The Influence of Biogeography and Mating System on the Ecology of Desert Annual Plants
Item Type text; Electronic Dissertation
Authors Gerst, Katharine Laura
Publisher The University of Arizona.
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THE INFLUENCE OF BIOGEOGRAPHY AND MATING SYSTEM ON THE ECOLOGY OF DESERT ANNUAL PLANTS by
Katharine Laura Gerst
______
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF ECOLOGY AND EVOLUTIONARY BIOLOGY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2011 2
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Katharine L. Gerst entitled “The Influence of Biogeography and Mating system on the Ecology of Desert Annual Plants” and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy.
______Date: October 28, 2011 D. Lawrence Venable
______Date: October 28, 2011 Judith Bronstein
______Date: October 28, 2011 Travis Huxman
______Date: October 28, 2011 Steve Smith
Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
______Date: October 28, 2011 Dissertation Director: D. Lawrence Venable 3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: Katharine L. Gerst 4
ACKNOWLEDGEMENTS
First and foremost I’d like to thank my advisor, Larry Venable. Larry provided me with the freedom to develop my research independently yet consistently encouraged me to approach my work through a critical and thorough lens. I am continually inspired by his vast knowledge of ecological and evolutionary theory and his enthusiasm for Sonoran desert natural history. I’d also like to thank my dissertation committee: Travis Huxman, Judie Bronstein, and Steve Smith. Travis’s optimism was the perfect remedy when I was in need of re-fueling and his insight into the world of plant physiology is priceless. Judie helped me to think critically about the implications of my work and encouraged me to share my knowledge with the GK-12 community, making me both a better scientist and a better science communicator. Steve pushed me to think broadly about my ideas and assumptions beyond the scales and systems I most often placed myself.
The current and former members of the Venable Lab have been there for constant support and camaraderie; thank you to Amy Angert, Sara Felker, Jenny Gremer, Jonathan Horst, Sarah Kimball and Eugenio Larios. This work couldn’t have been completed without hours of assistance in the lab by undergraduates Trevor Birt, Lauren Cmiel, Jackie Comisso, Nandi Devan, Adam Leitman, Tim Malan, and Huong Truong. My experiments in the greenhouse would not have been successful without the advice and assistance of Abreeza Zegeer.
I’d also like to thank my fellow graduate students past and present for your friendship and adventures here in Tucson, especially Greg Barron-Gafford, Anna Tyler, Erin Kelleher, Danielle Ignace, Kristen Potter, Claire Zugmeyer, Kevin Vogel, Joel Wertheim, Betsy Wertheim, Katie Massie and Matt Herron. It’s been a fabulous ride! My experience at the University of Arizona was made richer by the individuals who taught me about teaching: Kevin Bonine, Mitch Pavao-Zuckerman, and the BioME GK-12 coordinators and teacher mentors, particularly Stacey Forsyth, Kathleen Walker, and Carolyn Hollis. Finally, thanks to the EEB office staff for assistance with what seemed to be unending paperwork, travel plans, and broken computers.
I am incredibly grateful for my warm and supportive family. My mom and dad, Ellie and Ken Gerst, have always enthusiastically nurtured my pursuit of science- I could never thank you enough for all you’ve done to help me achieve my dreams. A special thanks to the entire Gerst, Feig, and Hunter families for all your love and encouragement. My husband Brian Hunter has shown me true patience, cheer, and partnership. Brian spent countless hours keeping me motivated while measuring plants, counting seeds and traveling to field sites. Finally, our son Isaac has provided me with endless joy and inspiration.
This work would not have been possible without the generous support of the University of Arizona Department of Ecology and Evolutionary Biology, Biosphere 2, Garden Club of America Desert Studies Award, California Desert Legacy Fund, McGinnies Scholarship in Arid Land Studies, NSF GK-12 BioME fellowship, and the Marshall Foundation Dissertation Fellowship. 5
DEDICATION
To my husband, Brian Hunter: Your love and support is unwavering, and your presence
calming. I share this accomplishment with you.
To our son, Isaac Hunter: Your smile lights up my world. Dream a dream little bear! 6
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………….7
I. INTRODUCTION…………………………………………………………...………….9
II.PRESENT STUDY…………………………………………………………………....16
REFERENCES…………………………………………………………………………..20
APPENDIX A. EFFECT OF RANGE POSITION ON DEMOGRAPHIC
VARIABILITY IN DESERT ANNUAL PLANTS…………….…………………….....28
APPENDIX B. PHENOLOGY MEDIATES POLLINATOR AND HERBIVORE
INTERACTIONS IN SELFING AND OUTCROSSING DESERT
ANNUALS………………………………………………………………………………68
APPENDIX C. DROUGHT EFFECTS ON PHENOLOGY AND PHYSIOLOGY IN
SELFING AND OUCROSSING DESERT ANNUAL PLANTS …………….……….106
APPENDIX D. RESOURCE LIMITATION AND PLANT PERFORMANCE IN
SELFING AND OUCROSSING DESERT ANNUAL PLANTS……………………...154 7
ABSTRACT
A major challenge in plant ecology is in understanding how species strategies mediate interactions between the environment and fitness. Variation in niche strategies that affect phenological, physiological, and reproductive traits will allow species to partition resources differently in space and time, allowing for coexistence of many species and strategies within a community. How species differentially respond to variable environments will ultimately influence their population dynamics and geographic distribution. This dissertation approaches this topic from two perspectives:
(a) examining the interaction between biogeography and variable demographic strategies in desert annual plants, and (b) examining the costs and benefits of contrasting reproductive strategies in co-occurring selfing and outcrossing desert annuals. Firstly, I tested the abundant center model to determine the role of range position on plant population dynamics. I examined how the geographic and climatic position of 13 desert annuals found at a common location, the Desert Laboratory at Tumamoc Hill in Tucson,
Arizona, related to their demography over a 25-year time span. I found that species for which the Desert Laboratory was close to the center of their geographic range have less variable long-term survival and fecundity compared to species for which the Desert
Laboratory was further from the center of their range. Secondly, I studied how related species with contrasting mating systems respond to variable environments to affect plant performance. In a three-year field study I investigated how inter-annual variation in plant reproductive phenology affects synchrony with pollinators and herbivores. Since selfing species are guaranteed to reproduce in the absence of pollinators, seasonal and annual 8
variation in phenology resulted in less variable plant reproductive success compared to outcrossing species. Greater variation in reproduction in outcrossing species resulted from asynchrony in some years between plants and pollinators. In a greenhouse study examining the interaction between mating system and drought, I found that the physiological functioning and survival of outcrossing species was more strongly negatively affected by drought conditions, suggesting that selfing species have an advantage in more arid environments. These studies demonstrate how plant reproductive and physiological strategies can play a critical role in influencing fitness, population dynamics and geographic distribution.
9
I. INTRODUCTION
One key challenge of plant ecology is to understand the mechanisms promoting the coexistence of species (Shmida and Ellner 1984, Chesson 2000, Chave 2004,
Agrawal et al. 2007, Weiher et al. 2011). Species within a community occupy a range of spatial and temporal niches in which they may employ different strategies encompassing their life history, phenology, species interactions, and physiology (Wright 2002, Tilman
2004, Adler et al. 2010). This variation in ecological strategies allows for the partitioning of resources over time and space between species (Schoener 1974). How species interact with the environment to affect fitness and coexistence has been a fruitful topic in the literature (e.g., Pake and Venable 1995, Chesson and Huntly 1997, Loehle 2000, Russo et al. 2008, De Frenne et al. 2009, Martinez-Vilalta et al. 2010). The diversity of strategies found in nature among plant species allows us to explore how variable environmental conditions may influence distribution and population dynamics. This dissertation approaches this topic from two perspectives: (a) examining the interaction between biogeography and variable demographic strategies in desert annual plants, and (b) examining the costs and benefits of contrasting reproductive strategies in co-occurring selfing and outcrossing desert annuals.
Research on desert annual plant communities has provided many valuable insights to the fields of physiological, population and community ecology. This work has contributed greatly to our understanding of bet hedging strategies (Philippi 1993, Erhman and Cocks 1996, Evans et al. 2007, Venable 2007), germination biology (Baskin et al.
1993, Clauss and Venable 2000, Adondakis and Venable 2004), competition (Kadmon 10
and Shmida 1990, Schwinning and Fox 1995), functional strategies (Forseth et al. 1984,
Gutierrez and Whitford 1987, Fox 1990, Ludwig et al. 2004, Angert et al. 2007, Huxman et al. 2008, Kimball et al. 2010), and species interactions (Inouye et al. 1980, Guo et al.
2000, Goldberg et al. 2001). Deserts tend to be highly variable environments in which plants depend on rainfall that is not consistent between years or sites (Schwinning and
Sala 2004, Bower 2005). Thus, species must have adaptations to cope with aridity as well as variability. The range of demographic and physiological strategies species use in this environment will influence both the niches they occupy in their community and their patterns of fitness within and between years and sites (Chesson et al. 2004).
Desert plant communities represent a diversity of biogeographic and evolutionary histories. For example, Sonoran Desert plants represent a convergence of taxa from multiple biogeographical regions, including the Madrean region and Mojave Desert
(McLaughlin 1986). Communities are composed of species with differences in range breadth and size that are related to their constraints in life histories, mating systems, and functional traits. The breadth and extent of species distributions can shed light on their adaptive strategies and plasticity; for instance, species with large vs. small ranges may differ in phenotypic plasticity (Sultan 2001, Chevin and Lande 2011). In addition, abiotic features that affect coexistence and population dynamics are expected to vary throughout species ranges (Antonovics 1976, Brown 1984, Kirkpatrick and Barton 1997, Guo et al.
2005). Strategies employed at the edge of species ranges have been shown to differ from those in the center as predicted by the abundant center model (e.g., Jump and Woodward
2003, Kluth and Bruelheide 2005, Samis and Eckert 2007, Krasnov et al. 2008), although 11
the ubiquity of this generalization has been called into question (Sagarin and Gaines
2002, Gaston 2003, Sexton et al. 2009). Aspects of range size and niche breadth are also found to be associated with reproductive traits, such as selfing rate and mating system
(Busch 2005, Lowry and Lester 2006, Randle et al. 2009).
Selfing and outcrossing mating systems represent two reproductive strategies with distinct evolutionary and ecological consequences in plants (Barrett 2003). Selfing occurs when ovules within a flower are fertilized by its own pollen, whereas outcrossing occurs when pollen is transferred from another individual. In co-occurring and related annual species, we find a continuum of reproductive strategies ranging from exclusive outcrossing to exclusive self-fertilization (Clay and Levin 1989). Given a much higher frequency of selfing in annuals than perennials (Lloyd 1980, Hamrick and Godt 1997), and the more frequent evolutionary transition from outcrossing to selfing (Barrett 2003), the annual life history strategy likely plays a predominant role in the evolution of self- fertilization (Aarssen 2000, Morgan and Wilson 2005). Autonomously self-fertilizing species are guaranteed reproductive assurance in the absence of pollinators (Baker 1955,
Stebbins 1957, Lloyd 1979, Lloyd 1992, Kalisz et al. 2004), but may suffer greater inbreeding depression (Charlesworth and Charlesworth 1987, Lloyd and Schoen 1992,
Herlihy and Eckert 2002). Despite the clear limitations of self-fertilization, it has obvious benefits, such as a genetic transmission advantage (Fisher 1941), a release from dependence upon biotic or abiotic pollination, and an advantage in colonization and range expansion (Baker 1955, Levin 2010). An outcrossing species benefits from the maintenance of higher genetic diversity and thus increased adaptive potential (Hamrick 12
and Godt 1996), but will lose opportunities to reproduce if it is not well synchronized to its pollinators or if pollinators are scarce (Miller-Rushing et al. 2010, Forrest and
Thomson 2011). These costs and benefits suggest tradeoffs between these mating systems that may provide a viable niche for both, and could help maintain coexistence in a variable environment.
Phenology, the timing of life events such as germination or reproduction, has strong associations with reproductive strategy and plays a crucial role in determining plant fitness. Phenological events can be triggered by climatic factors including temperature, rainfall and photoperiod (Bowers and Dimmit 1994) as well as habitat conditions, such as water availability (Aronson et al. 1992, Borchert 1994, Penuelas et al.
2004, but see Fox 1990). How species interactions are affected by variation in phenological strategies will be important in determining the distribution and dynamics of populations and communities (Gilman et al. 2010, Rafferty and Ives 2011). For desert annuals this is particularly crucial due to the inter-annual variability in rainfall that generally cues phenological responses (Beatley 1974, Crimmins et al. 2010) and the short period in which flowering takes place. Synchrony in species interaction, such as plants and their pollinators, is essential for the persistence of species dependent on those interactions (e.g., Waser 1979, Brody 1997, Suzuki et al. 2007). This presents a potential cost of an outcrossing strategy; depending on how synchronized pollinators are to flowering periods an outcrosser may not achieve reproductive success. The degree of synchrony plants and pollinators experience can result from both climatic triggers and resource availability to determine individual fitness and contribute to variable population 13
dynamics. Thus phenological dynamics can have profound effects on reproductive success and may function to promote or inhibit one strategy over the other in any given year, allowing for coexistence.
Plant physiology can mediate how plants interact with their environment and exhibit the strategies discussed above. Variation in physiological trait responses to environmental fluctuations is also expected to affect fitness and determine how plants cope with resource limitation (Aronson et al. 1993). Recent studies have investigated relationships between phenological and physiological traits associated with life history strategies, distributions, and habitats (e.g., Baskauf and Eickmeier 1994, Davies 1998,
Pohlman et al. 2005, Kudo et al 2008). In particular, a suite of physiological strategies have been identified that is strongly associated with the variation of demographic strategies in the Sonoran desert annual plant community at the Desert Laboratory,
Tucson, Arizona (Angert et al. 2007, Huxman et al. 2008, Angert et al. 2009, Kimball et al. 2010). This research has demonstrated that species that have high inter-annual demographic variability are more likely to have high relative growth rates and low water use efficiency compared to species that have more buffered population dynamics. Yet little is understood regarding how physiological strategies have accompanied evolutionary transitions in mating systems (but see Mazer et al. 2011).
The evolution of selfing has been associated with more rapid development and flowering time (Snell and Aarssen 2005, Elle et al. 2010) and is hypothesized to have an association with more arid habitats (Ivey and Carr 2011). A selfing strategy could evolve 14
as a response to colonization of more arid environments; alternatively the colonization of arid habitats might be facilitated by a greater functional plasticity in selfing species
(Chavin and Lande 2011, Evans et al. 2011). Thus, it has been predicted that selfing species will have physiological traits that allow them greater adaptation to drought
(Stanton et al 2000, Franks et al. 2007, Mazer et al. 2011), resulting in a more buffered strategy across variable environments. Since outcrossing species generally allocate to larger, longer-lived floral displays and greater nectar for pollinator attraction (Goodwillie et al. 2010), they may have fewer resources available to allocate to flower, fruit and seed production when resources are limited. Additionally they may be less able to alter their phenology to periods of high resource availability due to dependence on pollinators, affecting the timing and plasticity of their functional responses. These predicted differences in physiology and responses to resource limitation are expected to strongly influence fitness, population dynamics, and biogeography of selfing and outcrossing annual plants.
This dissertation was motivated by a desire to synthesize our understanding of the factors discussed above which affect the reproduction and fitness of desert annual plants.
Specifically, I was interested in how inter-annual variation in resources and climatic conditions influence species responses to their environment. Such variable responses between and within species can have profound influences on community and population dynamics through time and space. I approached this goal utilizing both a long-term population biology perspective to examine the role of range position on demographic 15
variability, and an experimental comparative perspective to examine how mating system and resource availability influence reproductive success and allocation.
Explanation of dissertation format
This dissertation consists of research investigating the ecological dynamics of desert annual plants and integrates biogeographic, phenological, reproductive, and physiological approaches and perspectives. The document is presented as four independent manuscripts. Appendix A examines how demographic variability in a guild of Sonoran desert annual plants is affected by their geographic and climatic position within their range. Appendix B consists of a three-year field study investigating how differences in reproductive success of co-occurring, related selfing and outcrossing annual species in the Mojave Desert are linked to their phenology and species interactions. Appendix C tests how phenological and physiological differences between two pairs of related selfing and outcrossing species under drought stress in the greenhouse. Lastly, Appendix D examines how resource stress affects reproduction and allocation between selfing and outcrossing species.
16
II. PRESENT STUDY
The methods, results, and conclusions of this study are presented in the papers appended to this dissertation. The following is a summary of the most important findings of these papers.
Appendix A studied how long-term demographic performance at a site relates to the position of a species within its geographic and climatic range. We tested hypotheses generated by the abundant center model (Brown 1984, Gaston 2003) which predicts that species abundance will decline from the centre towards the periphery of the geographic range, and that demographic variability will increase from the center towards the periphery. We examined long-term population dynamics at one location for 13 species of
Sonoran Desert annual plants that vary in their distances from their range centers. We looked at both the geographic distance to the range center from the Desert Laboratory as well as a climatic distance to the range center. Climatic distance was determined by calculating the Mahalanobis distance between the differences of the means of 19 bioclimatic variables of the range and the Desert Laboratory, which took into account a
Maxent probability distribution for each species and correlations between bioclimatic variables. This approach enabled us to find patterns across species using detailed demographic data from a single site and metrics of both geographic and ecological centrality. We found significant relationships between some geographic distance metrics and variance in fecundity, survival and per-germinant fecundity, but not germination fraction or population density. Thus, geographic distance from the center of the range of these annual plant species more strongly predicts their population dynamics than climatic 17
distance. This study reinforces the importance of examining vital rates and their variation in order to properly capture the effect of position within a range on population dynamics.
Appendix B examined the extent to which plants with contrasting mating systems are temporally synchronized with pollinators and herbivores, and how this synchrony affects plant fitness. We studied these patterns in three spring-blooming desert annuals, two self-incompatible outcrossing species and one selfing species, for three years in the
Mojave Desert. In each year of the study flowering time differed. The selfing species was not pollen-limited compared to the two outcrossing species and reproductive success did not differ early versus late in the season. However, when flowering occurred early in the season, the outcrossers were more successful late in the season and were found to have pollen-limited reproduction. Herbivory had the greatest impact in years with late season flowering phenology. This study demonstrates the costs associated with having an outcrossing reproductive strategy in a variable environment by documenting the risks associated with a relatively short annual life history that is dependent on pollinators for reproductive success, and subject to intense herbivory.
