of high quality (e.g., frequently burned, lacking agricultural histories) Longleaf Pine sites (Brudvig and Damschen 2011, Brudvig et al. 2014). Moreover, these species constitute major herbaceous life forms present in Longleaf Pine understory communities (graminoids and forbs), including the dominant bunchgrass, Wiregrass, which is often a focal species during understory reintroduction efforts (Aschenbach et al. 2010, Walker and Silletti 2005). Specifically, we asked: (1) What is the magnitude of self-compatibility in Sandywoods Chaffhead, Appalachian Blazing Star, and Wiregrass? and (2) Are populations of these species pollen limited? Southeastern Naturalist 407 M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 Field-site Description We performed this experiment at the Savannah River Site (SRS), a Department of Energy National Environmental Research Park located near Aiken, SC (33°12'N, 81°24'W). SRS contains thousands of hectares of upland Longleaf Pine savannas undergoing restoration through prescribed fire and overstory tree thinning. We worked at 5 locations at SRS, to evaluate 2 populations for each of the 3 study species. Both populations of Sandywoods Chaffhead and Appalachian Blazing Star and 1 population of Wiregrass were naturally occurring populations located within remnant (i.e., no known history of use as a plowed farm field) Longleaf Pine restoration areas that supported canopies with cover varying from 29.8% to 41.8% (mean = 36.3%; data from Brudvig and Damschen 2011). Our second Wiregrass population consisted of planted individuals used in seed-production efforts, located in a largely treeless field that is burned annually. Sites were underlain by sandy soil series: Ailey, Blanton, Lucy, Troup, and Vaucluse (USDA-NRCS 2012). Each population contained at least 50 individuals. Methods Study species We assessed pollen limitation and self-compatibility for 3 perennial herbaceous species that can be found in sandy uplands of the Longleaf Pine ecosystem: Sandywoods Chaffhead, Appalachian Blazing Star, and Wiregrass. Both Sandywoods Chaffhead and Appalachian Blazing Star are forbs that form basal rosettes, whereas Wiregrass is a bunchgrass; none of these species is known to be clonal. Based on plant family, floral morphology, and our field observations, Sandywoods Chaffhead and Appalachian Blazing Star are pollinated by insects, whereas Wiregrass is wind-pollinated. When mature, Sandywoods Chaffhead produces flowering stalks containing an average of 39.5 (max = 374) inflorescences, each of which contains an average of 20.6 (min–max = 2–71) flowers (based on n = 275 individuals; L.A. Brudvig, unpubl. data). When mature, Appalachian Blazing Star produces flowering stalks containing an average of 11.4 (max = 117) inflorescences, each of which contains an average of 13.3 (min–max = 5–28) flowers (based on n = 237 individuals at SRS; L.A. Brudvig, unpubl. data). When mature, Wiregrass produces culms containing an average of 35.1 (max = 279) inflorescences, each of which contains an average of 66.9 (min–max = 9–184) flowers (based on n = 286 individuals at SRS; L.A. Brudvig, unpubl. data). Sandywoods Chaffhead flowers from August through October, Appalachian Blazing Star flowers from September through October, and Wiregrass flowers from September through November (Radford et al. 1968). Experimental methodology Prior to anther dehiscence (opening of the anther for dispersal of pollen), we haphazardly selected 10 individuals within each population of each of our 3 focal species. To determine self-compatibility and degree of pollen limitation, we Southeastern Naturalist M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 408 applied each of the following 4 treatments to a randomly chosen inflorescences on each individual plant: (1) outcross-pollen supplementation, (2) non-manipulated natural pollination control (control for outcross pollen supplementation treatment), (3) pollination-exclusion bag, and (4) leaky bag control (i.e., a pollination-exclusion bag with holes to serve as a control for pollination-exclusion bag treatment). We monitored inflorescences every 2–3 d for anther dehiscence. We conducted our experiments from October 2009 through January 2010. We assessed pollen limitation by comparing the outcross pollen supplementation and non-manipulated natural-pollination control treatments (Bierzychudek 1981). We collected supplemental pollen from the anthers of open inflorescences of at least 10 plants located within each population, but at least 10 m away from experimental plants (Kearns and Inouye 1993). We did not collect supplemental pollen from individuals that had been selected for our pollen limitation and self-compatibility experiments. On the day it was to be used, we collected pollen by sweeping anthers of recently dehisced inflorescences with a paintbrush and allowing the pollen to fall into a petri dish. After homogenizing the pollen within the petri dish, we applied the supplemental pollen to the stigmas in the inflorescences pre viously marked for the outcross pollen supplementation experiment. We monitored supplemented and control inflorescences every 2–3 days until seeds were visibly mature. Upon maturation, we collected the inflorescences, counted all reproductive structures (achene or caryopsis), and determined whether or not each reproductive structure contained a seed by squeezing