Environmental niche partitioning among riparian sedges {, ) in the St. Lawrence Valley, Quebec

Laura Plourde

Master of Science

Department of Biology McGill University Montreal, Quebec, Canada A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of requirements of the degree of Master of Science August 31st, 2007 ©Copyright Laura Plourde 2007. All rights reserved. Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition

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To understand maintenance of the within-habitat diversity of closely related species, I investigated 11 Carex species growing along rivers in the south-western St. Lawrence Valley of Quebec. Microenvironments within a half meter of focal characterized for Carex comosa, C. crinita, C. grayi, C. intumescens, C. lacustris, C. lupulina, C. pseudocyperus, C. retrorsa, C. tuckermanii, C. typhina, and C. vesicaria revealed significant differences among the species in their environmental affinities. Species appear to fall into groups based on their tolerance of flooding and are secondarily differentiated on other environmental gradients such as insolation, soil pH and soil organic matter. Several traits were related to the environments that species inhabit: diaspore weight, diaspore floating duration, and root aerenchyma. The absence of any phylogenetic trend in niche differences for pairs of species supports the idea that evolutionary differentiation of the alpha-niche is the basis for coexistence of congeners. Resume Pour comprendre la preservation, a I'interieur d'un meme habitat, d'une diversite d'especes rapprochees phylogenetiquement, j'ai examine 11 especes de Carex vivant le long des rivieres dans le sud-ouest de la vallee du St-Laurent au Quebec. La characterisation de microenvironnements a I'interieur d'un demi metre de plantes focales characterises pour Carex comosa, C. crinita, C. grayi, C. intumescens, C. lacustris, C. lupulina, C. pseudocyperus, C. retrorsa, C. tuckermanii, C. typhina, and C. vesicaria a revele des differences significatives parmi les especes en ce qui concerne leurs affinites environnementales. Les especes sembleat s'agencer en groupes bases sur leurs tolerances face aux inondations et divises secondairement selon d'autres facteurs environnementaux tel I'insolation, le pH du sol, et le taux de matiere organique present dans le sol. Plusieurs traits ont ete relies a I'environnement que I'espece occupe: poids du diaspore, duree de flottation du diaspore et racine aerenchyme. L'absence de tendance phylogenetique en ce qui a trait aux differences entre les niches occupees par les differentes pairs d'especes supporte I'idee que la differentiation evolutive de I'alpha-niche est a la base de la coexistence d'especes appartenant a un meme genre.

ii Table of Contents Abstract i Resume ii Table of Contents iii List of Tables iv List of Figures ' v Acknowledgements vi Chapter 1: Introduction 1 The neutral theory: a null hypothesis 2 Niche conservatism, differentiation and phylogenetic hierarchy 2 Riparian communities and their environmental gradients 4 Study genus 6 Study species 7 Objectives. 12 Chapter 2: Methods 13 Study area 13 Study species and focal sample selection 13 Environmental data...... 16 Trait data 18 Statistical analyses 19 Chapter 3: Results ...22 Univariate analysis of environmental variables 22 Multivariate analysis of the environment 23 Trait analysis 23 Phylogenetic analysis of the niche 24 Chapter 4: Discussion 40 Filtering by flood tolerance and segregation along multiple environmental gradients 40 Upper swamp 41 Lower swamp 42 Marsh 43 Role of traits 44 Absence of phylogenetic signal 46 Summary and conclusion 48 References 49 Appendix I: Canopy Analysis 57 Photograph Collection: 57 WinsCANOPY Data: 58 Appendix II: Estimation of percent time flooded 60 Appendix III: Soil collection and preparation 62 Soil collection: 62 Considerations on sampling and soil preparation: 62 Soil crushing: 63 Quartering the crushed and sifted soil sample: 63 Appendix IV: Aerenchyma Estimation 64 Killing and preservation agent: 64 Field root collection, killing, and preserving: 64 Photography: 68 Appendix V: Electronic archive of thesis material 69 List of Tables Table 1: Matrix of patristic distance between pairs of species based on branch lengths (number of changes or mutations along each branch) in a parsimony analysis using two nuclear and four chloroplast non-coding DNA regions (tree shown in Figurel) 10 Table 2: Habitat occurrence of the 11 study species from comments in published floras and journals -. 11 Table 3: Site descriptions 14 Table 4: Environmental data collected 20 Table 5: Univariate statistics for environmental variables at each focal plot (n=229). Species grouped by elevation on floodplain 25 Table 6: Total-sample standardized canonical coefficients of environmental variables, P values (P>0) and percent variance explained for each axis 32 Table 7: Niche distances. Squared Mahalanobis distance among species are given above the diagonal, with associated p-values below the diagonal. Bold numbers indicate the few species pairings that are oot significantly different (p>0.01) 33 Table 8: Univariate statistics for trait data 35