Appendix C considers the topic of how plants with contrasting mating systems cope with stressful environments. Outcrossing desert annual species have smaller range distributions compared to their selfing relatives and may be more limited in the spatial and temporal niches where they can persist. Mating system may influence patterns of resource use and how plants respond to low water availability. Here we examined how drought stress affects growth and physiology of two pairs of related selfing and 18
outcrossing desert annuals. We tested the prediction that selfing species have growth patterns and physiological traits better adapted for drought stress compared to outcrossing species. We examined patterns of phenology and physiology in two pairs of sister selfing and outcrossing desert annual species in the family Onagraceae, Eremothera refracta and
Eremothera chamaenerioides, and Chylismia multijuga and Chylismia walkeri. Selfing species reproduced earlier than outcrossing species and had faster rates of leaf mass accumulation. In general, selfing species had greater carbon assimilation rates, greater stomatal conductance, and thicker leaves. Outcrossing species experienced greater negative effects of drought on their physiological functioning and survival compared to selfing species. These results supported our prediction that selfers are better adapted to drought stress than are outcrossers. This work demonstrates how understanding the interaction between mating systems and environmental stress will help us to better understand the distribution and dynamics of reproductive strategies in plants.
Appendix D tested for differences between selfing and outcrossing species in patterns of resource allocation to reproduction under drought and herbivore stress.
Outcrossing species have an increased resource investment in floral display and greater reproductive uncertainty due to their dependence on pollinators; these phenomena may influence patterns of biomass allocation to reproduction. We predicted that drought and herbivory would have a greater negative effect on performance for outcrossing species compared to selfing species, and that outcrossing species would experience greater resource re-allocation to fruits when fertilization was prevented in early flowers. We studied these patterns experimentally in two related pairs of selfing and outcrossing 19
species within the genera Eremothera and Chylismia (Onagraceae). Selfing species and outcrossing species did not differ in their final biomass or flower production; however, selfers made a greater number of seeds per fruit. Drought caused all species to have similar negative responses, and simulated herbivory did not affect flower production.
Preventing early fertilization did not affect future fruit re-allocation, suggesting compensation later in development. These results are in contrast with our previous metrics of performance that showed drought to cause outcrossing species experience a greater reduction in their physiological functioning for plants that survived drought to reach reproductive maturity. This study demonstrates the importance in examining multiple metrics of performance in evaluating how phenology, physiology, resource availability, and mating system may influence the distribution and fitness of desert annual plants. 20
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28
APPENDIX A
THE EFFECT OF GEOGRAPHIC RANGE POSITION ON DEMOGRAPHIC
VARIABILITY IN ANNUAL PLANTS
Published: Journal of Ecology (2011) 99: 591-599
29
30
The effect of geographic range position on demographic variability in annual plants
Katharine L. Gerst1, Amy L. Angert1,2, and D. Lawrence Venable1
1Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ
85721, USA
2Department of Biology and Graduate Degree Program in Ecology, Colorado State
University, 1878 Campus Delivery, Fort Collins, CO 80523, USA
31
ABSTRACT
1: The abundant centre model predicts that species abundance will decline from the centre towards the periphery of the geographic range. Thus, we expect to find decreases from the centre towards the edge in variables related to population dynamics such as population density and reproductive output. However, evidence for this pattern is contradictory, suggesting that geographically peripheral sites may not be ecologically peripheral. Populations may thrive in pockets of suitable habitat at the edge of the range or may be locally adapted to peripheral conditions.
2: This study examines how the position of a site within geographic and climatic ranges of 13 species is related to the population dynamics at one common location, The Desert
Laboratory at Tumamoc Hill, Tucson, Arizona, USA.
3: We used data on survival, fecundity, germination fraction and population density from a 25-year long-term data set on winter annual plants to determine if there was a relationship between distance to the centre of the range and population dynamics.
Geographic distance was calculated by determining the distance from the Desert
Laboratory to the centre of the observed range determined from locality records.
Climatic distance was calculated using the niche modelling software, Maxent, and subtracting the mean climatic profile for the species range from that of the Desert
Laboratory.
4: There was no relationship between mean population metrics and distance metrics. We found significant relationships between some geographic distance metrics and variance in 32
fecundity, survival and per-germinant fecundity, but not germination fraction or population density. We did not find a relationship with any metric of population dynamic variation and climatic distance.
5: Synthesis. Our results indicate that geographic distance from the centre of the range of
13 annual plant species more strongly predicts their population dynamics than climatic distance. This study reinforces the importance of examining vital rates and their variation in order to properly capture the effect of position within a range on population dynamics.
Key-words: abundant centre model, annual plants, demographic variability, geographic range, niche modelling, plant population and community dynamics, Sonoran Desert
33
INTRODUCTION
Elucidating the mechanisms shaping variation in population dynamics across species geographic ranges is key to understanding what creates geographic range limits and ultimately drives ecological and evolutionary processes such as speciation, extinction and coexistence. The abundant centre model has been invoked as a general rule in biogeography to describe the structure of populations across species geographic distributions (Antonovics 1976; Brown 1984; Kirkpatrick and Barton 1997; Gaston 2003;
Guo et al. 2005). This model assumes that the most favourable conditions will be found at the centre of a species range, and thus the centre will support greater abundances than geographically peripheral sites. Several additional predictions arise as corollaries of the abundant centre model. For example, central populations are expected to have high per capita fitness and may serve as demographic source populations, while peripheral populations may be demographic sinks (Kanda et al. 2009) that exhibit more temporally variable population dynamics (Nantel and Gagnon 1999; Williams et al. 2003) and are more prone to extinction (Gargano et al. 2007). However, while some species have decreased abundance towards the periphery (Woodward 1997; Alexander et al. 2007), many other species do not (Carey et al. 1995; Hodkinson et al. 1999), and recent literature reviews raise considerable doubt about the generality of the abundant centre pattern in nature (Sagarin and Gaines 2002; Gaston 2003; Sexton et al. 2009).
The lack of consistent evidence for the abundant centre model has caused researchers to question if geographically peripheral populations are necessarily 34
ecologically or climatically peripheral (Yakimowski and Eckert 2007; Duffy et al. 2009;
Jarema et al. 2009) and to call for the integration of biophysical variables into studies of species geographic range dynamics (Sagarin et al. 2006). Traditionally, studies have classified populations as being either geographically central or peripheral. Yet, habitat quality can be patchy throughout a range, and geographically peripheral populations may occupy pockets of locally favourable habitat. Conversely, populations may ‘spill over’ into ecologically marginal habitat at the geographic range centre. Using geography alone as a surrogate for the degree of ecological peripherality or centrality of a site, relative to the niche requirements of a species, potentially masks the importance of climate and microhabitat in shaping population dynamics. Furthermore, geographically peripheral locations may not be ecologically peripheral if current range limits are not at equilibrium with physiological limits or if geographically peripheral populations are locally adapted to range edge environments.
The kinds of response variables used to test the abundant centre model may also limit our ability to infer population dynamic differences across the geographic range.
Previous tests of the abundant centre model have focused on snapshot estimates of abundance (Enquist et al. 1995; Samis and Eckert 2007; Krasnov et al. 2008) or performance (Jump and Woodward 2003; Kluth and Bruelheide 2005). Such measurements do not necessarily capture the appropriate range of temporal variation in population dynamics. Although mean environmental conditions may be suitable at the geographic periphery, climatic extremes could exceed the species’ tolerance in occasional years (Olmsted et al. 1993, Bowman et al. 2005). This would result in equivocal patterns 35
of abundance or performance during most short-term studies, but greater variance at the geographic periphery over longer time periods. The data necessary to quantify demographic variability over meaningful temporal scales are difficult to attain. Most examples to date rely on harvest records (e.g. hunting licenses, fisheries stocks) or large- scale national surveys (e.g. North American Breeding Bird Survey; Coelho et al. 1994;
Curnutt et al. 1996; Mehlman 1997; Williams et al. 2003). These studies are valuable for examining patterns over broad spatial scales and for large numbers of species, but in most cases they are unable to provide detailed demographic information such as variation in vital rates. Moreover, few studies have integrated variation in multiple vital rates across the life cycle (Nantel and Gagnon 1999; Kluth and Bruelheide 2005; Angert 2009).
In this study we ask, how does long-term demographic performance relate to the position of a species within its geographic and climatic range? We have examined long- term population dynamics at one location for multiple species of Sonoran Desert winter annual plants that vary in their distances from their range centres. This approach enables us to look for patterns across species using detailed demographic data from a single site.
Moreover, cross-species comparisons can suggest how biogeography influences local dynamics and species coexistence (Ackerly 2003; Chesson et al. 2004). Desert annuals are characterized by high inter-annual population fluctuations driven by unpredictable and highly variable precipitation (Schwinning and Sala 2004; Bowers 2005). Although the population dynamics of all species are variable, species differ significantly in the magnitude of that variability (Venable 2007). 36
We hypothesized that differences among species in long-term mean and variance of survival, fecundity, per-germinant fecundity, germination fraction and population density are related to differences in species positions within their geographic and climatic ranges. Specifically, we predicted that species for which our study site is geographically and climatically central would have higher means and lower temporal variances in our measures of population dynamics than species for which our study site is farther from the species’ geographic range centre or climatic average. Because pockets of climatically suitable habitat may be found at the geographic periphery and unsuitable habitat may occur near geographic centres, we predicted that climatic variables would explain greater variance than geographic variables.
MATERIALS AND METHODS
Long-term demographic performance
Data on demographic performance (means and variability) come from a 25-year study of winter annuals at The University of Arizona Desert Laboratory on Tumamoc
Hill, initiated in 1982 with 14 1.2-m2 plots. Forty-two additional plots were added in
1992, and 20 in 1995, for a current total of 72 plots, half of which are in the open and half under shrubs. These plots have been visited every year following each autumn and winter rain event to document winter annual seed germination. Individual plants have been mapped at germination and followed until death for a total of 4-7 censuses per year to quantify lifetime survival and reproduction. The average number of seeds produced per survivor was determined as a pre-dispersal fecundity estimate (either direct count of 37
seeds or of reproductive structures later multiplied by the mean seed number per reproductive structure) of up to five individuals per species per cohort per plot. Dormant seeds were counted each year in 180 soil cores located on adjacent plots starting in 1990
(see Venable and Pake (1999) for methods). Several species were recorded only occasionally, so we restricted the analysis to 13 native species with consistent presence throughout the study period: Daucus pusillus (Apiaceae), Eriophyllum lanosum, Evax multicaulis, Monoptilon bellioides, Stylocline micropoides (Asteraceae), Eucrypta micrantha, Pectocarya recurvata, Lappula redowskii (Boraginaceae), Draba cuneifolia
(Brassicaceae), Erodium texanum (Geraniaceae), Plantago insularis, Plantago patagonica (Plantaginaceae) and Vulpia octoflora (Poaceae). For each species, these observations quantify population density (seedlings plus dormant seeds m-2), germination fraction (proportion of dormant seed bank that germinated each year) and lifetime per germinant fecundity, lb (calculated by multiplying the average survival from germination to reproduction, l, by the average number of seeds produced by survivors, b) for every germinated seedling for the past 25 years. Population density and germination fraction data cover 17 years rather than the entire 25-year study span, as the soil cores quantifying the seed bank were started in 1990. We calculated the long-term mean and variance in demographic success for each demographic parameter. We calculated the variabilities for mean survival (l) and germination fraction as relative standard deviations of means
(standard deviation/(mean*(1-mean))), which is appropriate for these binomially distributed variables (Morris and Doak 2004). Mean fecundity, per-germinant fecundity and population density were ln-transformed. Standard deviations of these values were 38
then transformed back to their original scale to calculate the geometric standard deviation of lb.
Distribution records and modelling
We compiled collection records from multiple online data bases and regional herbaria (see Appendix S1 in Supporting Information). We discarded all records without latitude and longitude coordinates, and duplicate records of the same locality, resulting in
370 – 1752 records per species (Table 1). We used these collection records to model the probability distributions of species occurrences using the maximum entropy ecological niche modelling program Maxent v. 3.2.19 (Phillips et al. 2006). This program requires only presence data and has been determined to perform well compared to other niche modelling methods (Elith and Graham 2009). We created a probability distribution of species occurrences based on known presence records and 19 ‘bioclim’ climatic variables taken from the Worldclim data set (www.worldclim.com, see Appendix S2). Climate variables are based on climatic conditions averaged over the period between c. 1950 and
2000. Maxent was run for 500 iterations per species. The convergence threshold was set to 1 × 10-5, a recommended value based on the North American breeding bird survey data and small mammal data from Latin America (Phillips et al. 2006). The accuracy of the niche models was evaluated by constructing the model using 70% of presence records as training points, with the remaining 30% used to validate the data set as testing points.
Model accuracy was determined using a receiver operating characteristic (ROC) analysis
(Phillips et al. 2006). The area under the ROC curve (AUC) of the resulting plot of 39
sensitivity vs. (1 − specificity) provides a measure of model performance. An optimal model that perfectly predicts a species’ occurrence would have an AUC of 1.0, while a model that predicted species occurrences at random would have an AUC of 0.5. After creating models for all 13 species using all 19 bioclim variables, we selected nine variables that were consistently the most influential in determining the distributions of all
13 species and then constructed reduced models using the same procedure outlined above. The nine variables used in reduced models were Bio1, Bio2, Bio3, Bio5, Bio6,
Bio9, Bio11, Bio16 and Bio18 (see Appendix S2 for descriptions).
Geographic and climatic distances between the Desert Laboratory and species ranges
We calculated several metrics of the position of the Desert Laboratory in each species’ range:
a. Climatic distance: We summarized climate profiles for each species following the methods described in Evans et al. (2009). We calculated the mean of each bioclim climate variable within the species’ range, weighted by the species’ predicted probability of occurrence. This was done by multiplying the bioclim value for each grid cell by the corresponding occurrence probability from the reduced-variable Maxent model, then summing these and dividing by the total number of grid cells. We then calculated the distance between the Desert Laboratory and the species’ distribution in multivariate climate space using Mahalanobis distance. Each Mahalanobis distance calculation incorporates the difference between the climate mean for the range and the climate at the 40
Desert Laboratory across the 19 bioclim variables. These values were calculated by multiplying the inverse covariance matrix describing the covariances among the 19 bioclim variables with the array of arithmetic differences between each bioclim variable and the Desert Laboratory. Since this multivariate metric takes into account the covariation between climate values, it was preferred over a multivariate Euclidean distance calculation.
b. Geospatial distance: We calculated the distance between the Desert Laboratory and the location of each species’ geometric range centre. We estimated the range centre as the centroid of the minimum convex polygon surrounding observed collection records using DIVA GIS (Hijmans 2002). For statistical analyses, we took the natural log of these distance values.
c. Geographic distance: For each species, we calculated differences between the latitude of the Desert Laboratory and the mean latitude of the set of observed collection records. We also calculated analogous differences using longitude values. We used latitude and longitude separately because these may capture different gradients across species ranges (e.g. temperature versus precipitation). This allowed us to better quantify the relative importance of directionality in determining the role that distance from the centre of a range plays in influencing demography.
Range size
To determine if range size had a potentially confounding effect on range position at the Desert Laboratory, we calculated the size of each species’ observed range. This 41
was done by calculating the area of the minimum convex polygon around all observed collection records using DIVA GIS 5.2 (Hijmans 2002). For statistical analyses, we took the square root of range area measured in kilometres2.
Statistical analysis
Relationships between explanatory variables (geographic and climatic distances, range size) and response variables (mean and variance in per germinant fecundity, fecundity, survival, germination fraction, and population density at the Desert
Laboratory) were analysed with simple linear regression in SAS version 9.1 (SAS
Institute, Cary, North Carolina, USA). We then used hierarchical partitioning analysis to calculate the independent effects of correlated distance metrics (geographic centroid distance, climate distance and range size) on measures of population dynamic variance.
Variables in which zero-order correlations were near zero were excluded from this analysis (Murray and Conner 2009).
RESULTS
There were no significant relationships between the mean metrics of population dynamics (per germinant fecundity (lb), survival (l), fecundity (b), germination fraction and population density) and any geographic or climatic distance metric (all P > 0.1, Table
2). The following results all relate to the variance of population dynamic metrics.
Demographic variation and climatic distance from mean of range 42
Maxent model results performed well (AUC ranging from 0.940 – 0.998; Table 1) with the reduced nine-climate-variable model (see Appendix S3 for a brief description of model results and Fig. S1 for modelled distribution maps). We found no relationship between any metric of population dynamics (all P>0.09) and the climatic distance
(Mahalanobis distance) between the Desert Laboratory and the mean climate for the species ranges (Table 2). The Mahalanobis climatic distance had a positive relationship with the geospatial distance from the centre of the range to the Desert Laboratory
(slope=4.58, intercept=-18.4, R2 =0.656, P=0.008).
Demographic variation and geographic position within range
We found a significant positive relationship between variance in lb (per germinant fecundity) and the geographic distance between the Desert Laboratory and the centre of a species range (the centroid of a polygon around the observed and the predicted range)
(slope=3.8, intercept=-14.9, R2 =0.339, P=0.037, Fig. 1). We also found a significant positive relationship between variance in l (survival) and the geographic distance (R2
=0.359, P=0.030), as well as a marginally significant relationship between variance in b
(fecundity) and the geographic distance (R2 =0.278, P=0.064, Table 2). There were no relationships between germination fraction variance or population density variance and distance from the range centre (Table 2).
Differences between the Desert Laboratory and observed latitude and longitude of species ranges show that some species at the Desert Laboratory had distributional peaks which were more central along the latitude axis but not longitude (e.g. Eriophyllum 43
lanosum), some species were more central along longitude but not latitude (e.g. Lappula redowskii), and others did not have distributions which peaked at the Desert Laboratory on either axis (e.g. Plantago insularis; Figs. 2 and 3). Furthermore, while the Desert
Laboratory was closer to the southern periphery of the distribution for some species, such as Eucrypta micrantha, it was closer to the northern periphery of the distribution of others, such as Evax multicaulis (Fig. 2). Likewise with longitude, the Desert Laboratory was closer to the western periphery of the distribution for some species (e.g. Evax multicaulis) but closer to the eastern periphery of the distribution for others (e.g.
Monoptilon bellioides) (Fig. 3). So while any two species may both be peripheral at the
Desert Laboratory relative to the centre or edge of their distribution, they may be along different geographic axes of their distributions and hence spatial marginality can encompass different ranges of climatic marginality. There were no species for which the
Desert Laboratory represented a central point along both longitudinal and latitudinal axes.
There were no significant relationships between differences in the latitude of the
Desert Laboratory relative to the mean latitude of the species range and variance in lb, l, b, germination fraction or population density (Table 2). There was a significant positive relationship between lb variation and the distance of the Desert Laboratory from species’ mean observed longitude (R2=0.372, P=0.027). When analysed separately, there was also a marginally significant positive relationship with variance in l (R2 =0.295,
P=0.055), and a significant positive relationship with variance in b (R2 =0.464, P=0.010)
(Table 2). That is, there is an increase in variation in survival, fecundity and per germinant fecundity as observed species distributions are more eastward relative to the 44
Desert Laboratory. There was no significant relationship between variance in germination fraction or population density and longitudinal distances of the observed range (Table 2).