it with forceps. We concluded that individuals within each population were pollen limited when outcross-pollen–supplemented inflorescences produced more seeds or fruit than non-manipulated controls (Bierzychudek 1981, Kearns and Inouye 1993, Young and Young 1992). We assessed self-compatibility with an experiment to compare the use of pollination-exclusion bags with self pollen applied to the inflorescences within (hereafter, referred to as pollination-exclusion bag) and leaky-bag control treatments (hereafter referred to simply as control treatments) (Kearns and Inouye 1993). To prevent outcross pollen from coming in contact with floral structures by pollinator or wind, we covered inflorescences with pollination-exclusion bags made of white chiffon fabric. For Sandywoods Chaffhead and Wiregrass, we attached the pollination-exclusion bags to a wire frame affixed to a bamboo stake that had been pushed into the ground in order to prevent the pollination-exclusion bags from weighing down the inflorescence. We attached the pollination-exclusion bags directly to Appalachian Blazing Star inflorescences without a wire frame because the stems were sturdy enough to support them. Also, Appalachian Blazing Star inflorescences are typically located along the plant’s stem attached with little to no petiole, which made it difficult to attach a wire frame. After installing the pollination-exclusion bags on previously designated inflorescences prior to dehiscence, we monitored the inflorescences for anther dehiscence every 2–3 days. Upon dehiscence, we hand-pollinated the inflorescence with self-pollen to ensure the transfer of self pollen from anther to stigma. For the control treatment, we attached a leaky bag to another randomly selected inflorescence. As in the pollen limitation Southeastern Naturalist 409 M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 experiment, we monitored inflorescences every 2–3 days for seed maturation and collected inflorescences when seeds matured. We compared the number of seeds formed on the inflorescences in the pollination-exclusion bag to those formed in the leaky bag control to assess levels of self-compatibility for each species. We did not supplement outcross pollen to the leaky bag control treatment; thus, pollen limitation could complicate interpretation of this comparison. Statistical analysis We tested for treatment effects on the number of developed seeds using mixed model ANOVA with a completely randomized block design for each species. We designated “plant” as a random block effect and “treatment” as a fixed effect. We also performed separate mixed model ANOVAs on each population of each species to determine if results were consistent among populations. We did not include population as a random effect in these models because of a lack of replication of populations; however, when included as a fixed effect in the model, we detected similar results. We used Tukey post hoc tests to determine significant differences among treatments. Specifically, we determined the degree of pollen limitation by comparing the number of seeds in the non-manipulated natural pollination control inflorescence with the number seeds in the outcross pollen supplementation treatment. We assessed self-compatibility by comparing the number of seeds produced by the inflorescences with pollination-exclusion bags to those covered with leakybags. To adhere to normality assumptions of ANOVA, we square-root–transformed the number of developed seeds. However, we provide untransformed data in our figures for ease of interpretation. We conducted all statistical analyses in R version 3.0.2 (R Core Team 2014). Appalachian Blazing Star individuals were located in a restored Longleaf Pine savanna that was subject to mowing and overstory tree thinning prior to the end of our experiment. As a result, we lost 3 Appalachian Blazing Star individuals from each population; thus, analyses are based on 7 individuals at each of these populations. Results Sandywoods Chaffhead When pooling populations, the mean number of seeds developed by Sandywoods Chaffhead inflorescences differed among treatments (F3,53 = 47.1, P < 0.001; Fig. 1) with inflorescences that received the pollination-bag treatment producing 90% fewer seeds than those that received the leaky bag control treatment (Tukey: P < 0.001; Fig. 1). This finding suggests that Sandywoods Chaffhead generally requires out-cross pollen for successful pollination in the populations we studied. Unbagged inflorescences that received the outcross pollen supplement treatment produced a similar number of seeds as the non-manipulated control (Tukey: P > 0.05, Fig. 2) indicating Sandywoods Chaffhead was not pollen limited in the populations we studied. Similar results were apparent for each population of Sandywoods Chaffhead. The mean number of seeds produced by inflorescences differed among treatments Southeastern Naturalist M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 410 (Population A: F3,25 = 33.7, P < 0.0001; Population B: F3,25 = 19.7, P < 0.0001; Fig. 1). The mean number of seeds produced by non-manipulated controls did not differ from inflorescences receiving outcross supplemental pollen in both populations of Sandywoods Chaffhead (Tukey: P > 0.05 in both cases; Fig. 2). Inflorescences inside a pollination-exclusion bag produced 82% and 94% fewer seeds than the