iv List of Figures Figure 1: Phylogram of riparian sedge species (Waterway unpublished). Bootstrap values based on 100 replicates are shown on branches with >50% support. The dashed line collapses in a strict consensus tree 9 Figure 2: Site Locations: CSJ: Parc-Nature du Cap-St-Jacques, DIL: Dowker Island, HUL: Hull, HUM: Huntingdon Marsh, MAS: Masson, OKA: Pare National d'Oka, PLS: Pare National de Plaisance, RIG: Pointe-Riviere-a-la-Raquette, VAU: Vaudreuil-Sur-Le-Lac; Gauging Stations: 1) Hull Dam, 2) Carillon Dam, 3) Summerstown, 4) Ste-Anne-De-Bellevue, 5) Pointe-Calumet, 6) Pointe-Claire 15 Figure 3: Boxplots of species elevation from the average river level (in cm) displaying habitat groupings used in the discussion of ecological differences among the study species. Dots are outliers, whiskers are the 10th and 90th percentiles. The box is defined by the 25th and 75th percentiles. The bar within the box is the median. In this compilation and comparison of foal plant data sampled across sites the average water level from 2003-2005 is standardized at zero elevation for each site 30 Figure 4: Biplots of species distribution on the first two axes of a Canonical Variates Analysis based on 9 environmental variables for 11 riparian Carex species. The first and second canonical axes explain 63 and 17% of the variation in this data set, respectively. Circles represent individual focal plots. Outer ellipses are the 95% confidence interval of the species distribution; the 95% confidence interval of the mean is the inner ellipse. Sets of biplots grouped within bold lines contain species of the same riparian habitat type; from top to bottom of the figure these are upper swamp, lower swamp, and marsh. Smaller boxes within each grouping denote pairs of sister species ....31 Figure 5: Biplots of species by habitat groups in canonical space. Canonical axes in each biplot are those which best visually separate the species for that particular group. Crosses (+) are species means, ellipses are 95% bivariate confidence intervals of focal plots. IN: Carex intumescens, GR: C. grayi, TY: C. typhina, CR:C. crinita, RE: C. retrorsa, LU: C. lupulina, TU: C. tuckermanii, VE: C. vesicaria, CO: C. comosa, LA: C. lacustris, PS: C. pseudocyperus 34 Figure 6: Scatterplot of species average location on flooding gradient in relation to floating duration. Species acronyms are as in Figure 5. Triangles indicate marsh species, squares: lower swamp, and diamonds: upper swamp 36 Figure 7: Scatterplot of patristic distance (Table 1) vs. difference in floating time 36 Figure 8: Scatterplot displaying negative correlation between diaspore mass and diaspore floating duration (1^=0.60). Species acronyms are as in Figure 5. Triangles indicate marsh species, squares: lower swamp, and diamonds: upper swamp 37 Figure 9: Scatterplot of diaspore mass vs. insulation. Species acronyms are as in Figure 5. Triangles indicate marsh species, squares: lower swamp, and diamonds: upper swamp 37 Figure 10: Representative images (90X) of upper root cross-sections of 11 riparian Carex species, ex, exodermis; ae, aerenchyma; en, endodermis; co, cortex. All species display at least some aerenchyma formation 38 Figure 11: Scatterplot of distance matrices. Patristic distance (Table 1), Niche difference (Table 7) 39 Figure 12: Example of surveying lines to estimate the elevational difference between a focal plant and the river water level. 60 Figure 13: Quartering 63 Figure 14: Wax Histology 65