Range size
Observed species distributions (Table 1) ranged from small and restricted to south-western deserts (i.e. 399 600 km2, M. bellioides) to large, covering much of North
America (11 161 900 km2, V.octoflora) (see Fig. S1 for distribution maps). Species with the largest ranges had range centres (the centroid of minimum convex polygons) farthest from the Desert Laboratory (slope=1411.5, intercept=-7111.4, R2=0.544, P=0.004). There was a significant positive relationship between range size and variance in l (R2 =0.336,
P=0.038, Table 2). However, there were no significant relationships between range size and variance in lb, b, germination fraction or population density (Table 2).
Hierarchical partitioning of variance
No hierarchical partitioning was done on variables related to germination fraction or population density since all had near-zero correlations. The factor that had the strongest independent contribution to variance in lb was geographic distance (59.7% independent contribution to variance in lb), followed by range size (22.2% independent contribution) and climate distance (18.1% independent contribution). Likewise, the factor that had the strongest independent contribution to variance in l was geographic distance (42.2% independent contribution to variance in l), followed by range size
(36.9% independent contribution), and climate distance (20.9% independent contribution). Lastly, the factor that had the strongest independent contribution to 45
variance in b was geographic distance (71.2% independent contribution to variance in b), followed by climate distance (16.4% independent contribution), and range size (12.4% independent contribution).
DISCUSSION
We found no relationships between mean metrics of population dynamics (lb, l, b, germination fraction and population density) and distance between the Desert Laboratory and the centre of a species range. This is perhaps not unexpected because species may have innate differences in their population structure or life histories that make interspecific comparisons difficult. For example, different species may have different characteristic population densities or fecundities (although we log-transformed mean values to minimize differences in scale).
We found the predicted positive relationship between variance in per germinant fecundity (geometric standard deviation of lb) at the Desert Laboratory and the geographic distance to the centre of each species ranges. When analysed separately, variance in survival (l) was also related to geographic distance and variance in fecundity
(b) was marginally related to distance from the range centre. Thus, as the distance between the Desert Laboratory and the centre of species ranges increases, the inter- annual variability in both survival and fecundity at the Desert Laboratory becomes greater. This indicates that species may be buffered against mortality effects and extreme plastic responses in fecundity if they are near the centre of their range. Conversely, if they are farther from the centre of their range, species may be more likely to have 46
relatively larger survival and fecundity responses in favourable years and poor responses in other years, suggesting they may not be as locally adapted to this site. Again, our metrics of variance are standardized to reflect proportional deviations from each species’ mean and thus permit comparisons across species. We did not detect a relationship between variance in germination fraction and any measure of geographic distance.
However, while seed dormancy is a trait influenced by climate and affecting fitness, variation in this trait more likely reflects a bet-hedging strategy than a poor response to environmental conditions. Likewise, we did not detect a relationship between variance in population density and any measure of geographic distance. This is noteworthy, as population density is predicted to be more variable towards the edge of a range, and density is the most commonly used measure of population dynamic differences between central and peripheral populations (Brown et al. 1995; Krasnov et al. 2008; Fuller et al.
2009). The lack of associations detected for population density variation suggests that variation in per-germinant fitness does not necessarily match variation in population density over time. However, this is not necessarily surprising as population density is strongly correlated with the history of previous years, while per capita measures are not.
Also, these species make long-lived seed banks, which buffer population density against year-to-year variation in reproductive success. Thus, our results highlight the importance of studying vital rates (in this case, survival, fecundity and per-germinant fecundity) in addition to population density in order to accurately describe the effects of range position on population dynamics. Since this approach utilizes a long-term data set of population 47
dynamics rather than a snapshot, we have been able to maximize our ability to quantify the overall demographic characteristics of these species at this site.
Our separate analyses of mean latitudinal and longitudinal axes relative to the
Desert Laboratory indicate that variation in fecundity, survival and per-germinant fecundity is associated with longitudinal, but not latitudinal, distance from the geographic range centre. Specifically, the further east a species’ centre of distribution is from the
Desert Laboratory the greater the l, b and lb variation. This result suggests that species with more mesic centres of distribution have greater demographic variance when growing at the more arid Desert Laboratory site. Interestingly, this tends to support the climatic distance hypothesis.
We originally hypothesized that climatic distance would be a stronger indicator of demographic variation than geographical distance, but our analysis of climatic distance using a multivariate descriptor of climate did not support this, despite the previously described indirect support from the longitudinal analysis above. In the multivariate analysis, no significant relationships were found between population dynamic variation and climatic distance. However, it should be noted that in this study geographic and climatic distances were highly correlated and their overall trends were similar. The hierarchical partitioning analysis suggested that climate distance had an independent effect on the l, b and lb variation that was less than half that of geographic distance. It is possible that our relatively coarse quantification of climate may have weakened our ability to detect a pattern. A site that is considered peripheral in either a geographical or 48
a macroclimatic sense could still potentially be in a favourable microhabitat (Dinsdale et al. 2000; Lee et al. 2009). Alternatively, our result could be due to local adaptation to differing environmental conditions (Conover and Schultz 1995; Gonzalo-Turpin and
Hazard 2009) throughout the range. These possibilities could be further explored through transplant studies and comparative studies on physiological tolerances throughout ranges
(Angert 2006; Kimball and Campbell 2009). Furthermore, other aspects of climate, such as the maximum differences between climate layers, might better capture the climatic distance between the Desert Laboratory and a species range as whole.
The location of the Desert Laboratory within North America has an interesting effect on these results. Species with a large range cannot have the Desert Laboratory close to their range’s longitudinal centre due to the proximity of the Desert Laboratory to the west coast. Hence, by default the Desert Laboratory is at the western but not necessarily the northern or southern periphery of a large species range. Another consequence of the Desert Laboratory’s location is that climatic variation within the range increases with range size: Small ranges can be included within the desert south- west, while larger ranges necessarily include additional biomes. Thus, desert endemic species have smaller ranges and are closer to their centres of distribution than widespread species, for which the desert is a peripheral environment within their range. Species with small ranges may be more likely to have less absolute variation in the climatic characteristics within their range, and be more able to adapt to them and buffer variation
(Maliakal-Witt et al. 2005). Widespread species, on the other hand, are more likely to occupy a large range of environments and thus may be more likely to be demographically 49
variable in any one environment. This, of course, depends on the degree to which a wide-ranging species achieves its broad distribution via generalization, plasticity and local adaptation to particular habitats within its diverse range (Gaston et al. 1997; Pandit et al. 2009). However, with the exception of variation in survival, range size was generally not important in contributing to the relationships driving demographic variation.
To our knowledge, this is the first effort to utilize niche modelling to quantify the climatic peripherality of a site and how that relates to spatiotemporal population dynamic variation. However, this work is complementary to recent studies elucidating the environmental and genetic components of range dynamics in migrating tree populations
(Wang et al. 2010). Additionally, a niche modelling approach is being increasingly used to study biological invasions and the potential demographic consequences of niche specialization in potential invaders (Rodder and Lotters 2009; Medley 2010). The results presented here suggest that geography plays a strong role in generating patterns of survival, fecundity and per-germinant fecundity variation, but not germination fraction or population density variation, within this guild of desert annual plants.
ACKNOWLEDGMENTS
Funding was provided by NSF grants BSR 9107324, DEB 9419905 (LTREB), DEB
0212782 (LTREB) DEB 0717466 (LTREB), and 0817121 (LTREB) to D.L.V.. The authors thank D. Hearn for assistance in analysing Maxent output, J. Donohue for GIS 50
assistance, and S. Felker, E. Larios, J. Horst, S. Kimball, J. Paul and S. Sheth for comments on the manuscript.
51
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57
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article.
Appendix S1 Sources of locality data.
Appendix S2 Wordclim variables derived from monthly data between ~1950-2000.
Appendix S3 Maxent model results and important variables.
Figure S1: Maxent generated maps showing predicted ranges based on occurrence records.
58
TABLES
Table 1: Species used for this study, number of points used for model generation, AUC
(area under ROC curve), and range size. ROC analysis (receiver operating characteristic) was used to determine model accuracy (Phillips et al. 2006).
Number of occurrence points Maxent Observed range Species used to generate AUC area (km2) Maxent model
Daucus pusillus 1369 0.972 6,146,300
Draba cuneifolia 1256 0.967 4,145,200
Eriophyllum lanosum 380 0.997 433,700
Erodium texanum 671 0.990 1,905,800
Eucrypta micrantha 370 0.94 1,004,900
Evax multicaulis 371 0.989 1,582,500
Lappula redowskii 721 0.968 7,894,000
Monoptilon bellioides 414 0.998 399,600
Pectocarya recurvata 504 0.998 458,900
Plantago insularis 783 0.996 2,165,900
Plantago patagonica 1205 0.974 6,384,600
Stylocline micropoides 298 0.997 705,500
Vulpia octoflora 1752 0.955 11,161,900
59
Table 2: Linear regression statistics for relationships between distance metrics and demographic variables (per germinant fecundity (lb), survival (l), fecundity (b), germination fraction and population density).
60
FIGURES
16 VUOC
14 DRCU
12 EVMU
10 LARE STMI ERLA 8 PERE MOBE PLPA DAPU PLIN ERTE 6
Geometric standard deviation of lb deviation standard Geometric 4 EUMI
2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2
Ln (distance between the Desert Laboratory and the center of species range (km))
Figure 1: Variation in per-germinant fecundity (lb) increases with greater distance between the Desert Laboratory and the centroid of the species’ range based on observed records ((SD(lb)=-14.9 + 3.8(distance), R2=0.339 , P= 0.037). Each four-letter code represents one species coded by the first two letters of the species and genus name. See
Table 1 for a full species list.
61
45 DAPU 45 DRCU 45 ERLA 40 40 40
35 35 35
30 30 30
Latitude
25 25 25 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5
45 ERTE 45 EUMI 45 EVMU 40 40 40
35 35 35
30 30 30
Latitude
25 25 25 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5
45 LARE 45 MOBE 45 PERE 40 40 40
35 35 35
30 30 30
Latitude
25 25 25 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5
45 PLIN 45 PLPA 45 STMI 40 40 40
35 35 35
30 30 30
Latitude
25 25 25 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 Proportion Proportion 45 VUOC 40
35
30
Latitude
25 0.1 0.2 0.3 0.4 0.5 Proportion
Figure 2: The relative frequency of presence records at different latitudes. Dotted line represents the latitude for the Desert Laboratory. Each four-letter code represents one species coded by the first two letters of the species and genus name. See Table 1 for a full species list. 62
0.5 0.5 0.5
0.4 DAPU 0.4 DRCU 0.4 ERLA
0.3 0.3 0.3
0.2 0.2 0.2
0.1 0.1 0.1
P roportion
0.0 0.0 0.0 -120 -100 -80 -120 -100 -80 -120 -100 -80
0.5 0.5 0.5
0.4 ERTE 0.4 EUMI 0.4 EVMU
0.3 0.3 0.3
0.2 0.2 0.2
0.1 0.1 0.1
P roportion
0.0 0.0 0.0 -120 -100 -80 -120 -100 -80 -120 -100 -80
0.5 0.5 0.5 MOBE PERE 0.4 LARE 0.4 0.4
0.3 0.3 0.3
0.2 0.2 0.2
0.1 0.1 0.1
P roportion
0.0 0.0 0.0 -120 -100 -80 -120 -100 -80 -120 -100 -80
0.5 0.5 0.5 PLPA STMI 0.4 PLIN 0.4 0.4
0.3 0.3 0.3
0.2 0.2 0.2
0.1 0.1 0.1
P roportion
0.0 0.0 0.0 -120 -100 -80 -120 -100 -80 -120 -100 -80
0.5 Longitude Longitude
0.4 VUOC
0.3
0.2
0.1
P roportion
0.0 -120 -100 -80 Longitude
Figure 3: The relative frequency of presence records at different longitudes. Dotted line represents the longitude for the Desert Laboratory. Each four-letter code represents one species coded by the first two letters of the species and genus name. See Table 1 for a full species list. 63
Appendix S1: Sources of locality data.
Database
Southwest Environmental Information Network (SEINet) Jepson Online Interchange Institute of Natural Resource Analysis and Management Gateway to New Mexico Biodiversity (INRAM) World Biodiversity Information Network (REMIB) Digital Flora of Texas Flora of the Southeastern United States Flora of Texas Consortium National Biological Information Infrastructure Synthesis and Analysis of Vegetation Across Scales (SALVIAS) Global Biodiversity Information Facility (GBIF) Missouri Botanic Garden W3 Tropicos University of Washington Herbarium Colorado State University Herbarium University of Connecticut Herbarium University of South Florida Herbarium Louisiana State University Herbarium Brooklyn Botanic Garden New York Botanic Garden Oregon State University Herbarium University of Alaska Herbarium Utah Valley State College Herbarium 64
Intermountain Herbarium University of Nevada Las Vegas Herbarium University of Wisconsin Herbarium University of Idaho Herbarium University of Oklahoma Herbarium
65
Appendix S2: Wordclim variables derived from monthly data between c. 1950-2000. Bolded variables are the ones that were included in the reduced variable Maxent model.
BIO1 Annual Mean Temperature
Mean Diurnal Range (Mean of monthly (max temp - BIO2 min temp))
BIO3 Isothermality (P2/P7) (* 100)
BIO4 Temperature Seasonality (standard deviation *100)
BIO5 Max Temperature of Warmest Month
BIO6 Min Temperature of Coldest Month
BIO7 Temperature Annual Range (P5-P6)
BIO8 Mean Temperature of Wettest Quarter
BIO9 Mean Temperature of Driest Quarter
BIO10 Mean Temperature of Warmest Quarter
BIO11 Mean Temperature of Coldest Quarter
BIO12 Annual Precipitation
BIO13 Precipitation of Wettest Month
BIO14 Precipitation of Driest Month
BIO15 Precipitation Seasonality (Coefficient of Variation)
BIO16 Precipitation of Wettest Quarter
BIO17 Precipitation of Driest Quarter
BIO18 Precipitation of Warmest Quarter
BIO19 Precipitation of Coldest Quarter
66
Appendix S3: Maxent model results and important variables.
For seven out of the 13 species (Eriophyllum lanosum, Erodium texanum, Eucrypta micrantha, Monoptilon bellioides, Pectocarya recurvata, Plantago insularis, and
Stylocline micropoides), ‘Bio5’, the maximum temperature of the warmest month, was the climatic variable that gave the highest gain in the model when used in isolation. These species stand out as being restricted to deserts and having small range areas (less than
2,200,000 km2). For four species, ‘Bio11’, the mean temperature of the coldest quarter, was the variable that gave the highest gain in the model when used in isolation (Daucus pusillus, Draba cuneifolia, Evax multicaulis, and Vulpia octoflora). All of these species had larger range areas (greater than 2,200,000 km2) with the exception of E. multicaulis.
E. multicaulis was notable in having a small range that is primarily south of the Desert
Laboratory, and thus appears restricted in its northern distribution. For two of the species
(Lappula redowskii and Plantago patagonica) ‘Bio3’, the isothermality index (mean diurnal range/temperature annual range), gave the highest gain in the model when used in isolation. Both of these species had their range distributions clustered north of the Desert
Laboratory and had ranges greater than 6,300,000 km2.
67
Figure S1: Maxent generated maps showing predicted ranges based on occurrence records (shown as white points) using 19 Bioclim variables.
68
APPENDIX B
PHENOLOGY MEDIATES POLLINATOR AND HERBIVORE INTERACTIONS IN SELFING AND OUTCROSSING DESERT ANNUALS
69
Phenology mediates pollinator and herbivore interactions in selfing and outcrossing desert annuals
Katharine L. Gerst1 and D. Lawrence Venable1
1Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ
85721, USA
70
ABSTRACT
By examining costs and benefits associated with the evolution of selfing and outcrossing reproductive strategies in plants, we can understand the circumstances that allow these strategies to persist and coexist in variable habitats. A major cost of having an outcrossing strategy is dependence on pollinators; plant reproduction may be more likely to be pollen limited when pollinators are unreliable or scarce. Conversely, species with a self-fertilizing strategy are guaranteed to reproduce in the absence of pollinators.
The degree of phenological synchrony between plants and their pollinators, and between plants and their herbivores, plays an important role in influencing plant fitness. Mating system likely mediates the effect of these interactions. In this study, we predicted that outcrossing species would have more variable pollen limitation and reproductive success than selfing species because of greater sensitivity to periods of asynchrony with pollinators. We also predicted that synchrony with herbivores would be detrimental to both selfing and outcrossing species and would influence the timing of successful reproduction. We tested these predictions in three spring-blooming desert annual plants, two outcrossing species and one selfing species, for three years in the Mojave Desert. We found that the selfing species was never pollen limited and that reproductive success did not differ early versus late within a season. The two outcrossing species were most strongly pollen limited in the year when plant reproductive phenology was earliest in the season, and pollination intensity and fruit set were lower early compared to late in the season for the outcrossers in the early year as well. Herbivore damage was highest for both selfers and outcrossers when plant reproductive phenology was the latest in the 71
season. Thus, for both selfing and outcrossing species, there is a cost to late reproduction as a result of synchrony with herbivores. However, for outcrossing species there is also a cost of early seasonal flowering, most likely resulting from asynchrony with pollinators.
72
INTRODUCTION
Exploring how variation in reproductive systems has arisen and diversified is a fertile area of study in evolutionary biology. Plants exhibit great diversity in their mating systems
(Barrett 2003), such as self-fertilizing and outcrossing strategies. One factor thought to be driving this diversity is pollen limitation, which results when there is not sufficient pollen available to fertilize all ovules on a plant (Ashman et al. 2004). Evidence suggests that pollen limitation can play a significant role in the evolution of mating systems (Lloyd 1979, Holsinger
1996), particularly in the transition from outcrossing to self-fertilization (Morgan and Wilson
2005, Porcher and Lande 2005). One explanation for the maintenance of outcrossing species is that they benefit from the maintenance of higher genetic diversity and thus increased adaptive potential. However, they will lose opportunities to reproduce if not well synchronized to its pollinators or if pollinators are scarce. A self-fertilizing species benefits from reproductive assurance, its ability to reproduce in the absence of pollinators, but may suffer from inbreeding
(Lloyd and Schoen 1992).