leaky control bag in populations A and B, respectively (Tukey: P less than 0.05 in both cases; Fig. 1) suggesting again that Sandywoods Chaffhead rarely exhibits self-compatibility. Appalachian Blazing Star Although the results pooled across populations indicate that Appalachian Blazing Star is not pollen limited and has the capacity for self-fertilization (F3,34 = Figure 1. Mean number of Sandywoods Chaffhead seeds present in pollination-exclusion bags with self pollen applied versus leaky control bags to test for self-compatibility. Error bars represent standard error around the mean. An asterisk (*) denotes significant differences between treatments. Southeastern Naturalist 411 M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 5.4, P = 0.004; Figs. 3, 4), these results diverged for individual populations. For population A the number of seeds produced differed among treatments (F3,15 = 7.0, P = 0.004; Figs. 3, 4), but this pattern was not evident in Population B (F3,16 = 2.8, P = 0.07; Figs. 3, 4). We did not find that either population of Appalachian Blazing Star exhibited pollen limitation, however inflorescences in population A that received the outcross pollen supplement produced 66% fewer seeds than the nonmanipulated, unbagged control (Tukey: P = 0.003; Fig. 4). The 2 populations of Appalachian Blazing Star also diverged in their rates of self-compatibility. Population A produced 78% fewer seeds in the pollination-exclusion bag treatments than control bags (Tukey: P = 0.03; Fig. 3), while population B produced a similar quantity of seeds in both (Tukey: P > 0.05; Fig. 3). These results suggest that population B appears to exhibit a degree of self-compatibility, while individuals in population A do not. Figure 2. Mean number of Sandywoods Chaffhead seeds present in unbagged inflorescences that received outcross supplemental pollen versus a natural-pollination control to test for pollen limitation. Error bars represent standard error around the mean. None of the differences between treatments was significant. Southeastern Naturalist M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 412 Wiregrass We did not find differences in Wiregrass among treatments for the number of seeds produced when pooling data for all individuals (F3,51 = 1.9, P = 0.14; Figs. 5 , 6). This evidence suggests that Wiregrass is self-compatible, and not pollen limited.These patterns were also apparent in each Wiregrass population. Figure 3. Mean number of Appalachian Blazing Star seeds present in pollination-exclusion bags with self pollen applied versus leaky control bags to test for self-compatibility. Error bars represent standard error around the mean. An asterisk (*) denotes significant differences between treatments. We do not present pooled results here because of the difference between populations. Figure 4. Mean number of Appalachian Blazing Star seeds present in unbagged inflorescences that received outcross supplemental pollen versus a natural-pollination control to test for pollen limitation. Error bars represent standard error around the mean. An asterisk (*) denotes significant differences between treatments. We do not present pooled results here because of the difference between populations. Southeastern Naturalist 413 M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 For population A, we detected no differences among treatments for number of seeds produced (F3,23 = 1.8, P = 0.17; Figs. 5, 6). Although we did find differences among treatments for population B for number of seeds produced (F3,25 = 3.835, P = 0.02; Figs. 5, 6), this result appears to be due to a significant difference between the pollination-exclusion bag and the non-manipulated control (Tukey: P < 0.05), and not a difference between the former and the leaky bag control (Tukey: P > 0.05). Discussion We found no evidence of pollen limitation in Sandywoods Chaffhead, Appalachian Blazing Star, or Wiregrass in the populations we studied. This result is surprising given the fragmented nature of Longleaf Pine savanna systems (Frost 2006) and the high incidence of pollen limitation found in other study systems (Ashman et al. 2004, Burd 1994, Knight et al. 2005, Larson and Barrett 2000). Our pollination-exclusion bag experiment indicated that while Figure 5. Mean number of Wiregrass seeds present in pollination-exclusion bag with hand self-pollination versus leaky control bags to test for self-compatibility. Error bars represent standard error around the mean. An asterisk (*) denotes significant differences between treatments. Southeastern Naturalist M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 414 Sandywoods Chaffhead likely requires out-cross pollen for successful pollination, both Appalachian Blazing Star and Wiregrass appear to exhibit at least low levels of self-compatibility. However, for Appalachian Blazing Star, we detected population-level variability in self-compatibility; 1 population exhibited selfcompatibility, but the other did not. Pollen limitation is a prevalent phenomenon in many ecological systems (Ashman et al. 2004, Burd 1994, Knight et al. 2005, Larson and Barrett 2000), but pollen limitation was not evident in any of the species in our study. While our Longleaf Pine savanna study system is severely fragmented after being reduced to about 3 percent of its former range, it is possible that sufficiently large pollinator