v Acknowledgements First I would like to thank my supervisors Martin J. Lechowicz and Marcia J. Waterway for all of their guidance and patience throughout the past two years. Both Marcia's attention to detail and Marty's perspective on the big picture make-them a complementary pair of advisors. A second thanks is extended to Marcia Waterway for access to her published and unpublished Carex phylogenetic data. This work would not have been possible without the funding from the FQRNT Team grant to Martin Lechowicz, Graham Bell, and Marcia Waterway and my Tomlinson Fellowship. Thanks to committee members Daniel Gagnon and Brain McGill I extend my thanks to lab workers Kevin Gibbons, Chantal Gagnon, Marie-Audray Oullette, and Jordane Roy-Leblanc for all their help. GIS work for surveying site locations and for maps included in this thesis was completed with the tremendous help of Claire Weil. Claire also communicated with people in French for me. Thanks Claire! Michel Truong and Joelle Guillet translated this thesis's abstract to French. Special thanks to Jordane Roy-Leblanc for his assistance in fieldwork and for being my personal translator and guide to Quebec. His laid-back attitude and sense of humor helped us through the strangest of days. Thank you to the various landowners who allowed me to collect data on their land. Thanks to Claire Cooney and Danielle Donnely for tutoring me in the tedious world of wax histology and to Tamara Western for permission to use her lab's microtome. Peter Barry was very helpful in teaching Jordane and I how to conduct elevation surveys. I would like to thank the members of the Waterway and Lechowicz labs for advice and support, especially Tyler Smith for introducing me to Carex. I also extend extra thanks to Tracy Eades and her father George. Thanks to my friends and family (including Brian's family) for all of their support, especially my parents for taking my late night/ early morning phone calls, for understanding my priorities and for giving me a quiet place to finish my thesis. Thanks Mom and Dad! I would like to especially thank my soon-to-be husband and best friend, Brian Lapierre, who has not only given me emotional support though my masters, but has been with me through nearly my entire higher education. He is an incredibly patient and understanding person. And he believed in me all along! Finally, I would like to dedicate this thesis to the memory of my Pepere, Harvey S. Plourde, for encouraging me in my childhood to ask questions about the world around me.

vi Chapter 1: Introduction

The concept of the environmental niche is an important theory for most of ecology, from the organism to ecosystem scale (Chase and Leibold 2003). The environmental niche is commonly understood as the range of environments in which a species can exist and reproduce. Many species often coexist within a community. In theory this can happen because species essentially subdivide the habitat by exploiting their specialized niches. This sort of niche partitioning is the most strongly supported hypothesis explaining the co-occurrence of many species of plants (Silvertown 2004). Understanding the environmental constraints on a species may aid in its conservation, whereas understanding how species partition habitats and how organisms evolve specialized niches brings us closer to understanding the maintenance of plant diversity. The word niche first appeared in scientific literature at the beginning of the 20th century when it was defined as a geophysical spatial unit within which a species can exist (Grinnell 1917). Ten years later, with a bias towards animal studies, Charles Elton described the niche as a functional unit (Elton 1926) where the niche could be defined by a species' food resources and the species' relation to its enemies. This Eltonian definition was the basis for what became one of the most longstanding ecological principles: competitive exclusion (Gause 1934). This principle states that no two species with the same resource requirements can coexist because one should competitively exclude the other over time (Gause 1934). This implies that species living in the same area at the same time have different resource requirements, as in different Eltonian niches. G.E. Hutchinson observed that in fact many species do have very similar resource requirements, and often times these species coexist (Hutchinson 1961). Hutchinson's observation of this paradox was the inspiration for coexistence models. Niches are an important assumption in many models attempting to explain local coexistence (Chase and Leibold 2003). The most notable models are: resource competition (Tilman 1982), spatial environmental heterogeneity (Pacala and Roughgarden 1982), the intermediate competitors hypothesis (Huisman and Weissing 1999), consumer resource cycles (Armstrong and McGhee 1976), and the storage effect (Chesson 1994). In all these models unique niches allow each species to be a superior competitor in its specific niche space, which may be defined in either a spatial or temporal sense.

1 The question of the role of niches in the coexistence of plants is particularly interesting because all plants, unlike animals, apparently need the same resources (light, water, carbon dioxide, mineral nutrients). Plants in turn are often the source of niche differentiation for animals that feed on or require shelter in vegetation, making animal diversity inherently dependent on plant diversity (Silvertown 2004). Since plant niches are a foundation of community diversity, they may be the most important factor in understanding the maintenance of diversity in an area.

The neutral theory: a null hypothesis Neutral theory (Hubbell 1997, Bell 2001, Hubbell 2001) takes the contrary view that niches in fact are not important in structuring community assembly and the maintenance of diversity. The theory states that species are all ecologically equivalent and all variation in species numbers in a community is due to stochasticity, often related to dispersal processes. Patterns of community assembly have been explained by these simple assumptions (Bell et al. 2006) without including the often intricate ecological differences among species. These neutral theory assumptions cannot be disproved by examples of species environmental differentiation alone because environmental change is confounded with distance, which in turn is confounded with dispersal processes (Gilbert and Lechowicz 2004). Furthermore, if species do not need adaptive traits to gain entry into a community, then the phytogeny of a neutral community should not have any ecological trends (Bell et al. 2006). Hence, if it can be shown that species diversify through time to adapt to differing conditions along gradients, than there will be support for the niche theory (Silvertown 2004, Bell et al. 2006).