The interaction of breeding system with factors limiting reproduction is expected to play an important role in determining plant fitness and population dynamics, and will influence the distribution and evolution of breeding systems within variable habitats. When pollinators and resources are variable, we expect reproductive success to exhibit temporal variation, both within and between years (Campbell and Halama 1993). Given that selfing and outcrossing plants may have significantly different resource allocation patterns (Cruden and Lyon 1985,
Parker et al. 1995, Goodwillie et al. 2010) they may experience different effects of pollen and resource limitation on their reproduction. Resources available for reproduction have been 73
shown to decrease through the reproductive period, such that a plant is capable of investing more resources to ovules and seeds of earlier flowers (Herrera 1991, Brunet 1996, Buide 2004,
Kliber and Eckert 2004). Therefore, the timing of when plants are reproducing and the availability of resources will be essential in influencing plant performance. This study examines how the timing of reproductive phenology and the timing of the interactions of plants with their pollinators and insect herbivores differ between selfing and outcrossing species throughout their flowering period to ultimately determine their reproductive success.
Phenology, the timing of life history events such as germination or reproduction, plays an important role in influencing the fitness of plants (Galen and Stanton 1991) and their distribution (Chuine 2010). The timing of spring flowering has been shown to be strongly affected by variation in weather and climate change (Price and Waser 1998, Cleland et al.
2007, Clark and Thomson 2010, Crimmins et al. 2010, Neil et al. 2010) with a general, but not ubiquitous, pattern of progressively earlier spring flowering with climate warming (Bowers
2007, Inouye 2008). Of particular interest is how the timing of growth and reproduction interacts with the timing of abundance and activity of species affecting plant fitness (Yang and
Rudolf 2010), such as pollinators (Memmott et al. 2007, Kaul and Koul 2008) and herbivores
(Brody 1997, van Asch and Visser 2007, Fabina et al. 2010). For instance, reproductive success of outcrossing species may depend on how synchronized pollinators are with the timing of flowering (Fleming 2006, Hegland et al. 2009). By understanding how these interactions are mediated by phenology, we can better understand the costs and benefits of various plant strategies, such as outcrossing versus self-fertilization. 74
Plants respond to cues such as rainfall, temperature, photoperiod and soil moisture to trigger events such as germination and reproduction. Pollinating and herbivorous insects are ectothermic and are constrained in their emergence and activity by cold temperature (Louda and Potvin 1995). The exact timing on these constraints depends on the timing of precipitation pulses and seasonal temperature increases and thus these cues may not always been synchronized in any given year, resulting in phenological mismatch (Miller-Rushing et al.
2010, but see Forrest and Thomson 2011). Frequent mismatch over time may affect how species respond to climate variability. For example, an experimental study by Rafferty and Ives
(2011) found that species that were constrained in their reproduction by the timing of their pollinators may be less likely to experience long-term changes in their reproductive phenology.
This suggests that outcrossing species may be more constrained in the timing of their reproduction in response to inter-annual variation of phenological cues compared to selfing species. How the timing of insect herbivore activity corresponds with differences in phenological trajectories of plant species with selfing and outcrossing strategies is also expected to have consequences for plant reproduction (Niesenbaum 1996, Krupnick 1999,
Singer and Parmesan 2010). These aspects of synchrony may have strong effects on fitness if reproduction is disrupted, and may result in variable success of different reproductive strategies across years. If differing reproductive strategies experience variable reproductive success across spatial and temporal scales, such variation in phenology may promote the maintenance of communities with multiple coexisting mating systems.
Annual plants frequently are assumed to avoid dependence on pollinators by having self-fertilizing breeding systems in order to colonize new or disturbed habitat (Baker 1955). In 75
general, most selfing species are annuals (Aarssen 2000). However, desert annual plants are distinct from other guilds of annuals in that they are not fugitive but persist over the long term in relatively undisturbed desert communities (Venable and Pake 1999). This large guild of plants, consisting of at least 50% of the flora in the Sonoran Desert and 70% in the Mojave
Desert displays a full spectrum of sexual reproductive strategies, from obligate self- incompatible outcrossers to cleistogamous selfers; many are biotically pollinated (e.g. Delph
1986, Schemske and Bierzychudek 2001). The relatively high frequency of outcrossing within the Sonoran and Mojave deserts is curious, since outcrossers experience strong constraints on the timing of reproduction: they must complete their reproduction in a relatively short time period. The exact timing of flowering varies each year, depending on the arrival of winter rainfall and temperature patterns. This variable nature of rainfall and temperature in the desert during the winter months leads to large fluctuations in population dynamics (Schwinning and
Sala 2004, Bower 2005) and a short reproductive season for winter annual species. The short window of time available to reproduce means that desert annual plants with an outcrossing reproductive strategy may be more vulnerable to mismatches with pollinators than perennials, which can be buffered against mismatches by reproducing over multiple years. Desert winter annuals germinate, grow, and complete their life cycle in the relatively cooler months when soil moisture is generally more available. They are limited by heat and drought which constrain photosynthesis and ultimately determine the end of the winter annual growing season (Venable and Pake 1999, Huxman et al. 2008).
This study examines how the timing of pollinator and herbivore activity, pollen limitation, and reproduction differ for plant species with outcrossing and selfing mating 76
systems, and how variable phenology affects fitness during three spring flowering seasons. The goals are to enhance our understanding of the costs and benefits of selfing versus outcrossing by exploring: (a) the degree of plant-pollinator-herbivore synchrony for two self-incompatible and one self-compatible related species, and (b) the role of pollen limitation in determining seed production through the flowering season for these species. We predicted that when plant and pollinator phenology are asynchronous, outcrossing species will be more pollen limited and have reduced fruit and seed set compared to selfing species. Additionally, we predicted that when there is greater plant- pollinator synchrony, damage to plants from insect herbivores would be greater compared to asynchronous years, because insect herbivores are expected to respond to the same phenological cues as pollinators. Lastly, we predicted that selfing species would have higher performance when plants and insects are asynchronous; this allows them to escape herbivory without a reproductive cost, since they are not dependent on pollinator emergence. We tested these predictions using three native Mojave Desert plants: two self-incompatible outcrossing species, Eremothera refracta and Chylismia brevipes, and one self-fertilizing species, Eremothera chamaenerioides.
MATERIALS and METHODS
Study Site and Species:
The study species were the winter germinating annuals Chylismia brevipes,
Eremothera refracta, and E. chamaenerioides (Onagraceae). In the Mojave Desert, these species germinate after winter rainfall and complete their life cycle by April or May. E. 77
refracta and C. brevipes are both self-incompatible obligate outcrossers (Raven 1969). C. brevipes has morning-opening yellow flowers primarily visited by bees, while E. refracta has evening-opening white flowers primarily visited by small moths. Flowers of both species will stay open for more than four days when not fertilized. E. chamaenerioides is self-compatible. It is the putative sister species of E. refracta, based on systematic treatment by Raven (1969, 1979) with recent genera re-classifications by Wagner et al.
(2007). The flowers of this species are only open for one to two days and anthers deposit pollen on the stigma before flowers open.
The study site used in this research is located at the south end of the lower slopes of the Bristol Mountains in the Mojave Desert in San Bernardino County, California
(34o37’13.66 N, 115o40’30.05 W). In one year, 2008, a second site was included located in a wash near Goffs Butte, approximately 56 km northeast of the Bristol Mountain site
(34o51’48.17 N, 115o5’37.13 W). This site was added because in 2008 the selfing species, E. chamaenerioides, was present at Goffs Butte but not Bristol Mountain. In
2009 and 2010, no plants of any of the three species were present at the Goffs Butte site.
The habitats were wide sandy washes characterized by the presence of Hyptis emoryi,
Acacia greggi, and Encelia farinosa. Temperature and precipitation data were retrieved from the Amboy, CA weather station (for the Bristol Mountain site; approximately 13 km south and 500 meters lower in elevation than field site) and the Needles, CA weather station (for the Goffs Butte site; approximately 39 km east and 700 meters lower in elevation than field site) using the National Climate Center Data Center online database
(http://www.nesdis.noaa.gov/). 78
Plant Phenology
Data were collected twice, approximately monthly, during the flowering season
(early, late) on dates that differed across years and sites as a function of the flowering period. Plants were included in the study if they had at least three open flowers.
Depending on how many plants were present and in flower at the site, anywhere from 10-
80 individuals were monitored per species per time period. The total number of flowers, fruits, and flower buds were recorded, as well as the fruit set (proportion of flowers that set fruit). The corolla falls off 1 to 5 days after opening, but the inferior ovary remains on the plant whether the ovules have been fertilized (exhibiting “swollen” fruit) or not
(exhibiting “thin” fruit), allowing us to calculate the total number of flowers that had been produced and the fruit set. Plants were marked with a small label around the base of the stem and the GPS location was taken for relocation. Three to four weeks after each treatment period, plants were re-visited to record phenological status and fruit set. The majority of plants completed their reproduction within this three to four week period.
In addition to the above measurements, at each time period, transects across the study area were established to count the number of individuals that were pre-flowering, flowering, or post flowering.
Pollen tube density
For each marked individual monitored early and late in the season, the stigma from the oldest open flower on the primary flowering stalk was collected and immediately placed in 70% ethanol. Excised styles were then fixed in a 3:1 solution of 79
70% ethanol and 45% glacial acetic acid for 24 hours. They were then stored in 70% ethanol for 1-2 weeks. Styles were softened for one hour in sodium hydroxide and then rinsed in distilled water. Styles were then stained for one hour with a 1% aniline blue solution in tri-potassium orthophosphate. The stained styles were squashed under a cover slip and observed under a fluorescence microscope. The number of germinated pollen grains with visible pollen tubes was estimated to the nearest multiple of 50 for each style to assess pollination intensity and frequency at that time period.
Pollen limitation
In order to determine if flowers were pollen limited in their reproduction, we compared fruit set of flowers that were allowed to be naturally pollinated to fruit set of flowers that we hand-pollinated. This allows us to quantify differences between the maximum possible seed set possible given available resources with natural seed set. The youngest opened flower was marked on the fruit with a small blue paint mark (the
“control” fruit). Pollen was added to the stigma of the second most recently opened flower by rubbing dehiscent anthers on the stigma that had been removed from non-study plants with forceps. The pollen came from at least three plants ≥10 m away to reduce relatedness and the fruit was marked with a small pink paint mark (the “crossed” fruit).
During the return visit to these plants, fruits were collected and the number of fertilized seeds and unfertilized ovules were later counted. Seed to ovule ratios were calculated as the number of fertilized seeds divided by the total number of both fertilized and unfertilized ovules. 80
Herbivory
For each plant, insect herbivore damage on leaf, stem, and fruit tissue was assessed at each visit on a scale from 0-3 where 0 = no damage, 1= minimal damage (<
25% of tissue), 2= substantial damage (25-75%) 3: intense damage (>75% of tissue).
Damage was deemed to be caused by herbivores if visible caterpillar or beetle chewing was evident.
Statistical Analysis
Statistical analyses were performed in SAS 9.1.3 (SAS Institute, Cary, NC, USA).
For each species, proportional fruit to flower ratio (fruit set) and seed to ovule ratio (seed set) data were analyzed using a generalized linear model assuming a binomial distribution and logit link function in PROC GLIMMIX to quantify differences in reproduction between early and late in the season and between years. This tested the prediction that reproduction was different when phenology wasearly versus late. For each species, pollen tube density class, the number of seeds within control fruit, and herbivory score were each analyzed separately using PROC GLM to quantify differences between early and late in the season and between years for reproduction and herbivore damage. Prior to analysis, pollen tube density was log-transformed. Year and season (early versus late) were treated as fixed effects. Differences in individual means were tested using Tukey-
Kramer HSD comparisons within treatments and within species. In separate analyses using only the two sister taxa in the genus Eremothera, “species” was added as a fixed effect to test for differences between mating systems. This allowed us to test the 81
prediction that reproduction differed with mating system. In these analyses 2008 data from the Goffs site were included for the purpose of comparing the two species in each year where they occurred together; unless otherwise noted, all other analyses were performed using only data from the Bristol Mountain site. Interaction terms were included if at least one main effect was significant. Lastly, pollen limitation was assessed using paired t-tests between seed number per fruit of control, crossed, and paired fruits for each species and time period.
RESULTS
In all three years, Hyles lineata (Lepidoptera: Sphingidae) larvae were the predominant herbivores on all three species. In 2010, an adult chrysomelid leaf beetle was also an abundant leaf consumer of all three species. Hyles larvae were observed consuming leaf, stem, flower, and fruit tissue. It was not uncommon to observe larvae consume an entire plant.
Inter-annual variation in phenology
Reproductive phenology differed in each year of the study, and began progressively later from 2008 to 2009 to 2010 (Figure 1; See Table 1 for which species were present in each year as well as rainfall and temperature differences between years).
Seasonal variation in reproductive success
For the two outcrossing species, Chylismia brevipes and Eremothera refracta, there were significantly more germinated pollen grains on stigmas late in the season 82
compared to early in the season in 2008, the year with the earliest plant reproductive phenology (see Table 2 for means, Table S1 for ANOVA results). Pollen tube data were not collected on the selfing species Eremothera chamaenerioides. In C. brevipes there were no significant differences in fruit set, the proportion of flowers that set fruit, early versus late in the season for any of the three years (Tables 3, S1). In E. refracta, fruit set was significantly greater early compared to late in the season in 2008 and 2009, and significantly lower late in 2010. There was no effect of early versus late season on fruit set in the selfing species E. chamaenerioides. These differences in early versus late pollen deposition and fruit set are likely to reflect greater plant-pollinator asynchrony in
2008, the year with earliest plant reproductive phenology, and greater plant-pollinator synchrony in 2009 and 2010, the years with intermediate and late plant reproductive phenology.
Seed to ovule ratio is a metric of the within-fruit fertilization rate. For C. brevipes, the seed to ovule ratio was significantly lower all three years early compared to late in the season (Tables 4, S1). In E. refracta, there was no consistent pattern with season. In the
Bristol Mountains, seed to ovule ratios were significantly lower early in 2008 and 2010 but significantly higher early in 2009; at the Goffs site, they were significantly higher early in 2008. In E. chamaenerioides, seed to ovule ratios were significantly higher earlier in the season compared to late in 2008 and 2010.
Chylismia brevipes matured significantly fewer seeds per fruit early compared to late in the season in 2009 (Tables 4, S1), while E. refracta and E. chamaenerioides 83
showed no differences in the number of seeds per fruit between early and late in the season.
There was significantly greater number of seeds per fruit when flowers were hand-pollinated with outcrossed pollen compared to naturally open-pollinated flowers for early 2008 and late 2010 in C. brevipes, and at all points in the season (early and late) for
E. refracta (Table 5) in the Bristol Mountain site. This result is indicative of consistent pollen limitation within and between years. There were no significant differences between control and hand-pollinated crossed fruits for E. chamaenerioides in any year or season, although there were no data for 2010 because of the lack of sufficient plants on which to perform cross-pollination.
Inter-annual variation in reproductive success
The number of germinated pollen grains in styles was significantly higher in 2008 compared to later years for both C. brevipes and E. refracta (Tables 2, S1). This indicates that pollen delivery was highest in the year with the earliest plant reproductive phenology. However, this pattern was driven by the high numbers late in the season in
2008. There were no significant differences in fruit set between years in any species
(Tables 3, S1).
The seed to ovule ratio, or within-fruit fertilization rate, was significantly higher in 2008, the year with early plant reproductive phenology, compared to 2009 and 2010 for C. brevipes. However, for both E. refracta and E. chamaenerioides it was significantly higher in 2009, the year with intermediate timing of plant reproductive 84
phenology. Both C. brevipes and E. chamaenerioides had significantly more seeds per fruit in 2008 compared to 2009 and 2010, while E. refracta showed no difference between years (Tables 4, S1).
Differences between selfing and outcrossing species
The selfing species E. chamaenerioides had consistently greater seed to ovule ratio (Figure 2a), seed number per fruit (Figure 2b), and fruit set (Figure 2c) compared to its sister outcrossing species E. refracta (Table S2).
Herbivory
For all three species, herbivory was significantly lower for early flowering plants in 2008, the year with the earliest flowering phenology (see Table 6 for means, Table S1 for ANOVA results). These differences were significant in C. brevipes at the Bristol site and E. chamaenerioides at Goffs, and almost significant for E. refracta at Bristol (P =
0.06) and Goffs (P = 0.07). For the two years in which reproduction occurred later in the season, the differences in herbivory for early versus late flowering plants were small and non-significant.
DISCUSSION
In three spring flowering seasons in the Mojave Desert, we observed different plant phenological patterns. In 2008, germination-triggering winter rainfall was early, leading to particularly early plant reproductive phenology, whereas in the following two years germination-triggering rainfall did not occur until later in the season, resulting in 85
progressively later growth and reproduction for all three species. In 2010, rainfall did not occur until mid-January, resulting in the latest peak flowering in mid-April. In this study, we hypothesized that a consequence of asynchronous phenology between pollinators and outcrossing plant species would be a reduction in reproductive opportunities leading to more variable reproduction compared to selfing species, that synchrony with herbivores would reduce plant reproductive success, and that selfing species would have greatest reproductive success when plants were asynchronous with their interacting insects in order to avoid herbivory. We found evidence that asynchrony between plants and pollinators occurred when plant reproductive phenology was early. Additionally, we found evidence that when plant reproductive phenology was late in the season, there was greater synchrony between plants and herbivores. Finally, we found support for the prediction that performance of the selfing species was not affected by pollinator-plant synchrony, but was negatively affected by synchrony with herbivores.
For plant flowering that occurred early in 2008, pollinator activity (inferred from the number of germinated pollen tubes in styles of open flowers) was lower early in the season compared to later for the two outcrossing species, C. brevipes and E. refracta.
Previous research has shown that insect development, activity, and spring emergence is strongly tied to temperature (Tauber et al. 1986, de Jong et al. 1996, Roy and Sparks
2000, Gordo and Sanz 2006). Thus, the depression in activity of the primary insect visitors to these plants during this period of peak flowering was likely due to cold temperatures during February whereas rainfall was driving plant phenology.
Analogously, we found greater overall herbivore damage in the two years with late 86
reproduction in which peak flowering temperatures occurred when temperatures were warmer than in 2008, the year with early flowering phenology. It is important to note that this study focused on only the insect activity that is present when focal plants were in their reproductive phase and not during periods when plants were not being monitored.
This result emphasizes the need to better understand the environmental cues associated with insect phenology in order to fully understand how interacting insects and plants respond to climate variability (van Asch and Visser 2007, Miller-Rushing et al. 2010).