populations existed at our study sites, or that plant–pollinator interactions existed in highly nested networks that provided insurance for plants relying on biotic pollinators for reproductive success (Bascompte et al. 2003). In a landscape-fragmentation experiment located near our study sites, Brudvig et al. (2015) found that fragmentation Figure 6. Mean number of Wiregrass seeds present in unbagged inflorescences that received outcross supplemental pollen versus a natural-pollination control to test for pollen limitation. Error bars represent standard error around the mean. An asterisk (*) denotes significant differences between treatments. Southeastern Naturalist 415 M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 had no effect on the rate of pollination for the species in our present study, as well as others. Breland et al. (2018) showed that Longleaf Pine savanna restoration at our study location rapidly increased pollinator diversity and abundance, but had no effect on rates of pollination for a model mustard species. However, as neither Breland et al. (2018), Brudvig et al. (2015), nor our study included all flowering plants in the diverse plant community characteristic of Longleaf Pine savanna, it is difficult to make generalizations about the overall prevalence of pollen limitation in this system. Additional pollen-supplementation experiments on a wider range of species would provide a better portrait of the state of pollen limitation in Longleaf Pine savanna. For 1 population of Appalachian Blazing Star, we found that inflorescences receiving outcross supplemental pollen produced fewer seeds compared to control inflorescences. Although this finding might potentially indicate an issue with the methodology (i.e., floral structures may have been damaged in the process of receiving supplemental pollen), it is not necessarily an uncommon result (Young and Young 1992). The addition of supplemental pollen may represent an unnaturally high pollen load which may lead to competition or interference among pollen tubes or the attraction of pollen thieves that then remove the supplemental pollen (Ashman et al. 2004, Young and Young 1992). We suspect that this anomaly does not represent an issue with the methodology because we only detected it in a single population of 1 of 3 focal species; however it could be representative of a small sample size. All outcross pollen-supplementation experiments were conducted by the same person. The chronic occurrence of pollen limitation in ecological systems may lead to the development of self-compatibility over time (Vallejo-Marín and Uyenoyama 2004). Possible evidence of this possibility is in the lack of both pollen limitation and self-compatibility observed in Sandywoods Chaffhead. We also did not detect pollen limitation in the populations of Appalachian Blazing Star or Wiregrass in our study, though we found that individuals of these species developed seeds with self pollen. Our study, however, provides only a snapshot of a potentially temporally variable trait, and it is possible that past pollen limitation led to the evolution of self compatibility in these 2 species. Our study provides a detailed assessment of the reproductive biology of a few Longleaf Pine savanna herbs; however, we still know little about the prevalence of self-compatibility and pollen limitation across Longleaf Pine savanna plant communities. Although pollen limitation was not evident in the 3 plant species in our study, land-managers may still need to implement restoration methods to mitigate the pollen limitation in other species. Additional multi-year supplementation experiments on more species in the diverse Longleaf Pine understory are needed in order to assess the potential temporal variation in pollen limitation. With almost 90% of plants pollinated by biotic vectors (Ollerton et al. 2010) and the number of pollinators decreasing globally (Burkle et al. 2013, IPBES 2016), additional studies on the reproductive biology of plants at the scale of communities are necessary. In this way, we can better predict the potential effects of habitat fragmentation, habitat Southeastern Naturalist M.A. Burt and L.A. Brudvig 2019 Vol. 18, No. 3 416 loss, climate change, and the introduction of invasive species, all of which are current threats to Longleaf Pine savanna ecosystems (Knight et al. 2018). Acknowledgments We thank the National Science Foundation (DEB-0613701, DEB-0613975, and DEB- 0614333) and the USDA Forest Service and the Department of Energy at the Savannah River Site (Interagency Agreement DE-A09-00SR22188) for funding. We also thank the USDA Forest Service and the Department of Energy at the Savannah River Site for use of field sites and facilities at SRS. More specifically, we thank J. Blake, C. Hobson, A. Horcher, E. Olson, J. Segar, T. Thomas, and K. Wright. We are also grateful to the Corridor Research Group for helpful feedback on design and results including E. Damschen, M. Habenicht, N. Haddad, D. Levey, J. Orrock, J. Tewksbury, and S. Wagner. We received friendly and thoughtful reviews that improved this manuscript from T. Bassett and C. Zirbel. We thank 2 anonymous reviewers for feedback that helped us to revise an earlier version of this manuscript. Literature Cited Aguilar, R., L. Ashworth, L. Galetto, and M.A. Aizen. 2006. 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