Niche conservatism, differentiation and phylogenetic hierarchy: How might a species evolve to occupy a particular environmental niche? There are many studies showing the segregation of plant species along environmental axes, often including shading, soil pH, soil texture, soil fertility, altitudinal gradients, and most commonly soil moisture gradients (Grace 1981, Menges and Waller 1983, Wilson and Lee 1994, Sultan 1998, Silvertown et al. 1999, Vellend et al. 2000, Cavender-Bares et al. 2004a, Dabros and Waterway in press). There are even more studies showing that a plant's ability to tolerate different environments is determined by its adaptive traits (Woodward 1987). Species must have evolved traits that allow them to grow in a particular environment. Traits evolve in response to selection pressures by means of

2 mutations that favour greater offspring survival, allowing those genes for favourable traits to be selected and, over the long term, to lead to species that differ in their adaptations along environmental gradients. Therefore modifications to a common ancestral trait can underlie differences in habitat preferences (Webb et al. 2002). Understanding how species came to have their present environmental affinities would contribute valuable new information to the understanding of diversity (Bazzaz 1991, Webb et al. 2002). There are two mainstream hypotheses related to habitat selection of species through time: niche conservatism (Peterson et al. 1999) and niche differentiation (Webb et al. 2002). These hypotheses are based on distinct community processes termed environmental filtering (Weiher and Keddy 1995) and interspecific competition (Gause 1934), respectively. Environmental filters constrain site membership by permitting only species with traits that allow survival and reproduction in that environment to establish (Holdaway and Sparrow 2006). Related species commonly have more similar traits than random species pairs and are thus likely to have similar environmental tolerances. From this perspective, related species are expected to co-habit the same environment, conserving their ancestral habitat requirements. Many empirical studies support the niche conservatism hypothesis (Wilson and Lee 1994, Tofts and Silvertown 2000, Prinzing 2001, Wiens and Graham 2005). If on the other hand adaptive trait evolution is driven by competition, then related species should compete more intensely than random species pairs. This stronger competition would cause related species to have no more similar niches than random species pairs or even more different niches than random pairs (niche differentiation). Despite all of the focus on competition in community ecology (Chase and Leibold 2003), there are few empirical studies that have demonstrated niche differentiation or lack of pattern between closely related species using phylogenetic analysis (Losos et al. 2003, Cavender-Bares et al. 2004a, Silvertown et al. 2006b). Niche conservatism and differentiation were first tested by Elton (1946) and Williams (1947) simply by tallying species:genus ratios in communities. Using the same data set but different analyses, Elton supported niche differentiation and to the contrary Williams supported niche conservatism (Elton 1926, Williams 1947, Harper et al. 1961)! We are still finding support for both hypotheses. The truth must be that species are subject to both competition and environmental filtering in an area to determine their occurrence in a community (Ackerly 2003). If this is the case, then why are we seeing

3 any phylogenetic trends in the niche at all? Would it not make sense for competition and environmental filtering to cancel out any evolutionary trends in the niche? Silvertown and his colleagues hypothesized that such discrepancies in phylogenetic pattern are a scaling issue (Silvertown et al. 2006a). It has long been suggested that diversity can be described on differing scales (Whittaker 1975). Whittaker described three scales of diversity that are commonly explored today: within- habitat, between-habitat, and geographical. These are termed alpha, beta, and gamma diversity, respectively. Since species diversity can be studied at differing scales (Whittaker 1975), it makes sense to also look at the niche at different scales (Pickett and Bazzaz 1978). Gamma niches are those that concern a species' geographical range; the beta niche is the habitat niche. Alpha niches are what Grinnell (1917) first described, the within-habitat niche. Silvertown et al. (2006a) proposed that species composition at the beta diversity scale is regulated by environmental filtering that allows similarly adapted species to coexist; whereas alpha diversity, which is at the scale of local coexistence, is regulated by competition that only allows species with differing niches to coexist. They pointed out that only studies concerned with environmental factors sampled at the beta niche scale have reported niche conservatism (Prinzing 2001, Silvertown et al. 2006b). Silvertown et al. (2006a) also hypothesized that the hierarchical structuring of the niche is consistent with hierarchy within the phylogeny. Congeners should occur in similar beta niches, but species pairs at branch tips should have diverse within-habitat alpha niches. The question of interactions among congeners within a habitat has been contemplated since Darwin (Darwin 1859, Harper et al. 1961), but these recent discoveries and an increase in phylogenetic data have made these questions more testable than ever before. More empirical studies focusing on niches of congeneric species within a habitat especially are needed to further explore Silvertown et al.'s (2006a) hierarchical evolutionary niche hypothesis. To test this hypothesis, the genus under study needs to have several species co-habiting the same community to have enough replication to be statistically testable.