For all three species, we observed high population density in 2008 compared to subsequent years, which has been shown for some species to lead to greater pollinator visitation (Allison 1990, Kunin 1997, Fausto et al. 2001). Thus there were environmental factors, including high and frequent early rainfall, which contributed to a “good” year in
2008. The number of germinated pollen grains in styles during this early period in 2008 was equivalent to levels found in 2009 and 2010. While pollinators visited less early in the season in 2008, they were no less active compared to subsequent years when flowering was delayed. Yet, the decrease in activity early relative to late in 2008 represents a cost of this early phenology, since fruit set may have been higher if pollinators had equivalent activity compared to later in the season, particularly because
2008 had the greatest overall apparent pollination intensity. Patterns of fruit set generally corresponded well to the pollen tube density results. In C. brevipes, fruit set was not different in any season or year; however, in E. refracta it increased from early to late in both 2008 and 2009, but decreased in 2010, the year with very late reproduction. Two potential explanations for a decrease in fruit set in 2010 in the absence of any difference 87
in pollen tube are increased fruit herbivory late in the season, or reduced resource availability. To tease out the mechanisms causing late season decreases in fruit set in this species, and to determine how the magnitude of late seasonal patterns could potentially balance the cost of early seasonal reproduction, subsequent field and greenhouse experiments will be necessary.
The timing of herbivory can be a major factor affecting plant reproduction
(Pettersson 1991, Pilson 2000). In general, herbivory was greater later in the season in
2008 for all three species; this was significant for C. brevipes and E. chamaenerioides and marginally significant for E. refracta. In 2009 and 2010 herbivory levels were consistent across the season, with the exception of C. brevipes in 2009, which experienced higher herbivory later in the season. Additionally, herbivory was higher in those years with later reproduction. This supports our prediction that while greater plant- pollinator synchrony is beneficial for outcrossing species, there may be cost of concurrent plant-herbivore synchrony. Indeed, when phenology was shifted the latest in 2010, we observed herbivores to be abundant consumers before many plants had even begun flowering. The selfing species was equally as affected by herbivores as the outcrossing species. It is possible that, in the absence of constraints on pollinator activity, selfers will benefit most from an earlier and a quicker phenology to avoid late season stressors such as herbivory or drought (Ehrlen and Munzbergova 2009, Forrest and Thomson 2010, Ivey and Carr 2011). 88
The selfing species showed no difference in fruit set in any year or season. When we compared the sister taxa E. refracta and E. chamaenerioides, we found that the selfing species consistently had larger and less variable seed to ovule ratios, seeds per fruit, and fruit set across years and seasons. This points to selfing as a “safer” strategy when climatic conditions influencing phenology and pollination are variable, and provides evidence for the benefits conferred by reproductive assurance. Whereas previous studies have shown that flowering phenology can have a significant influence on plant fitness in outcrossing species (e.g., LeBuhn 2004, Fleming 2006, Kameyama and
Kudo 2009), this work demonstrates that phenological variation has a lesser impact on the fitness of a self-fertilizing species compared to its outcrossing relative.
Both the seed to ovule ratio and the number of seeds per fruit showed a general and sometimes significant decrease from early to late in the season in all three species, regardless of year. Here we saw a contradiction between the patterns for plant-level fertilization rate (fruit set) compared to within-fruit resource allocation. Therefore, the factors limiting flower fertilization rate (i.e., how many flowers receive pollen by visitors) are separate from the factors limiting resource allocation within fertilized fruits
(i.e., how many ovules are fertilized within visited flowers). This can occur from reproduction being limited by seasonal variation in availability of resources, such as soil moisture, that are not associated with pollen availability. Since plants were included in the study based on being early in their reproduction both early and late in the season, we attribute this to external resource limitation in the environment and not to internal resource depletion that may occur as the plant reaches the end of its reproductive period. 89
We examined pollen limitation of reproduction throughout the season by hand- pollinating flowers and comparing the fruit set to naturally pollinated flowers (Burd
1994). The seed number per fruit of hand-pollinated flowers was higher in all years and seasons compared to naturally pollinated fruits for E. refracta, and during 2008 and late
2010 for C. brevipes. Thus, E. refracta was always found to be pollen limited, and C. brevipes was pollen limited in the year with early rainfall and at the end of a year with late-season flowering. The selfing species E. chamaenerioides was never found to be pollen limited. These results indicate that an outcrossing reproductive strategy can be risky in a time-limited variable environment, leading to more variable reproduction.
To summarize, we have shown that in two obligate outcrossing desert annual species, reproduction is more variable within and between years compared to a selfing species, indicating that outcrossing is a more risky strategy in variable environments.
Additionally, outcrossing species were found to frequently experience pollen limitation, whereas selfing species did not; selfers benefited from reproductive assurance in the absence of pollinators. When reproductive phenology was early in the season, there was greater asynchrony between both plants and pollinators and between plants and herbivores compared to when reproductive phenology was late in the season. In years and seasons in which asynchrony occurs, it is possible that outcrossing species are buffered against timing variation by having longer reproductive periods or adaptations, such as low ovule number, to natural variability in pollinator communities (Artz et al.
2010). However, if the frequency of years in which asynchrony between plants and pollinators increases in the future, as has been demonstrated in other species (Hegland et 90
al. 2009), it is plausible to expect outcrossing species to experience a disadvantage compared to selfing species in the same environment. The increased risk of herbivory with late season reproductive phenology was also evident in this study, demonstrating a potential conflict for outcrossing species in their optimal phenological responses to climate variability. It is clear that it is beneficial for a selfing species to complete reproduction early, but successful reproduction in outcrossing species will be dependent on how well they are buffered against the inter-annual variability of their interactions with pollinators and herbivores. Additionally, desert annuals have the ability to buffer against the risk of asynchrony with pollinators by having long-lived seed banks and the potential for bet hedging via germination strategies (Venable 2007).
This study is unique in its investigation of the role plant-pollinator-herbivore phenology plays in determining fitness in related selfing and outcrossing taxa. We have shown that variable environmental conditions associated with desert annual phenology can contribute to the reproductive success of species based on the timing of their reproduction, and that this in turn is influenced by mating system.
ACKNOWLEDGEMENTS
We thank Tasha LaDoux and Jim Andre at the University of California Granite Mountain
Desert Research Center for assistance and advice in setting up this study. We also thank
Brian Hunter for assistance in the field and in the lab. Judie Bronstein and Steve Smith provided extensive helpful comments on earlier drafts of this manuscript. This work was 91
supported by the Garden Club of America Desert Studies Award and the California
Desert Legacy Fund.
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TABLES
Table 1: Study sites with winter rainfall, temperatures for peak flowering month, and species present in the three years of the project. CHBR=Chylismia brevipes (outcrosser),
ERRE=Eremothera refracta (outcrosser), ERCH=Eremothera chamaenerioides (selfer).
Table 2: Mean and standard error values for the ln-transformed pollen tube densities from stigmas early and late in the season for the two outcrossing species. Superscript letters represent significant Tukey-Kramer HSD differences (P < 0.05) within species.
C. brevipes E. refracta mean (ln (pollen tube mean (ln (pollen tube Year Season density)) ± SE N density)) ± SE N early 3.98 ± 0.18A 70 3.60 ± 0.21AC 118 2008 late 5.52 ± 0.16B 60 4.86 ± 0.17B 98 early 3.27 ± 0.34ACD 40 3.31 ± 0.30AC 40 2009 late 4.50 ± 0.22ABD 22 3.78 ± 0.19A 80 early 2.54 ± 0.32C 42 2.87 ± 0.19AC 76 2010 late 2.69 ± 0.28CD 46 2.56 ± 0.20C 80 98
Table 3: Mean and standard error for the number of fertilized fruits per flower (fruit set) for each year, species, site, and season (early vs. late). Significant Tukey-Kramer HSD comparisons differences (P < 0.05) are represented by superscript letter differences within species only.
C. brevipes E. refracta E. chamaenerioides mean fruit set mean fruit set mean fruit set± Year Season Site ± SE N ± SE N SE N A A 2008 early Goffs ------0.240 ± 0.066 60 0.805 ± 0.031 51 late Goffs ------0.473 ± 0.068B 41 0.821 ± 0.022 A 37 early Bristols 0.656 ± 0.136 A 70 0.496 ± 0.026 B 59 ------2008 late Bristols 0.685 ± 0.150 A 60 0.616 ± 0.025 C 56 ------early Bristols 0.681 ± 0.210 AB 40 0.572 ± 0.051 C 39 0.738 ± 0.065 A 38 2009 late Bristols 0.541 ± 0.357 AB 19 0.735 ± 0.023 D 74 0.790 ± 0.054 A 45 early Bristols 0.727 ± 0.220B 42 0.670 ± 0.042 D 77 0.969 ± 0.020 A 25 2010 late Bristols 0.703 ± 0.037AB 46 0.600 ± 0.029 C 76 0.942 ± 0.000 A 10
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Table 5: Paired t-test of seed number for control and hand-pollinated crossed fruits early and late in the season for all three species. Bolded results had significant differences between seed numbers in control and crossed fruit.
*Note: 2008 E. chamaenerioides data are from Goffs site whereas all other data are from
Bristol site. No data are presented for E. chamaenerioides in 2010 because there were not enough plants available to perform cross pollinations.
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Table 6: Mean and standard error of herbivory score for each year, site, species, and season (early vs. late). Significant Tukey-Kramer HSD comparisons differences (P <
0.05) are represented by letter differences within species only.
Herbivory score for leaves, stem and fruit: 0 = no damage, 1= minimal damage (< 25% of tissue), 2= substantial damage (25-75%) 3: intense damage (>75% of tissue).
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FIGURES
Figure 1: Annual phenological status of each species and population visited. Walking transects determined the proportion of individuals that were pre-reproductive (no flowers or fruits), reproductive (flowers present), or post-reproductive (fruit, no flowers or buds). 105
Figure 2a-c: Comparison of selfing (grey bars) and outcrossing (black bars) sister species for the following reproductive metrics: (a) Seeds per ovule, (b) Seeds per fruit, and (c)
Fruit set.
* represents significant differences between the two species in each year and season (P <
0.05).
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APPENDIX C
DROUGHT EFFECTS ON PHENOLOGY AND PHYSIOLOGY IN SELFING AND OUTCROSSING DESERT ANNUALS
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Drought effects on phenology and physiology in selfing and outcrossing desert annuals
Katharine L. Gerst1, Travis Huxman1,2 and D. Lawrence Venable1
1Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ
85721, USA
2Biosphere 2, University of Arizona, Tucson, AZ 85721, USA
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ABSTRACT
How plants cope with stressful environments will impact their distribution, population dynamics, and interactions. In particular, drought stress strongly influences growth and physiology. Mating system may also affect patterns of resource use and how plants respond to low water availability. Plants with outcrossing reproductive strategies invest more resources into floral displays compared to their self-fertilizing relatives, and their phenology is likely to be strongly influenced by the timing of their pollinators.
Because selfing species are not tied to pollinator phenology, they should be able to match their activity to periods of greater water availability to avoid drought stress. In addition, when species have physiological traits that promote greater water use efficiency these traits may provide a mechanism for the tendency of selfing species to colonize a greater range of habitats and occupy more arid habitats compared to their outcrossing relatives.
In this study, we tested the prediction that selfing species would have growth patterns and physiological traits better adapted for drought tolerance compared to outcrossing species.
We examined patterns of phenology and physiology in two pairs of sister selfing and outcrossing desert annual species, Eremothera refracta and Eremothera chamaenerioides
(Onagraceae), and Chylismia multijuga and Chylismia walkeri (Onagraceae). We found that selfing species reproduced earlier than the outcrossing species and had faster rate of leaf mass accumulation. In general, selfing species had greater carbon assimilation rates, greater stomatal conductance, and thicker leaves. Outcrossing species experienced a greater negative effect of drought on their physiological functioning compared to selfing species. These results supported our predictions that selfers are better adapted to drought
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stress than outcrossers. This work demonstrates how understanding the interaction between mating systems and environmental stress will help us to better understand the distribution and dynamics of reproductive strategies in plants.
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INTRODUCTION
Two common evolutionary trends in plants are the transition from self- incompatibility to self-compatibility and from non-autogamous to autogamous fertilization (Stebbins 1957, Antonovics 1968, Holsinger 1996, Goodwillie 1999, Barrett
2003, Willi and Maattanen 2010, Busch and Urban 2011). A prolific area of research in evolutionary biology is the exploration of the circumstances and processes that select for an autogamous or selfing strategy (Lloyd 1992, Schoen et al. 1996, Charlesworth and
Vekemans 2005, Busch et al. 2010). Research on transitions to selfing has focused on the genetic mechanisms related to the loss of self-incompatibility (e.g., Georgiady 2002,
Fishman and Stratton 2004) as well as ecological drivers of selfing in natural populations.
One driver of the evolution of selfing that has been well studied is a release from pollen limitation and the benefit of reproductive assurance. Selfing increases the probability of reproduction in the absence or scarcity of pollinators when there is not sufficient pollen available for maximum fertilization (Whisler and Snow 1992, Goodwillie 2001, Kalisz and Volger 2003, Willi 2009). Many studies have focused on the relative extent of pollen limitation in populations of species with mixed mating systems when there is variation in the frequency of selfing between individuals and populations. This research has identified a common association between a higher frequency of selfing and pollen limitation (e.g.,
Davis and Delph 2005, Moehler 2006, Weber and Goodwillie 2009), as well as an increase in selfing towards the edge of species ranges (Busch 2005, Herlihy and Eckert
2005).
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The transition to a selfing strategy from outcrossing relatives or populations is most often accompanied by changes in floral morphology (Dudley et al. 2007,
Goodwillie et al. 2010, Sicard and Lenhard 2010, Thiess et al. 2010). These include smaller, less showy flowers (Elle and Carney 2003, Goodwillie 2005, Tomimatsu and
Ohara 2006), a reduction in available nectar (Galetto et al. 1997), reduced floral longevity
(Snell and Aarssen 2005), reduced herkogamy or anther-stigma distance (Armbruster et al. 2002, Barrett 2003), and reduced dichogamy, or temporal separation of sexual functions (Routley et al. 2004). Given the cost of resources allocated to pollinator attraction, a reduction in investment towards pollinator attraction may result in changes to allocation to growth and reproduction, whether it is via fewer flowers, more ovules, or greater vegetative biomass and photosynthetic capability. Within plants, there is evidence for a trade-off between flower size and number (Sargent et al. 2007), thus the evolution of smaller flowers resulting from a transition towards selfing may allow for a shift to greater flower number (Aarssen 2000, Hechel and Riginos 2005).
Sex allocation theory predicts differing reproductive investment in females versus males in dimorphic species (Obeso 2002, Leigh and Nicotra 2003, Delph et al. 2005,
Caruso and Yakobowski 2008, Van Etten et al. 2008) often resulting in differing reproductive allocation, functional traits and niche occupation by gender. Likewise, within hermaphrodites, theory predicts a shift in allocation away from male function with an increase in selfing (Brunet 1992, Damgaard and Abbott 1995, Parachnowitsch and
Elle 2004, Mazer et al. 2007). This shift away from male allocation (e.g., reduced pollen/ovule ratios), coupled with reduced floral allocation for pollinator attraction, may
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have consequences for the functional traits and niche occupancy of selfers compared to their outcrossing relatives, as large flowers are costly in resource-limited environments
(Galen et al. 1999, Galen 2000). Since patterns of resource allocation vary with mating system as well as with environmental conditions (e.g., Galen 2000, Fabbro and Korner
2004), we expect that different reproductive strategies will be suited to particular habitats based on species phenology and physiological functioning.
A selfing strategy is predicted to promote the colonization of novel habitats or niches (Baker 1955, Pannel and Dorkin 2006, Levin 2010, but see Busch 2011), allowing for the expansion into habitats that were not accessible to their ancestral outcrossing sister taxa. Such an expansion may be driven by various mechanisms; while the evolution of selfing occurs as a result of reproductive considerations, such as pollen limitation, it is also likely that selfing may evolve as a result of climatic drivers, such as aridity. Researchers have observed that selfing populations of species with mixed mating systems are more likely to be found in drier habitats than outcrossing populations (Elle et al. 2010) or related outcrossing species (Mazer et al. 2011). In addition, drought stress has been shown to select for earlier flowering in at least two species of annual grasses
(Sherrard and Maherali 2006, Franks et al. 2007) and in a wild mustard (Stanton et al.
2000).
There is also evidence that selfing species develop more rapidly than outcrossers
(Aarssen 2000, Snell and Aarssen 2005, Ivey and Carr 2011, but see Hill et al. 1992), particularly in annual species. Accelerated development would be particularly
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advantageous in habitats that experience drought, especially for annual species with a temporally limited growing season to complete their lifetime reproduction. Faster growth and reproduction could allow an annual species to concentrate its life history during times of water availability and avoid later periods of drought stress (Mooney et al. 1976,
Dudley 1996a, Dudley 1996b, Ackerley et al. 2000, Artz and Delph 2001). Outcrossers may be more sensitive in the timing of their reproduction in order to match pollinator phenology, rather than grow and reproduce in optimal timeframes for resource availability (Miller-Rushing et al. 2010). This is particularly true when reproduction is time limited or they depend on specialized pollinators. Thus they may be limited in the habitats or niches they can successfully occupy not only by pollinators but by environmental conditions. Therefore outcrossers may be under weaker selection for phenological adaptation to drought compared to their selfing congeners or be less likely to occupy the drier habitats or temporal niches.
Selection on ecophysiological traits can be particularly strong when water is limiting and variable (Geber and Dawson 1990, Chaves et al. 2003, Molina-Montenegro et al. 2001, Gianoli et al. 2005). Selection for drought avoidance in selfers that are unconstrained by pollinator requirements could favor physiological strategies such as high growth rate or high CO2 assimilation rate (Chapin et al. 1993, Ackerley et al. 2000,
Stanton et al. 2000, Artz and Delph 2001, Ludwig et al. 2004, Donovan et al. 2007, Wu et al. 2010). Likewise, if selfers are more able to occupy drier habitats, they are predicted to exhibit traits associated with increased water use efficiency, such as thicker leaves and higher Carbon-13 isotope discrimination (Wu et al. 2010, Ivey and Carr 2011, Mazer et
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al. 2011). A drought tolerant and a drought avoidance strategy are not mutually exclusive, and each could promote the success of selfers in a wider range of habitats compared to outcrossers, facilitated by their more flexible resource allocation strategy.
However, few studies have explored the variation in growth and physiological strategies that have accompanied mating system evolution.
In this paper, we examine growth and physiology of two pairs of annual selfing- outcrossing sister species, Eremothera chamaenerioides and Eremothera refracta, and
Chylismia walkeri and Chylismia multijuga (Onagraceae). Previous field studies of
Eremothera demonstrated that inter-annual and seasonal reproductive success was more variable for outcrossing species compared to selfing species, and was sensitive to the snychrony of plant reproductive phenology, pollinator activity, and herbivore activity
(Gerst and Venable, in prep). However, we do not know if the timing of selfing and outcrossing species growth and reproduction differs as a result of differences in development time or germination cues. Here we tested the hypothesis that the selfing taxa are able to allocate relatively more resources to growth and are physiologically better adapted to drought than their outcrossing congeners. We tested the following predictions: (1) selfing species will have faster growth rates and reach flowering more rapidly relative to outcrossers, (2) selfing species will have higher carbon assimilation rates than outcrossers, (3) selfing species will have traits associated with greater water use efficiency compared to outcrossers, and (4) selfing species will have higher relative performance in drought conditions compared to outcrossing congeners.
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MATERIALS AND METHODS
Study species:
Two pairs of sister species were used in this experiment: Eremothera refracta, an outcrossing self-incompatible species, and the self-pollinating species Eremothera chamaenerioides (Onagraceae); and Chylismia multijuga, an outcrossing self- incompatible species, and the selfing Chylismia walkeri (Onagraceae). Each selfer is recognized to be derived from its congeneric outcrosser, based on Raven (1969, 1979) with recent generic re-classifications in Wagner et al. (2007). Self-incompatibility in E. refracta and C. multijuga and self-fertilization in E. chamaenerioides and C. walkeri were confirmed by bagging individuals and observing lack of seed production in the former, and with full fruit set of bagged individuals in the later (Gerst, personal observations). All species are annuals found in North American deserts on slopes and in sandy washes and the sister pairs are known to grow sympatrically. While range distributions of sister taxa overlap, the two selfing species have larger ranges
(approximately 2x greater) that include the ranges of their outcrossing pair (Raven 1969).
Individuals within each species pair are comparable in size, growth habit, and leaf size and shape.
Seeds from both E. refracta and E. chamaenerioides were collected in a wash adjacent to Goffs Butte in the Eastern Mojave desert, California (34o51’48.17 N,
115o5’37.13 W). Seeds from C. multijuga were collected in Red Rock Canyon, Nevada
(36°24'30.54"N, 114°33'29.84"W) and seeds from C. walkeri were collected near Primm,
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Nevada (35°36'42.63"N, 115°23'40.77"W). These sites are all relatively central in each species range. All seeds were collected in the Spring of 2009.
Study Design:
This study was carried out in a greenhouse at the University of Arizona.
Nighttime temperatures were maintained between 50-60oC and daytime temperatures were maintained between 60-75oC. 100-200 seeds were planted per mother plant from six mothers per species. Seeds were germinated in 2% agar in petri dishes and transferred to cone-tainers (with 3-5 individuals per cone-tainer) when roots were at least 1 cm long.
Plants were grown in Stuewe and Sons D40 Deepot cone-tainers (2.5 in diameter, 10 inch depth, 40 in3). Soil composition was a 3:1 mixture of Sunshine mix #2 (Sun Gro
Horticulture Canada Ltd.) and 30 grit sand. Once seedlings were transferred to cone- tainers, they were covered with plastic sheets to keep soil and air moist until plants had two sets of true leaves to confirm establishment. Once seedlings were established, pots were thinned to one per pot for a total of 30 individuals per mother per species.
Seeds were planted in agar the week of November 16-20, 2009, and transferred to pots January 4-15, 2010. Plants were arranged in cone-tainer trays by species and mother, with ten plants per tray of the same mother spread over three trays, for a total of
30 individuals. These were distributed across three greenhouse tables with one tray per species per mother represented on each table. Trays within tables were arranged randomly and all trays were shuffled between tables twice throughout the experiment.
Only every other tray slot was filled with a plant to ensure no competition for above-
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ground space or light between individuals; the experiment consisted of 18 trays of 10 per species and 72 trays total.
High water treatment plants were saturated every week, which allowed soil to drain completely between watering; this was sufficient to prevent any signs of water stress in plants. Low water treatment plants were watered every two weeks which resulted in partial wilting but did not induce high mortality.
We determined general patterns of floral allocation by examining a small subset of flowers from individuals in the high water treatment. Because of small weight values, allocation to floral tissue was determined by combining the weight of 10-20 early- produced flowers of each species spread across mothers, and dividing to determine the average per flower weight; this was repeated five times. Nectar volume of freshly opened flowers, petal length, and floral longevity (no fertilization permitted in outcrossers) were determined from 15 early-produced flowers. Nectar volume was measured with 0.5µl 3.2 cm capillary tubes placed in nectar tubes on the first day that flowers were open, prior to flower hand-fertilization.
Growth and Time to Flowering
To determine the time to flowering, we checked each plant weekly for opening of the first flower. During the first two weeks of March 2010, before plants entered their reproductive phase, we counted the number of leaves on each individual and assessed plant mortality. We calculated leaf production rate as the number of leaves produced over the number of days since planting. We then estimated leaf mass accumulation rate
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by multiplying that value by the mean species leaf mass from plants used in physiological measurements (see below). Finally, we estimated biomass accumulation rate by extrapolating leaf production rate to the time of reproduction (leaf production rate * days to reproduction), multiplied by the final ratio of leaf+stem mass/leaf mass for an estimate of the combined leaf and stem biomass at reproduction. We then divided that value by the days to reach reproduction for a metric of biomass accumulation rate.
Physiological measurements
To examine performance differences between selfers and outcrossers and the effect of water treatment, we measured physiological traits on a subset of individuals in the greenhouse. During the final two weeks of February and the first two weeks of
March 2010, before the reproductive phase, we carried out a series of physiological measurements using a LI-COR 6400 portable infrared gas exchange analyzer system (LI-
COR, Lincoln, Nebraska, USA). On 12-16 individuals per water treatment per species, we measured the carbon assimilation (A) relative to the internal CO2 concentration (Ci) to construct A-Ci curves to determine ambient (Anet) and maximum (Amax) carbon assimilation rates and ambient stomatal conductance (gs). We calculated these gas exchange measurements on both a leaf area and a leaf mass basis. CO2 cuvette concentrations (ppm) used to develop curves and reach maximum rates were: 375
(ambient), 400, 300, 200, 100, 50, 400, 600, 800, 1200 (maximum); we used a pre- programmed setting allowing for periods of acclimation between measurements. We used the standard chamber with the red-blue light source (LI-6400-02b) with the block
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temperature set to 25oC, a flow rate of 500 µmol s-1, and a light level of 1200 PAR. All measurements were taken between 0900 and 1300. Measurements were carried out on the most recently produced mature leaf.
Leaves used for physiological measurement were collected and scanned for area using the LI-3100 Leaf Area Meter (LI-COR, Lincoln, Nebraska, USA) and their dry weight measured to obtain specific leaf area (SLA, cm2 g-1). Dried leaves were ground to a fine powder in the laboratory for carbon and nitrogen analysis at the University of
New Hampshire Stable Isotope Lab to obtain carbon and nitrogen content and δ C13 isotope ratio. Leaf nitrogen percentages were used to calculate nitrogen mass per unit leaf area (N m-2= %N/SLA). δ C13 isotope ratio values were converted to carbon discrimination values (Delta, Δ) (Farquhar et al. 1989). Delta is a proxy for time- integrated water use efficiency (WUE) with lower discrimination values indicative of higher WUE.
Field measurement of stomatal conductance
In order to compare performance of greenhouse plants to plants in the field and examine differences between selfers and outcrossers, we examined the physiology of E. refracta and E. chamaenerioides at a site where they co-occurred in the Sacramento
Mountains in the Eastern Mojave Desert, California (34°50'6.87"N, 114°45'34.46"W).
On April 2nd, 2010 between 10am and 12pm, we used a Decagon SC-1 Leaf Porometer
(Decagon Devices, Inc) to measure stomatal conductance (gs) in 30 pre-reproductive E. refracta and 30 pre-reproductive E. chamaenerioides plants. The conditions were full
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sun with temperatures ranging between 25oC and 28oC. Carbon and nitrogen isotopes and specific leaf area were collected following the same protocol described above.
Statistical analysis
All statistical analyses were performed in SAS 9.1.3 (SAS Institute, Cary, NC,
USA). To examine differences in leaf size we constructed mixed models using water treatment (dry vs. wet) as a fixed effect and mother as a random effect for each species.
For differences in survival in all four species, we used an ANOVA to test for the effects and interaction of mating system and water level with survival modeled as a binomial variable. To examine differences in our three metrics of growth rate and time to flowering, we constructed mixed models using water level and species as fixed effects and mother nested within species as a random effect with separate analyses for the two sets of species pairs.
To analyze physiological variation between the species of each pair, we used a
Generalized Linear Model (PROC GLM) to determine the effect of species (selfer or outcrosser), water level (dry or wet), and species*water interaction for the following
2 -2 dependent variables: Anet, Amax, gs, Delta, SLA, nitrogen per m (N m ), % leaf nitrogen
(%N) and relative stomatal limitation (Amax-Anet/Amax). Anet, Amax and gs were analyzed from both area-based and mass-based calculations. Relative stomatal limitation was arcsine-squareroot transformed for data analysis.
Using data from all pooled individuals sampled for physiological measurements, we carried out simple linear regressions to determine how nitrogen per leaf area, stomatal
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conductance, and SLA relate to Anet and Delta, and how these patterns differed between selfers and outcrossers.
For field measurements of conductance in E. refracta and E. chamaenerioides, we
2 tested for differences in gs, SLA, %N, N/m and Delta using Student’s t-tests. We also
-2 compared pooled greenhouse and field measurements of gs, SLA, %N, N m and Delta within each species using Student’s t-tests to determine how differences in physiological functioning between greenhouse and field plants.
RESULTS
Floral allocation
We examined allocation to flowers of selfing and outcrossing species in greenhouse plants growing in identical high water conditions in order to confirm our assumption of greater allocation to floral display in outcrossers. The measurements of floral traits related to resource allocation (petal size, flower weight, nectar volume and floral longevity) demonstrated a pattern of significantly greater floral investment in outcrossing taxa and confirming our assumptions of floral resource allocation (Table 1).
Survival
We predicted that drought would have a greater negative effect on outcrossers than selfers. Selfing species had a higher survival to reproduction compared to the outcrossing species (P = 0.001) (selfers: 89% and 95% vs. outcrossers: 62% and 72% for
Eremothera and Chylismia respectively, Table 2). While water treatment (wet vs. dry)
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did not affect survival (P = 0.3197), there was a significant mating system*water interaction (P = 0.0383) such that drought had a greater negative effect on survival for outcrossers compared to selfers, confirming our prediction.
Plant growth and development
We predicted that selfers would have faster growth and time to reproductive than outcrossers. Selfers and outcrossers did not differ in their leaf production rate for either pair of sister species (Table 3, Figures 1a and 1b), but in Eremothera there was a higher rate of leaf production in the wet treatment, which was of greater magnitude in the outcrosser E. refracta (Figure 1a; Table 4). For both genera, selfers had greater leaf mass accumulation rates than outcrossers, with rates for outcrossers being more reduced by drought (Figure 2a and 2b; Table 3). In Eremothera the selfer also had greater biomass accumulation rates (Figures 3a and 3b; Table 3) with the dry treatment resulting in a lower biomass accumulation rate than did the wet treatment. In Chylismia biomass accumulation rate was higher for the selfer compared to the outcrosser, but the difference was not significant. The interaction between water level and species was nearly significant (P = 0.06), with the outcrosser growth rate being more reduced than the selfer by dry conditions. Thus, we found support for our prediction that selfers would have more rapid growth than outcrossers and we found that outcrossers were somewhat more negatively affected by drought.
For both genera, the selfing species flowered earlier than their outcrossing sister species by approximately two weeks (Table 3, Figures 4a and 4b). Plants in the dry
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treatment flowered earlier than in the wet treatment for both species, with the effect of water level in Eremothera being stronger in the outcrosser than in the selfer, confirming our predictions that both time to reproduction and drought would have a greater negative effect on outcrossers.
Physiological responses
Greenhouse measurements
We predicted that selfing species would have higher carbon assimilation rates and water use efficiency compared to outcrossers. For both genera, carbon assimilation rate at ambient CO2 (Anet) was greater in the selfing species, and greater in the wet compared to the dry treatment (see Table 4 for means, Table 5 for model results). In Chylismia, there was a greater relative reduction in Anet in low water for the outcrossing species, C. multijuga compared to the selfing species C. walkeri. Maximum assimilation rate, Amax, was also greater in the selfing species E. chamaenerioides compared to E. refracta, but not influenced by water level. For both genera, stomatal conductance, gs, was lower in dry treatments. In Chylismia, gs was lower in the outcrossing species than in the selfing species, and in Eremothera there was a greater depressive effect of the dry treatment on gs in the outcrossing species. These results indicate that gas exchange rates are generally greater for selfers than outcrossers and that drought had a greater negative effect for outcrossers, consistent with our predictions.
For Eremothera the outcrossing species, E. refracta, had greater SLA (thinner leaves) and higher leaf nitrogen than did the selfing species E. chamaenerioides. These
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traits are related to greater investment in photosynthesis. However, no pattern was detected for these variables in Chylismia (Tables 4 and 5). There was no effect of water level on SLA or leaf nitrogen in either pair of sister species. Delta, our integrated metric of water use efficiency, did not differ between species or water treatment in Eremothera although there is a trend for greater values, consistent with lower WUE, in the wet treatments. In Chylismia Delta was higher in the wet treatments and in the selfing species, C. walkeri, suggestive of lower WUE. The effect of water level on Delta was relatively larger in the outcrossing species consistent with greater sensitivity to water stress in this species. Relative stomatal limitation was greater for both outcrossers compared to selfers, and greater in the dry treatments. These results are contrary to our prediction that selfers are more water use efficient than outcrossers, and appear to result from outcrossers shutting down their gas exchange more readily than selfers due to water stress.
Relationships between physiological traits
In order to determine intra-species patterns of water use efficiency and photosynthetic performance, we pooled data for high and low water treatments to determine the nature of the relationships between traits within species. For all four species, there were positive significant relationships between stomatal conductance, gs, and carbon assimilation, Anet. In Eremothera, the selfer had a higher slope and intercept when examined on a per area-basis (Figure 5a), demonstrating that it was able to achieve higher assimilation rates with the same magnitude of stomatal regulation. However, this
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pattern was not present in Chylismia (Figure 5b), nor when calculated on a per mass-basis
(Tables S1 and S2). SLA was also a good predictor of Anet (mass-basis), with the selfers having higher slopes than the outcrossers. These results suggest that thin leaves have relatively greater carbon gain in selfers compared to outcrossers, indicative of greater resource use efficiency.
In Eremothera, both SLA and gs (mass-basis) were good predictors of Delta
(Table S1). The slope of the positive relationship with SLA was higher in the selfer, so thinner leaves result in relatively less isotope discrimination. The selfer also had a higher slope for gs showing a relatively greater effect of increasing stomatal conductance on reducing isotope discrimination (Figure 5c). In Chylismia, gs was also a good predictor of Delta, but with a higher slope in the outcrosser (Figure 5d). Leaf nitrogen per unit area was not a strong predictor of Anet or Delta (Tables S1 and S2).
Field measurements of conductance
In the field, stomatal conductance was significantly higher in E. refracta than E. chamaenerioides (area based: t60 = -3.258, P = 0.0019; mass-based: t60 = -2.309, P =
0.0246) and there was a marginally significant trend towards higher Delta in E. refracta
(t57 = -1.868, P = 0.066), indicating that E. chamaenerioides had greater WUE. These result from field plants are consistent with our predictions, but not with the greenhouse measurements. E. refracta had a significantly higher percent nitrogen in leaves (t57 = -
3.023, P = 0.0037) and nitrogen per leaf mass (t57 = -2.281, P = 0.0263) but no difference in SLA (t60 = 1.677, P = 0.100) (see Table 4 for means).
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When field and pooled greenhouse measurements were compared for E. chamaenerioides and E. refracta, we found that greenhouse grown plants of both species had significantly greater SLA and higher percent leaf nitrogen than field plants (Table 6).
Nitrogen per leaf area was significantly higher in the greenhouse for E. chamaenerioides, while there was no difference for E. refracta. E. refracta in the field had significantly greater stomatal conductance (both area- and mass-based) than individuals in the greenhouse, but mass-based conductance measurements for E. chamaenerioides were lower in the field and there was no difference in area-based measurements. Finally, field measurements of Delta were significantly lower (indicative of greater water use efficiency) compared to the greenhouse plants. In general, plants of both species in the field had thicker leaves, less nitrogen, and greater WUE compared to greenhouse grown individuals (see Table 4 for means).
DISCUSSION
Ecological and evolutionary processes, such as reproductive strategies, are strongly influenced by environmental stresses such as drought (Chapin et al. 1993,
Chaves et al. 2003, Wu et al. 2010). In this study we examined the growth and physiological characteristics of two sister pairs of desert annual selfing-outcrossing species by experimentally altering water availability. Selfing species show lower resource allocation to floral displays compared to their outcrossing relatives, which may increase their ability to function when resource availability is low. We hypothesized that, for this reason, selfing species would be superior at coping with water stress than their
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outcrossing relatives. Specifically, we predicted that they would grow and reproduce faster to avoid drought and have traits promoting water use efficiency. Selfing species had greater survival in this study, and drought had a greater negative effect on the survival of outcrossers compared to selfers.
We found support for our first prediction, that selfers would have faster growth rates and reach flowering more rapidly than outcrossers. Rates of leaf mass and total biomass accumulation were greater in selfing species relative to their related outcrossers regardless of water treatment. These results are consistent with a pattern found in a multi- species meta-analysis by Snell and Aarssen (2005), as well as in mixed-mating populations of Collinsia parviflora (Elle et al. 2010) and of Mimulus selfing-outcrossing species pairs (Ivey and Carr 2011). In addition, water stress caused an increase in the onset of reproduction, consistent with previous research on Mediterranean and desert annual plants (Aronson et al. 1992). The faster growth and time to reproduction in selfers provides support for the prediction that selfers can more readily escape late season drought conditions. Rapid development also provides indirect support for the notion that selfers may be well adapted to colonize and occupy a wider range of habitats (Baker
1955), since they will not be as strongly affected by late season environmental conditions such as high temperatures or low soil moisture.
Our second prediction, that selfers would have higher rates of carbon assimilation than outcrossers, was also supported by these results. This is consistent with findings by
Mazer et al. (2011), who found increased photosynthetic rates as well as earlier flowering
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times in selfing taxa compared to their outcrossing relatives in Clarkia. The more rapid growth in selfers is likely a direct result of higher rates of Anet. We then asked, how do selfers achieve a higher assimilation rate? Allocation to leaf nitrogen correlates with investment into photosynthetic capability (Evans 1989). However, despite higher instantaneous photosynthetic rates (Anet and Amax) in selfers, percent leaf nitrogen and leaf nitrogen per leaf area were higher in outcrossing species compared to selfing species.