Riparian plant communities, and their environmental gradients: Riparian areas are ecotones between aquatic and terrestrial habitats along rivers (Naiman and Decamps 1997) that provide a useful venue for the study of niche differentiation. Riparian zones have a spatial and temporal environmental mosaic that is

4 rarely matched in other systems (Naiman and Decamps 1997). The gradient from shore to upland, the hydrogeomorphic processes, the longitudinal forces (from headwaters to deltas), and yearly flood pulses in rivers all result in environmental heterogeneity and stress on plant growth in riparian habitats (Vannote et al. 1980, Junk et al. 1989, Steiger et al. 2005). Possibly because of this temporal and spatial heterogeneity, riparian zones generally have greater species diversity then adjacent upland habitats (Naiman and Decamps 1997). Plant assemblages along rivers have typically been explained by the flooding regime, pH, canopy gap fraction, soil particle size and percent organic matter in the soil (Menges and Waller 1983, Jean and Bouchard 1993, Lyon and Sagers 1998, Siebel and Bouwma 1998, Sluis and Tandarich 2004). Hydrological influences create latitudinal, longitudinal and vertical gradients along a river edge (Naiman and Decamps 1997) including differences in litter accumulation, nutrient availability, and soil texture (Johnston et al. 2001). The frequency of waves and the amount of river ice accumulation along the shores both allow trees to establish only in higher elevations on the floodplain of most large rivers (Siebel and Bouwma 1998, Prowse 2001, Turner et al. 2004). This barrier to tree growth creates an extreme light gradient from the forest edge to open marsh, influencing the amount of coarse woody debris and pit and mound topography in an area. Depending on whether a particular channel is slow or fast flowing, the yearly flood pulse can either contribute to or remove organic matter, nutrients and fine soil particles from a section of riparian habitat (Junk et al. 1989). The variation of these materials in turn can affect plant community structure. To persist in different sections of the riparian zone, plants have developed different morphological strategies. Plants cope with extended flooding in a variety of ways. Flooding can cause stress to plants because it prevents oxygen from diffusing to cells (Armstrong 1979). Many plant species have developed aerenchyma (tissue with interstitial air space) in their leaves, stems and roots to allow oxygen to reach the flooded sections of the plant (Armstrong 1979). Although a few plants are able to germinate in flooded conditions or after diaspore submergence, many seeds cannot tolerate these conditions (Baskin and Baskin 1998). At the same time, the seasonal flooding can increase the chances of diaspore survival by dispersing floating seeds along the river corridor. Seed buoyancy and dispersal by water (hydrochory) have been recorded for many wetland species (Middleton 2000, Lopez 2001, van den Broek et al. 2005). Due to the wide range of hazards in riparian habitats, riparian plants may trade off seed size and

5 seed number. They may produce few diaspores, each with abundant reserves, to aid in germination and establishment, or many smaller diaspores with more opportunity for colonization of new sites, but less likelihood that any particular seed will establish (Westoby et al. 2002). In conclusion, there are certain environmental variables that are more likely to influence species distributions in riparian habitats because of both the known habitat heterogeneity and the limitations of plant physiology. These environmental axes are: flooding, light availability, soil mineral nutrients and organic matter. In this thesis, I focus on the influence of these factors on species distribution.

Study genus: The species richness of the riparian habitat offers many congeneric species with which to study niche-related questions. A suitable study genus needs to 1) be species rich 2) have co-occurring species in riparian habitats and 3) have a well-defined phytogeny to test for niche differentiation. Sedges in the genus Carex (Cyperaceae) meet these requirements. Carex is the most species-rich plant genus in North America, with more than 200 species in eastern North America and at least 2000 species worldwide (Ball 1990, Gleason and Cronquist 1991). There are many locations where many Carex species co-occur in a local habitat. For example, more than 55 species occur in the 1100 hectare Gault Nature Reserve at Mont St. Hilaire, Qc (Bell et al. 2000). The species richness and ecological diversity of Carex, along with recent advances in its phytogeny (Waterway and Starr 2007, Waterway et al. in press, Waterway unpublished data) makes this an ideal group to study the maintenance of high diversity in closely related species living in the same area. Recently, Waterway et al. (in press) explored the phylogenetic pattern of ecological specialization among species of Carex subgenus Carex. They observed both niche conservatism and differentiation. Conservatism was shown in flooding tolerance with the subgenus Carex containing a high proportion of wetland species, specifically in two sister clades. Waterway et al. (in press) also gave examples of sister species within subgenus Carex that showed differentiation along gradients of soil pH, moisture availability and organic matter concentration (Vellend et al. 2000, Dabros and Waterway in press, Latremouille and Waterway unpublished data). Although this work is only the beginning of studies in the evolutionary ecology of Carex, the results already suggest support for Silvertown et al.'s (2006a) hierarchical niche hypothesis.