Selfers are therefore achieving high carbon assimilation rates more efficiently than outcrossers per unit nitrogen allocation. In Eremothera, we also see evidence for greater resource use efficiency for the selfer in our linear regression analyses of area-based gs and Anet via a greater assimilation rate per water loss. In general, plants with thinner leaves and high conductance have relatively greater assimilation rates and less carbon discrimination (greater slopes) in selfers compared to outcrossers. This suggests that selfers may capitalize more in the high resource conditions that promote thinner leaves with less stomata closure. The extra construction costs associated with tissue allocation to flowers and flower maintenance in outcrossers may drive this difference by having fewer relative resources available to allocate towards carbon assimilation (Ashman and
Baker 1992, Aronson et al. 2003, Kudo and Ida 2010). Alternatively, this pattern may result because of the timing in which we captured the physiology of selfing species being during a particularly optimal period in their development, which may differ from that of outcrossers given the difference in the rate of growth and reproduction (Farnsworth 2004,
Maherali et al. 2011). Future efforts to characterize the ontogenetic variation in
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physiological performance throughout the developing of selfers and outcrossers would help elucidate the interactions between phenology, physiology and fitness.
Lower stomatal conductance and higher relative stomatal limitation in outcrossing species points towards greater stomatal regulation compared to selfing species. Also, the low water treatment had a greater negative effect on performance of the outcrossing species in survival, Anet, gs, and Delta more than it did for the selfing species. Integrated
WUE of carbon isotope discrimination showed that the outcrossing Chylismia species grown in the low water level was more water use efficient than the selfing species.
Although selfing taxa have thicker leaves allowing for greater diffusional protection against water loss, our results do not support our prediction that selfers will have traits associated with greater water use efficiency, and actually suggest that outcrossers tend to be more water use efficient by reducing water loss, evident by both an instantaneous metric of stomatal regulation (low gs) and integrated metric (low Delta). In experiencing greater drought-induced stress, outcrossers were shutting down their stomata, effectively reducing opportunities for carbon assimilation. Thus, in the context of understanding the persistence of selfing and outcrossing reproductive strategies, we see that the tendency towards water use efficiency in outcrossers may be a mechanism for survival in conditions in which they are not able to optimally perform physiologically rather than an adaptation to thrive in dry habitats (Galen 2000, Kelly et al. 2008). Selfers, on the other hand, appear to be adapted to maintain high levels of physiological performance in both high and low resource conditions. Thus, they may be more flexible in their ability to persist in a wider range of habitats, allowing for greater range distributions and
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colonization (Baker 1955, Busch 2005, Randle et al. 2009, Evans et al. 2011). Given that outcrossers had lower survival than selfers, particularly in the dry treatment, we see persuasive evidence for this interpretation due to this direct fitness effect of drought via mortality.
Our field survey of conductance, SLA, Delta and leaf nitrogen between the selfing and outcrossing Eremothera plants growing together in the field provided conflicting patterns compared to those observed in the greenhouse. E. chamaenerioides had lower conductance and lower Delta than E. refracta, suggesting greater water use efficiency, whereas we found outcrossers to generally be more water use efficient in the greenhouse.
One possible explanation for the reversal in water use efficiency between field and greenhouse observations is that the field plants were under less stress than plants in the greenhouse despite lower apparent nitrogen availability. Thus, outcrossers appeared to be more efficient at regulating water loss when resources are limited, which can limit their fitness and ultimate distribution. Selfers may have a more buffered conservative strategy in which they do not exhibit as large a range of plastic behavior in water use efficiency; rather they have more consistent stomatal regulation across environments.
However, this is not wholly consistent with patterns seen in the greenhouse plants, which had a steeper relationship between gs and Anet. One explanation for this discrepancy is that given the tendency by selfers to have advanced phenology they may have already surpassed their primary growing stage compared to co-occurring outcrossers since they were measured at the same time but not necessarily the same point in their growth curve.
When we compared physiological traits for Eremothera plants growing in the field with
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those from the greenhouse experiment we found that conductance for the outcrossing species were higher in the field and in general leaves were thinner, had higher nitrogen content, and had lower integrated WUE. However, our ability to detect variation between wet and dry treatments in a similar range of values as our field measurements indicates that our experimental results are realistic and informative to the natural variation within and between these four species.
This study demonstrated that selfing species reach reproduction faster, accumulate leaf mass faster, and have greater carbon assimilation rates compared to outcrossing species consistent with a drought avoidance strategy. We propose that these result from increased resource use efficiency by selfers due to reduced construction costs associated with showy floral displays. Contrary to our predictions, we found that outcrossing plants growing in the greenhouse were more water use efficient, particularly in dry conditions.
This may represent a plastic tendency by outcrossers to shut down physiologically in order to persist and minimalize water loss in suboptimal conditions. Plants in the field showed the opposite pattern, indicating that conditions were more favorable for outcrossers resulting in less stomatal constraints. Thus, while outcrossers may persist in both dry and wet years in the desert, our results suggest they are primarily adapted to function in good conditions. While increased water use efficiency may be an adaptation to occupy drier habitats, it may also be an adaptation to fill a temporal niche by functioning to tolerate drier years but only doing well in good years (Casper et al. 2005).
Selfers by contrast, may be more buffered to do well in both dry and wet years with greater resource use efficiency and advanced phenology. This would allow selfers to
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more successfully occupy drier niches than outcrossers, resulting in local adaptation and evolution within those environments (Lloyd 1992, Herford 2010, Levin 2010). This functional approach to understanding the costs and benefits of selfing and outcrossing will help to build upon traditional studies focusing on ecological drivers such as reproductive assurance and pollen limitation. In particular, we see compelling evidence to incorporate the role of resource limitation, in this case water availability, into studies on mating system evolution and reproductive ecology.
ACKNOWLEDGEMENTS
We thank Greg Barron-Gafford for assistance with the Licor 6400, Abreeza Zegeer for assistance in the greenhouse, and Trevor Birt, Nandi Devan, Brian Hunter, and Tim
Malan for assistance in data collection. We also thank Judie Bronstein and Steve Smith for extensive comments on earlier drafts of this manuscript. This work was supported by the Garden Club of America Desert Studies Award and the California Desert Legacy
Fund.
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TABLES
Table 1: Mean and standard deviation of floral allocation traits for the four focal species taken from greenhouse grown plants. S = selfer, O = outcrosser. Letters represent significant Student’s t-test differences (P < 0.05) within traits within species pairs.
Mating dry floral nectar petal length Floral longevity species System weight (mg) volume (µl) (mm) (days)
E. chamaenerioides S 0.43 ± 0.09A 0.08 ± 0.03A 2.47 ± 1.03A 1.7 ± 0.4A
E. refracta O 2.72 ± 1.40B 0.64 ± 0.32B 6.16 ± 2.33B 6.1 ± 2.1B
C. walkeri S 0.88 ± 0.12A 0.09 ±0.06A 5.27 ± 1.89A 2.3 ± 0.5A
C. multijuga O 4.77 ± 2.02B 0.54 ± 0.27B 8.38 ± 2.94B 4.8 ± 1.9B
Table 2: Survival to reproduction of selfers and outcrossers in the greenhouse.
mating water species survival system level dry 0.91 E. chamaenerioides S wet 0.87 dry 0.58 E. refracta O wet 0.65 dry 0.96 C. walkeri S wet 0.94 dry 0.69 C. multijuga O wet 0.85
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Table 6: Student t-test results for variable differences observed in the field and the greenhouse. Significant P values are bolded (P < 0.05).
mating t- species system variable df statistic P SLA -2.238 0.0228 % N -5.968 <0.0001 N m-2 -2.341 0.0404 E. chamaenerioides S 51 gs (area) -0.250 0.8039
gs (mass) -2.155 0.0359 Delta -3.859 0.0003 SLA -7.892 <0.0001 % N -6.574 <0.0001 N m-2 1.158 0.2519 E. refracta O 53 gs (area) 2.780 0.0075
gs (mass) -2.150 0.0361 Delta -2.177 0.0339
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Table S1: Linear regression statistics for relationships between SLA, leaf nitrogen content, stomatal conductance, carbon assimilation rates, and Delta for selfer-outcrosser sister species within Eremothera.
explanatory dependent Eremothera variable variable intercept slope R2 P-value selfer ------0.023 0.481 SLA Anet (area) outcrosser ------0.023 0.456
selfer -2 ------0.040 0.345 N m Anet (area) outcrosser 10.54 0.041 0.183 0.033 selfer 15.272 22.170 0.345 0.003 gs (area) Anet (area) outcrosser 12.785 14.617 0.343 0.002 selfer -0.045 26.919 0.735 <0.0001 SLA Anet (mass) outcrosser -0.0277 20.166 0.592 <0.0001
selfer -2 ------0.120 0.097 N m Anet (mass) outcrosser ------0.071 0.197 selfer 0.125 40.354 0.624 <0.0001 gs (mass) Anet (mass) outcrosser 0.241 19.651 0.517 0.000 selfer 19.313 167.89 0.725 <0.0001 SLA Delta outcrosser 20.252 90.849 0.407 0.001 selfer ------0.027 0.442 N m-2 Delta outcrosser ------0.003 0.781 selfer ------0.026 0.452 gs (area) Delta outcrosser ------0.056 0.267 selfer 20.602 208.529 0.394 0.002 gs (mass) Delta outcrosser 21.546 72.089 0.230 0.023
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Table S2: Linear regression statistics for relationships between SLA, leaf nitrogen content, stomatal conductance, carbon assimilation rates, and Delta for selfer-outcrosser sister species within Chylismia.
explanatory dependent Chylismia variable variable intercept slope R2 P-value selfer 4.614 639.762 0.359 0.001 SLA Anet (area) outcrosser ------0.003 0.774
selfer -2 ------0.039 0.320 N m Anet (area) outcrosser ------0.022 0.473 selfer 10.502 16.819 0.425 0.000 gs (area) Anet (area) outcrosser 9.230 21.084 0.524 <0.0001 selfer -0.189 27.391 0.778 <0.0001 SLA Anet (mass) outcrosser 0.028 11.770 0.352 0.001
selfer -2 ------0.005 0.733 N m Anet (mass) outcrosser ------0.029 0.406 selfer 0.136 26.140 0.659 <0.0001 gs (mass) Anet (mass) outcrosser 0.129 27.377 0.491 <0.0001 selfer 20.044 96.230 0.172 0.028 SLA Delta outcrosser ------0.100 0.101 selfer ------0.012 0.583 N m-2 Delta outcrosser ------0.079 0.146 selfer 20.936 2.507 0.194 0.017 gs (area) Delta outcrosser ------0.074 0.179 selfer 21.059 112.004 0.209 0.014 gs (mass) Delta outcrosser 20.364 246.878 0.209 0.019
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FIGURES
Figure 1a-b: The mean and standard deviation for the rate of leaf production rate for the first two months of growth in dry and wet treatments for E. refracta and E. chamaenerioides (1a) and C. multijuga and C. walkeri (1b). Letter differences denote significant differences (Tukey-Kramer HSD, P < 0.05).
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Figure 2a-b: The mean and standard deviation for leaf mass accumulation rate in dry and wet treatments for the first two months of growth for E. refracta and E. chamaenerioides
(2a) and C. multijuga and C. walkeri (2b). Letter differences denote significant differences (Tukey-Kramer HSD, P < 0.05).
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Figure 3a-b: The mean and standard deviation for biomass accumulation rate in dry and wet treatments for E. refracta and E. chamaenerioides (3a) and C. multijuga and C. walkeri (3b). Letter differences denote significant differences (Tukey-Kramer HSD, P <
0.05).
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Figure 4a-b: The mean and standard deviation for the time to flowering in dry and wet treatments for E. refracta and E. chamaenerioides (4a) and C. multijuga and C. walkeri
(4b). Letter differences denote significant differences (Tukey-Kramer HSD, P < 0.05).
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Figure 5a-d: Plots 5a and 5b show linear regressions between stomatal conductance (gs) and. instantaneous carbon assimilation rate (Anet). Plots 5c and 5d show linear regressions between stomatal conductance and carbon isotope discrimination, Δ. Open circles = E. chamaenerioides (ERCH), Solid squares = E. refracta (ERRE), Open triangles = C. walkeri (CAWA), Stars = C. multijuga (CAMU). See Table S1 and S2 for linear regression statistics.
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APPENDIX D
RESOURCE LIMITATION AND PLANT PERFORMANCE IN SELFING AND OUTCROSSING DESERT ANNUAL PLANTS
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Resource limitation and plant performance in selfing and outcrossing annual plants
Katharine L. Gerst1, Travis Huxman1,2 and D. Lawrence Venable1
1Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ
85721, USA
2Biosphere 2, University of Arizona, Tucson, AZ 85721, USA
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ABSTRACT
Selfing and outcrossing plants differ in their patterns of resource allocation to floral display and investment to male and female function. Allocation to reproduction, such as flower or seed production, may be particularly sensitive to resource stress, such as drought and herbivory. How plants with differing mating systems cope with low water availability and tissue damage will influence their growth, fitness, and ultimately population dynamics and geographic distribution. Because of an increased resource investment in floral display and greater reproductive uncertainty due to their dependence on pollinators, outcrossing species may be more limited in their spatial and temporal niches where they can persist. In this study we predicted that drought and herbivory would have a greater negative effect on performance metrics of fitness (biomass, flower production, reproductive effort, fruit size, and seed and ovule number) for outcrossing species compared to selfing species. We also predicted that outcrossing species would experience greater resource re-allocation to fruits produced later when early flowers failed to be fertilized. We studied these patterns in two pairs of selfing and outcrossing taxa: Eremothera chamaenerioides, Eremothera refracta, Chylismia walkeri and
Chylismia multijuga. We found that selfers and outcrossers did not differ in their final biomass or flower production; however selfers make a great number of seeds per fruit.
Drought stress caused selfers and outcrossers to have similar negative responses.
Herbivory did not affect flower production and flower removal did not affect future re- allocation to fruit, suggesting compensation later in the plants development. These results
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are in contrast with previous results showing that physiological metrics of outcrossing species performance had a greater negative response in to drought.
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INTRODUCTION
Plant mating systems play an important role in influencing fitness by determining the processes by which reproduction occurs (Lloyd 1992, Barrett 2003, Harder et al.
2008). Mating system evolution is often driven by ecological conditions such as uncertain pollinator environments (Fishman and Wyatt 1999, Moehler and Geber 2005,
Morgan and Wilson 2005), climatic conditions, such as aridity (Elle et al. 2010, Ivey and
Carr 2011), or genetic factors, such as inbreeding depression (Holtsford and Ellstrand
1990, Johnston 1992, Willis 1993, Goodwillie and Knight 2006). The resources that are available to plants for growth and reproduction ultimately will determine performance in their environment; thus resource availability is expected to interact and/or drive these ecological, climatic, and genetic selective processes.
The evolution of mating systems towards selfing or outcrossing is accompanied by correlated evolution of floral traits (Cruden 1985, van Kleunen and Ritland 2004,
Gotzenberger 2006, Goodwillie et al. 2010). Specifically, evolution towards selfing frequently results in a reduction of investment in floral structures and male function
(Sicard and Lenhard 2001, Fishman and Willis 2008, Johnson et al 2010). Conversely, outcrossers have an increased resource investment to male function and to pollinator- mediated traits related to floral attraction, such as showy large corollas or increased nectar quantity (Lyons and Antonovics 1991, Parker et al. 1995, Delesalle et al. 2008,
Delesalle and Mazer 2009). Outcrossers generally make more attractive, larger flowers than selfers but this can mean they make fewer flowers (Sata and Yahara 1999) or fewer
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ovules per fruit. Such shifts in reproductive allocation can be non-linear, where there is a disproportionate increase in fitness with selfers by making proportionately more flowers with an increase biomass (Sakai 2000, but see Zhang 2000). Theoretical and empirical studies have demonstrated resource allocation shifts with mating system evolution
(Charlesworth and Morgan 1991), at multiple hierarchical levels, from within flowers to whole plants (Tomimatsu and Ohara 2006).
While allocation to showy floral structures is a frequently cited cost associated with outcrossing (Celedon-Neghme et al. 2007), another cost is pollination uncertainty
(Miller-Rushing et al. 2010). Studies have suggested that pollen limitation may be as a primary factor limiting seed set in nature due to the generally stochastic nature of pollinator services (Knight et al. 2005). Thus outcrossing plants may need to make many more ovules or flowers than will be fertilized in order to maximizing seed set (Burd 1994,
Holland and Chamberlain 2007); if fertilization events are rarer in outcrossing species, they can still result in high seed output (Burd et al. 2009). However, this uncertainty itself may select for a reduction in ovules per fruit if resources are limited and ovules and fruits are costly. Depending on how plastic a plant is in their resource allocation towards fertilized flowers (Wesselingh 2007) resources may be re-allocated away from unsuccessfully fertilized flowers towards successful ones. This could provide a mechanism for outcrossing plants to recoup some of the costs of employing a strategy dependent on uncertain pollinators. However, both pollinator uncertainty and investment in showy reproductive structures are costs that are likely exacerbated when resource availability is low.
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Variation in the environment is known to affect allocation, floral traits and mating system (Elle and Hare 2002, Zhao et al 2006). Recent literature has provided evidence that drought can play a role in the evolution of selfing by selecting for a reduction in reproductive allocation and development time (Snell and Aarssen 2005, Elle et al. 2010,
Ivey and Carr 2011, Mazer et al 2011). Drought conditions are also known to amplify effects of inbreeding depression (Hauser and Loeschcke 1996). In addition to drought, herbivory is a universal costly phenomenon that plants must cope with. Herbivory strongly affects the availability of resources for reproduction and affects both plant fitness and phenology (Brody 1997). Mating system traits and pollination rates are affected by herbivory (Letiha and Strauss 1999, Ashman 2002, Steets and Ashman 2004,
Steets et al. 2006, Kessler et al. 2010), and inbreeding depression may be amplified by herbivory (Carr and Eubanks 2002). While mating system and resource allocation are both influenced by water stress and herbivory, we do not know how the fitness costs and benefits associated with having a selfing or outcrossing reproductive strategies shift under conditions of low resource availability and herbivory in closely related taxa.
This study examines resource allocation and fitness in response to low resource availability and herbivory in two pairs of desert annual selfing and outcrossing sister taxa.
In one of these pairs, Eremothera refracta and Eremothera chamaenerioides, we have carried out field studies documenting how inter-annual phenological variation in pollination and herbivory affects fruit and seed production. In both pairs of sister taxa, we have previously found that selfers have more rapid development to reproduction as possible adaptation to drought escape since they are not dependent on pollinator
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phenology. Here we attempt to elucidate the role of resource availability in mediating these interactions in a greenhouse experiment.
In this study, we asked the following questions: (1) Do outcrossers have a stronger response in plant performance (final biomass and flower production) to water stress or simulated herbivory compared to selfers? (2) How is reproductive effort affected by drought and herbivory in selfers and outcrossers? (3) How do selfers and outcrossers allocate differently to fruits and ovules under drought? and (4) Do plants allocate more resources to fruit, ovule, and seed production when prior open flowers have failed to be fertilized?