6 While the phylogenetic relationships among species of Carex subgenus Carex are starting to be understood, there is very little quantitative information on the environments of Carex species. Most environmental data that have been collected for Carex species are from peatland habitats (Chapin and Oechel 1983, Gignac et al. 2004) and only a few recent studies have looked at upland Carex environments (Lieffers 1984, Bell et al. 2000, Vellend et al. 2000). Still fewer studies have attempted to quantify the environmental niches of Carex species (Vellend et al. 2000, Dabros and Waterway 2004, in press). The most logical next step in the study of Carex ecology is to look at the within- habitat (alpha) niche of the species from wetland habitats that cluster within two sister clades in the phylogeny of Carex subgenus Carex. Although many members of the wetland clades occur in. peatlands, there are also many that grow in riparian zones. Because much is unknown about the environmental affinities of riparian Carex, and the phylogeny of these clades is known for the species in southern Quebec (Waterway and Starr 2007, Waterway et al. in press), I have focused my research on riparian Carex in this region.

Study species: The specific species I chose to study were: Carex comosa Boott., C. pseudocyperus L, C. retrorsa Schwein., C. tuckermanii Dewey, C. vesicaria L. (all section Vesicariae), C. intumescens Rudge, C. grayi Carey, C. lupulina Muhl. Ex. Willd. (all section Lupulinae), C. crinita Lamark,( section Phacocystis) C. lacustris Willd. (section Paludosae), and C. typhina Michx.(section Squarrosae). All species are members of the subgenus Carex in the two "wetland clades" (Waterway et al. in press) and represent about a third of the Carex flora of floodplain habitats in south-western Quebec. A phylogram of the 11 species in my study with C. scabrata as an outgroup is shown in Fig. 1 (M. J. Waterway, McGill University, unpublished results, used herewith permission). The phylogram is based on internal and external transcribed spacer regions in nuclear ribosomal DNA (ITS and ETS 1f) and four non-coding chloroplast DNA regions (trnL intron, trnL-trnF intergenic spacer, rpL16 intron, and trnE-Y-D intergenic spacers). Four pairs of species in this phylogram are sister species or very closely related in a similar analysis that includes a much broader sampling of species within the two "wetland clades" (M. J. Waterway, personal communication). Species with a very close

7 relationship to C. lacustris are not found in riparian habitats in the region under study. Similarly, although C. crinita and C. typhina appear as sister pairs in this 12-species analysis (Figure 1), they in fact belong to distinct subclades with closest relatives in other habitats or geographic regions in the larger analyses (Waterway et al. in press, M. J. Waterway unpublished data). The larger phylogenetic distance between these three species and all the others studied is illustrated in their patristic distances (Table 1). The species studied here have been found growing in a variety of wetland habitats (Table 2), many of which can be found along edges of rivers, but the quantitative data on these species is very little. The ecological knowledge of Carex species growing in riparian habitats has not passed the stage of naturalist description. Not all of these species have been described as growing specifically in riparian zones, implying that no one has explored where some of these species occur along rivers. There clearly is work to be done if we are to understand quantitatively the distribution of Carex species along environmental gradients.

8 C. comosa r100- C. pseudocyperus -75- C. tupuiina -100- -•51- C. retrorsa C. tuckermanii -77- -95- C. vesicaria 92 C.iacmtm C grayi -85- C. imtumescem C. crinita •92- C.fyphma C. scabmia

Figure 1: Phylogram of riparian sedge species (Waterway unpublished) based on a parsimony analysis using two nuclear and four chloroplast non-coding DNA regions. Bootstrap values based on 100 replicates are shown on branches with >50% support. The dashed line collapses in a strict consensus tree.

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Table 1: Matrix of patristic distances between pairs of species based on number of changes or mutations along each branch in a parsimony analysis using two nuclear and four chloroplast non-coding DNA regions (cf. the tree shown in Figure 1).

C C intume c c c seu c c ' C crinita C aravi ' ~ - - - P ~ - - tucker- „ tvonjna comosa • 9 y scens lacustris lupulina docyperus retrorsa manii ' yp C. comosa

C. crinita 222

C. grayi 216 222 C. intumescens 190 196 90

C. lacustris 186 242 236 210

C. lupulina 122 244 238 212 208 C. pseudo- 74 216 210 184 180 116

C. retrorsa 96 218 212 186 182 50 90 C. tuckermanii 134 216 210 184 154 156 128 130

C.typhina 248 174 248 222 268 270 242 244 242

C.vesicaria 122 204 198 172 142 144 116 118 44 230 ^ ") )

Table 2: Habitat affinities of the 11 study species summarized from comments in published floras and journals (represented by numbers). 1:Bailey, 1887; 2: Gray, 1950; 3: Dansereau, 1959; 4: Voss, 1972; 5: Auclair, 1977; 6: Gauthier, 1980; 7: Thomas, 1982; 8: Wheeler & Ownbey, 1984; 9: Lieffers, 1984; 10: Flora of North America Editorial Committee, 2002; 11: Gleason & Cronquist, 1991; 12: Bernard & Seischab, 1994; 13: U.S. Department of Agriculture; 14: Marie-Victorin, 1995.

Wet mesic Species Marsh swamp shore ditch Bog Fen meadow forest 2,4,8, 10, 10,11 4,8, 10, 14 2,4,8, 10 8 4, 8, 14 12 C. comosa Boott. 11 2, 4, 7, 8, 3, 4, 7, 8, 4, 10, 14 4,8, 10, 14 4,8, 10, 14 8, 10 10, 14 C. crinita Lamarck 10, 14 10, 11, 14 2,7,8, 11, 14 7,8, 10 2, 14 C. grayi Carey 10 2,7,8, 10, C. intumescens 14 1,8,13, 14 8 10 13,14 2, 10,13 Rudge 11, 13, 14 2,8, 10, 11, 2,8, 10, 13, 8, 10, 11 8 8 10 5, 9, 10 C. lacustris Wilid. 13 14 2,8,7, 10, C. lupulina Muhi. 11, 14 8, 14 8, 14 8,10,11, 14 10, 14 Ex Willd. 11, 14 C. 2,8, 10, 11, 10, 14 2,8, 10, 14 8, 14 2,8, 11, 14 8 10, 14 pseudocyperus L 14

C. retrorsa 1, 10, 11, 14 7,8, 10, 11 10 8 10, 11, 14 2,8 Schwein

C. tuckermanii 10, 11, 14 10, 11 2, 10, 11 2, 14 Dewey 2, 4, 7, 10, 11 11 6, 14 2 C. typhina Michx. 11

10, 14 10, 11, 14 2, 10, 11, 14 10, 11 2, 10, 14 10, 14 C. vesicaria L. Objectives: Although niche differentiation has been documented among species within several different plant communities, few have focused on the niche relationships among co-existing congeneric species. With the strengthening in phylogenetic hypotheses, there has been a call to relate phytogenies to local communities (Webb et al. 2002) and to the hierarchical structure of the niche (Silvertown et al. 2006a). The species-rich and ecologically diverse genus Carex is an understudied group of species that is well suited to answering recent questions related to the concept of niche (Waterway et al. in press). In this study my main objective therefore is to define the environmental niches of Carex species that grow together in riparian habitats of the St. Lawrence and Ottawa Rivers in southern Quebec. I expect species to segregate along environmental gradients that are known to influence riparian plant community composition and therefore to occupy environmental niches that differ along at least one of these gradients. Along with this I will explore adaptive traits that may allow species to exist in different riparian habitats. My final objective is to explore phylogenetic structure in niche partitioning by comparing sister-species pairs with random pairs of species.

12 Chapter 2: Methods Study area: My studies were restricted to genuine riparian habitats with minimal human disturbance along the shores of the St. Lawrence River downstream from Dundee, Qc (45° 2' 30.08" N, 74° 26' 43.51" W) to the Sorel Islands (46° 5' 19.55" N, 72° 57' 15.76" W) and downstream along the Ottawa River from Hull, Qc (45° 25' 40.26" N, 75° 42' 37.76" W) to Lac des Deux Montagnes (45°26'38.77"N, 74° 2'4.73"W). I explored the entire region using records of species distribution, aerial photos, and visits to potential sites to find 9 sites (Figure 2) representing various floodplain habitats and including a good number of the focal species to ensure sufficient replication across sites.