We predict that resource limitation, via drought or simulated herbivory, will have a stronger negative effect on the fitness of outcrossing species (e.g., biomass, flower production, reproductive effort, and seed production) compared to selfing species because of their greater expenditure of resources on flowers, nectar, and floral longevity. We also predict that outcrossers will re-allocate resources from unfertilized flowers to subsequent fruit and seed as an adaptation to pollination uncertainty.
MATERIALS AND METHODS
Study species:
Two pairs of annual selfing-outcrossing taxa were used in this study: Eremothera refracta, the outcrossing self-incompatible sister species to the selfing Eremothera chamaenerioides (Onagraceae), and Chylismia multijuga, the outcrossing self-
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incompatible sister species to the selfing Chylismia walkeri (Onagraceae). Species are putative sister taxa based on Raven (1969, 1979) with recent genera re-classifications in
Wagner et al (2007). Self-incompatibility in E. refracta and C. multijuga was confirmed by bagging individuals and observing lack of seed production, while predominance of self-fertilization in E. chamaenerioides and C. walkeri was confirmed with prior experiments showing no difference in fruit set when hand-cross pollinated compared to bagged flower buds (Gerst, personal observations). All species are annuals found in
North American deserts; they are also known to grow sympatrically with their pair and are found on desert slopes and in sandy washes. While range distributions between sister taxa overlap, the two selfing species both have larger ranges (approximately 2x greater) that envelope the ranges of their outcrossing pair (Raven 1969). Each species pair is comparable in size, growth habit, and leaf size and shape. In addition to mating system, measurements on floral traits related to resource allocation (petal size, flower weight, nectar volume and floral longevity) differ between sister taxa and demonstrate a greater floral investment in outcrossing taxa (Dissertation Appendix C).
Seeds from both E. refracta and E. chamaenerioides were collected in a wash adjacent to Goffs Butte in the Eastern Mojave desert (34o51’48.17 N, 115o5’37.13 W).
Seeds from Chylismia multijuga were collected in Red Rock Canyon, Nevada
(36°24'30.54"N, 114°33'29.84"W) and seeds from Chylismia walkeri were collected near
Primm, Nevada (35°36'42.63"N, 115°23'40.77"W). These sites are all relatively central in each species range. All seeds were collected in the Spring of 2009.
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Study Design:
Study was carried out in a greenhouse at the University of Arizona. Nighttime temperatures were maintained between 50-60oC and daytime temperatures were maintained between 60-75oC. 100-200 seeds were planted per mother plant from six mothers per species. Seeds were germinated in 2% agar in petri dishes and transferred to pots (with 3-5 individuals per pot) when roots were at least 1 cm long. Plants were grown in Stuewe and Sons D40 Deepot cone-tainers (2.5 in diameter, 10 inch depth, 40 in3). Soil composition was a 3:1 mixture of Sunshine mix #2 (Sun Gro Horticulture
Canada Ltd.) and 30 grit sand. Once seedlings were transferred to pots, they were covered with plastic sheets to keep soil and air moist until plants had two sets of true leaves to confirm establishment. Once seedlings were established, pots were thinned to one per pot for a total of 30 individuals per mother per species.
Seeds were planted in agar the week of November 16-20, 2009, and transferred to pots January 4-15, 2010. Plants were arranged in cone-tainer trays by species and mother, with ten plants per tray of the same mother spread over three trays, for a total of
30 individuals. These were distributed across three greenhouse tables with one tray per species per mother represented on each table. Trays within tables were arranged randomly and all trays were shuffled between tables twice throughout the experiment.
Only every other tray slot was filled with a plant to ensure no competition for above- ground space or light between individuals; the experiment consisted of 18 trays of 10 per species and 72 trays total.
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Water treatments
Half of the plants within each mother group and within each tray were randomly assigned to a high water treatment and half to a low water treatment. High water treatment plants were saturated every week which allowed soil to drain completely between watering, but prevented signs of water stress in plants. Low water treatment plants were watered every two weeks allowing signs of water stress (e.g. wilting) but prevented high rates of plant mortality.
Simulated herbivory treatment
A simulated leaf herbivory treatment was applied to four plants per tray of 10 plants; two plants were selected per each of the two watering treatment. In early March, prior to switching into their reproductive mode, one third of the leaves spread evenly along the plant stem were removed while the remaining plants were left intact.
Pollination and Flower removal
Every flower on plants of the two outcrossing species was hand-pollinated. Prior experimentation confirmed full seed set in the two selfing species, thus flowers of these two species were not manipulated and were left to fertilize autonomously. We applied a flower removal treatment in which we removed the first two flowers on their first day open on four plants per tray; one plant was selected per each treatment combination
(intact+low water, intact+high water, herbivory+low water, and herbivory+high water).
The third flower was then either hand-pollinated (outcrossers) or allowed to fertilize
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autonomously (selfers). The fruit produced from the third flower on every plant was collected after maturation but prior to fruits splitting open in order to compare fruits with and without prior flower fertilization to test for resource re-allocation. The fruit length was measured and we counted the number of seeds and the number of unfertilized ovules in order to calculate total ovule production.
Plants were harvested as they died and the total number of flowers was calculated by the number of fruits present. Plant material was separated into fruit, stem, root, and leaf biomass and dried in an oven at 60o until desiccated at which time material was weighed. Flower weight was estimated by multiplying the total numbers of flowers produced by an average flower weight determined from a subset of flowers from each species (see Table S1) because flowers fell from plants shortly after fertilization and were difficult to retrieve with certainty of their mother plant. Reproductive effort was calculated as fruit, seed and flower biomass / total plant biomass. For Chlylismia, fruits often split releasing seeds before they were collected and thus we used a subset of fruit mass from unopened fruits to calculate the relationship between empty fruit and seed mass. Then we estimated seed mass based on empty fruit mass in order to calculate reproductive effort.
Plants that did not survive to reproduce were not included in any analyses (see
Dissertation Appendix C for survival results).
Statistical analysis
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Statistical analyses were performed in SAS 9.1.3 (SAS Institute, Cary, NC, USA).
Plants that did not survive to reproduction were excluded from analyses. To examine differences in flower production, we constructed mixed models using water treatment
(dry vs. wet), herbivory (simulated herbivory manipulation or control), species, species*water and species*herbivory interaction terms as fixed effects and mother as a random effect nested within species. For total biomass and reproductive effort, we excluded plants that had simulated herbivory (due to our own artificial vegetative biomass removal) and constructed models using water level (dry versus wet), species, and species*water interaction terms as fixed effects and mother as a random effect nested within species. For statistical analysis, reproductive effort was arcsine square-root transformed. For differences in third fruit length and ovule and seed number, we constructed models using flower removal (yes or no), water level (wet or dry), species, species*water and species*flower removal interaction terms as fixed effects with mother nested within species as a random effect. Herbivory was not included in this model because it did not have a effects on fitness metrics while water treatment did induce significant resource limitation. All pairwise comparisons within species pairs and response variables were analyzed with Tukey-Kramer HSD tests with p-values less than
0.05 considered significant. Separate analyses were performed for each genus and response variable.
RESULTS
Total biomass and flower production
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There was no difference in final biomass or total flower production between E. refracta and E. chamaenerioides (Table 1, Figure 1a-b). For both species, individuals grown in the high water treatment were significantly bigger and made more flowers, while simulated herbivory had no effect on total flower production. Neither the magnitude of the effect of water stress for biomass or flower production, nor the effect of simulated herbivory for flower production, was different between the selfer and the outcrosser. C. multijuga and C. walkeri demonstrated the same patterns for biomass and flower production responses as Eremothera (Table 2, Figure 1d-e).
Reproductive Effort
Reproductive effort was greater in the selfing species, E. chamaenerioides, compared to the outcrossing species E. refracta (Table 1, Figure 1c). Water stress also had no overall effect on reproductive effort, although the selfing species had increased reproductive effort in the low water treatment, whereas the selfing species had greater reproductive effort in the high water treatment. There was no difference in reproductive effort between C. multijuga and C. walkeri, and no significant effect of water stress or simulated herbivory on reproductive effort (Table 2, Figure 1f).
Fruit and seed allocation
Fruit length, ovule number, and seed number were all greater in both selfing taxa compared to their outcrossing paired taxa (Table 3). For Eremothera, fruits length and ovule number were greater in the high water treatment than the low water treatment.
However, within species this pattern only held for E. refracta fruit length. There was no
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direct effect of water on seed number, however there was a significant species*water interaction; the high water treatment had a greater proportional effect on seed number in the outcrossing species than the selfing species (Table 1). Within Chylismia species, fruit length, increased with the high water treatment but ovule and seed number did not. There was no significant interaction between water and species in this pair (Table 2).
The flower removal treatment precluding fertilization of the first two flowers resulted in no difference in fruit length, ovule number or seed number in the third fruit for in either species pair (Tables 1, 2, and 3).
DISCUSSION
In this study we examined resource allocation and fitness in two pairs of related selfing and outcrossing taxa that differed in their investment in floral display. We predicted that responses of selfers and outcrossers in their reproductive allocation and plant performance would differ under conditions of low water availability and herbivory.
Specifically, we hypothesized that outcrossers would be more negatively affected by stressful conditions because of their high resource investment in floral size and longevity.
Additionally, we predicted that due to the uncertainty that each flower would be fertilized outcrossers would mobilize resources to increase fruit size, ovule and seed production in fertilized flowers when prior flowers were not fertilized. This is in contrast to selfers who have low risks associated with fertilization due to reproductive assurance.
In the two Eremothera species, we found that total biomass and flower production did not differ between the selfing and outcrosser, and that they had similar negative
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responses to stress induced by low water availability. This contrasts with previous results on these plants indicating that the outcrossers responded more negatively in their physiological functioning as a response to drought compared to the selfers (Gerst
Dissertation Chapter 3). One possible explanation for this disparity is that there is a depression in fitness in outcrossers that was not captured by flower production or biomass metrics. This could be manifested as reduced seed number or quality, particularly in later fruits. Another possibility is that resource allocation to flower size and/or nectar is reduced in outcrossers under resource stress, particularly in later flowers, which could cause lowered pollinator visitation and reproductive success in the field
(Worley et al. 2000). Finally, it is possible that outcrossers are not functionally experiencing greater depression in fitness as a result of drought stress; if this is the case they may have demonstrated increased physiological differences due to variation in peak physiological activity with species phenology that was not captured in our measurements.
Importantly, previous results (Dissertation Appendix C) indicated that mortality was greater for outcrossers in drought conditions. Thus the most crucial fitness effects of drought may occur before plants reach reproductive maturity, and that survival, not reproduction, is the critical stage in determining the interactions between mating system and climate.
Simulated herbivory did not ultimately affect total flower production in
Eremothera. Like water limitation, it is possible that the consequences of herbivory as a resource stress may manifest later in a plants life, resulting in reduced fruit size, seed number or seed quality. They also may have smaller, shorter-lived flowers or altered
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plant architecture, which could lead to reduced pollinator visitation (Juenger and
Bergelson 1997, Suarez et al. 2009).
The selfing species had higher reproductive effort compared to the outcrosser, which was contrary to expectations. This comes about despite considerably less investment by selfers in floral tissue; the pattern is instead driven by selfers having heavier fruits with greater seed number. In addition, there was a significant interaction between species and water; the outcrosser decreased its reproductive effort with water stress, while the selfer increased its reproductive effort. Water stress did result in shorter fruits with fewer ovules and seeds in all species, and selfers had longer fruits with more ovules and seeds than outcrossers. The outcrossing Eremothera species had a slightly greater overall negative effect on seed number from water stress, with the selfer actually had a response in the opposite direction with higher seed number in the dry treatment compared to the wet treatment. The patterns within the two Chylismia species were almost identical to Eremothera, although there were no interactions between water and species in reproductive effort or total seed number. There were also no differences between selfers and outcrossers in reproductive effort, suggesting a compensative pattern driven by selfers having heavier fruits with greater seed number while outcrossers have larger floral investment.
Contrary to our predictions, we did not detect resource re-allocation to the third fruit when prior flowers experienced failed fertilization in either selfing or outcrossing taxa. It is likely that flexibility in resource allocation to fruits and ovules may come about
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later in flowering when plants are under greater resource stress, or could be manifested in other ways such as increased investment to seed quality, floral display, or photosynthetic tissue. Resource limitation may affect fitness and reproductive traits at multiple hierarchical levels of resource allocation (Tomimatsu and Ohara 2006) and in non-linear ways (Sakai 2000). For instance, a reduction in resources may have a disproportional effect on resource allocation away from seed or fruit production relative to floral tissue or vice versa.
Our previous results have suggested that selfers have greater photosynthetic rates and shorter development times than outcrossers, and outperform outcrossers in conditions of low water availability (Dissertation Appendix C) which was consistent with other recent studies (Mazer et al. 2010, Ivey and Carr 2011). Unexpectedly, these physiological and phenological patterns did not result in differences in total biomass between the taxa or flower/fruit production, and may function to facilitate a drought escape strategy in selfers. Outcrossers may not have as strong selective pressure against drought escape because they need to be well synchronized with their pollinators and would benefit from a later and more stretched out flowering season. Given that selfers have higher carbon assimilation but no increase in flower number, it is reasonable to assume that selfers may be able to allocate disproportionately more resources to seed and ovule number and quality rather than making proportionately more flowers and fruits.
While selfers and their related outcrossers make a similar number of flowers and potential fruits, overall, selfers have greater total seed production under similar resource
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conditions as outcrossers due to their greater number of seeds per fruit. So under what conditions is it beneficial to be an outcrosser? They have a riskier pollination strategy and produce fewer seeds per fruit than selfing species. Our previous field studies have shown similar within-fruit ovule fertilization rates (seed to ovule ratio) for selfers and outcrossers when they are hand-pollination, but a risk of low fruit production when plant phenology is shifted early in the season (Gerst Dissertation, Chapter 2). Thus the reduced ovule number in outcrossers may be an adaptation to pollinator uncertainty such that the increase in floral investment is correlated with a decrease in per fruit ovule investment
(Burd et al. 2009). Alternatively, outcrossers may have reduced ovule number due to selection for greater seed size, which can allow for greater offspring survival and fitness
(Stanton 1984). While seed mass is slightly greater (~1.5x) in the outcrosser in
Eremothera, this is not the case for Chylismia.
Another possible scenario explaining the maintenance of outcrossing is that outcrossers may outperform selfers in high resource and reliable pollinator environments.
When resources are not strongly limiting, outcrossers may reach a threshold in which they can invest proportionately greater resources towards flower production. This scenario is compatible with observed differences in species range distributions between selfers and outcrossers in which outcrossers occupy a smaller subset of the same range as their related selfer (Raven 1969, Randle et al. 2009) and consistent with studies demonstrating selection for reproductive assurance in extreme environments (Elle et al.
2010, Evans et al. 2011). Further work could elucidate whether the outcrossers are
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occupying the less resource limited subsets of those ranges, since selfers have greater resilience to water limited conditions (Dissertation Appendix C).
While herbivory appears to strongly affect plant fitness in the field, particularly in years with late reproductive phenology (Dissertation Appendix B), our treatment of removing leaves to simulate herbivory didn’t influence flower production in any of the four species. This lack of response is similar to results found from experimental clipping in Ipomopsis aggregata (Juenger and Bergelson 1997) which suggests that other factors must allow for compensation of damage induced by herbivory, and that herbivory often still can strongly impact plant-pollinator interactions by changing plant architecture, phenology, and floral display (Suarez et al. 2009). Herbivores in the field on Eremothera and Chylismia are heavy consumers of stems, fruits, and flowers, in addition to leaves, causing a direct negative effect on current and potential future fruits and flowers. More experiments are needed to understand the direct and indirect mechanisms by which leaf, stem, fruit and flower herbivory can influence fitness in these species, and how herbivory affects resource allocation patterns and re-allocation of resources (Steets et al. 2006).
Mating system transitions from outcrossing to selfing reproductive strategies are accompanied by shifts in reproductive allocation patterns and reproductive effort (Lyons and Antonovics 1991, Parker et al. 1995, Goodwillie et al. 2010). We hypothesized that resource limitation interacts with mating system to determine patterns of allocation and fitness in related selfing and outcrossing taxa. While our results did not support our prediction that outcrossers would be more negatively affected by reductions in resource
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availability, they illustrate the differences in reproduction at multiple hierarchical levels in selfers and outcrossers, and demonstrate that given the same resource environment selfers will have greater individual fitness via seed production compared to outcrossers.
To expand our understanding of the conditions which promote the maintenance of selfers and outcrossers in the same environment, we need to increase our understanding of the fitness benefits conferred on outcrossers from inbreeding avoidance and increased genetic variability, and the mechanisms driving variation in mortality.
ACKNOWLEDGEMENTS
We thank Trevor Birt, Lauren Cmiel, Jackie Comisso, Nandi Devan, Brian Hunter and
Adam Leitman for assistance in data collection and Abreeza Zegeer for assistance in the greenhouse. This work was supported by the Garden Club of America Desert Studies
Award and the California Desert Legacy Fund.
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TABLES
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FIGURES
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Figure 1a-f: Effect of drought and herbivory conditions on flower production, total biomass and reproductive effort for the selfer E. chamaenerioides (ERCH) and the outcrosser E. refracta (ERRE) (a-c), and for the selfer C. walkeri (CHWA) and the outcrosser C. multijuga (CHMU) (d-f). Letter differences represent significant pairwise differences determined by Tukey-Kramer HSD tests (P < 0.05). Plants with simulated herbivory were excluded from analyses on biomass and reproductive effort due to the inherent manipulation of these variables in removing leaves. Sample size (N) for biomass and reproductive effort for Eremothera: ERCH-dry: 49; ERCH-wet: 50; ERRE- dry: 33; ERRE-wet: 34. Sample size (N) for biomass and reproductive effort for
Chylismia: CHWA-dry: 52; CHWA -wet: 51; CHMU-dry: 35; CHMU-wet: 45.
Sample size (N) for flower production for Eremothera: ERCH-dry-noherb: 49; ERCH- dry-herb: 29; ERCH-wet-noherb:50; ERCH-wet-herb: 33; ERRE-dry-noherb: 33; ERRE- dry-herb: 20; ERRE-wet-noherb: 34; ERRE-wet-herb: 24. Sample size (N) for flower production for Chylismia: CHWA-dry-noherb: 52; CHWA -dry-herb: 35; CHWA -wet- noherb:51; CHWA-wet-herb: 34; CHMU-dry-noherb: 35; CHMU-dry-herb: 27; CHMU - wet-noherb: 45; CHMU-wet-herb: 32.