Study species and focal sample selection: I found 11 Carex species that occurred in at least two and usually more of my sites: C. comosa, C. crinita, C. grayi, C. intumescens, C. lacustris, C. lupulina, C. pseudocyperus, C. retrorsa, C. tuckermanii, C. typhina, and C. vesicaria. These species were selected because they are members of the wetland clades (Waterway et al. in press) and I was able to find a large enough sample of populations for each species. I stratified the sites by habitat type and selected focal plots randomly within each habitat. If a site had multiple patches of the same habitat type (i.e., the same plant associations and topography), one representative patch, with Carex in the understory, was selected randomly for sampling. This stratified sampling was used across all possible conditions where these species might grow in the riparian zone. I used focal sampling instead of plots to measure the microhabitat of each individual, obtaining approximately equal sample sizes of focal plants for both common and rare species. Between 17 and 27 focal samples were collected across sites for each of the 11 Carex species. Vouchers of focal plants are deposited in the McGill University Herbarium (MTMG, Ste-Anne-De- Bellevue, Qc).

13 \

Table 3: Site descriptions, coordinates, and water level gauging station used for estimating flooding time

Gauging Site Code Lat/Long Station Habitats Young Acer saccharinum floodplain forest, Pointe-Riviere-a- Scirpus dominated marsh and an oxbow la-Raquette RIG 45°29'27.69"N 74°14'39.48"W Carillon Dam Typha marsh

Huntingdon C. lacustris marsh, Alnus shrub wetland, Marsh HUM 45° 3'16.67"N 74026'44.77"W Summerstown Fraxinus nigra swamp Pare National Lac des Deux Late successional Acer saccharinum swamp, d'Oka - Grande Montagnes at Sagittaria sp. and Lythrum salicaria Baie OKA 45°29,21.69"N 74° 0'18.22"W Pointe-Calumet dominated marsh

Pare National de Acer saccharinum floodplain forest, Plaisance PLS 45°35'46.41"N 75° 6'7.99"W Hull Dam Acer/Fraxinus floodplain forest, Carex marsh

Dowker Island Lac Saint-Louis Alnus swamp, rocky marsh, young and (lie Lynch) DIL 45°24'6.37"N 73°53'27.92"W at Pointe-Claire mature floodplain forests

Parc-Nature du Cap-Ste-Jacques CSJ 45°27'31.32"N 73°56'40.67"W Carillon Dam Acer saccharinum forest, rocky marsh shore

Young floodplain forest with Populus, Masson MAS 45°31 '47.31 "N 75°23'55.47"W Hull Dam Fraxinus, and Acer, small marsh

Vaudreuil-Sur-Le- Sainte-Anne-de- Acer saccharinum dominated floodplain Lac VAU 45°25'14.76"N 74° 1'22.61 "W Bellevue forest, marshy shore Juglans cinerea-Tillia americana- Acer saccharinum levee and channel bottom Hull HUL 45°27'0.87"N 75°42'14.70"W Hull Dam floodplain forests, and a grassy cove. )

Figure 2 (Previous Page): Site Locations: CSJ: Parc-Nature du Cap-St-Jacques, DIL: Dowker Island, HUL: Hull, HUM: Huntingdon Marsh, MAS: Masson, OKA: Pare National d'Oka, PLS: Pare National de Plaisance, RIG: Pointe-Riviere-a-la-Raquette, VAU: Vaudreuil-Sur-Le-Lac; Gauging Stations: 1) Hull Dam, 2) Carillon Dam, 3) Summerstown, 4) Ste-Anne-De-Bellevue, 5) Pointe-Calumet, 6) Pointe-Claire Environmental data: I collected data on 18 environmental variables within 0.5 M of each focal plant (Table 4). These variables were selected to measure variation in insolation, hydrology, soil fertility, topography, and other microhabitat factors immediately around each focal plant. Other variables measured included: percent vegetation cover immediately around the focal plant, tree basal area, and wave action. Insolation regime: I used canopy photographs (Appendix 1) to interpret the light available to each focal plant throughout the growing season (1 May to 31 Sept). Canopy photographs were taken with a Canon EOS 30D digital SLR camera with a Costal Optics Systems, Inc. (West Palm Beach, FL, USA) 185° angle view Fisheye lens mounted in a Regent Instruments (Quebec City, Quebec, Canada) Universal O-Mount with NorthFinder attached to a tripod. The WinSCANOPY (Regent Instruments Inc. 2006) program was used to analyze: 1) gap fraction (%); 2) direct radiation received under the canopy; 3) diffuse radiation received under the canopy; and 4) total radiation received under the canopy. Radiation data were analysed in WinSCANOPY as an average over the growing season by estimating the sun position and radiation regime at five day intervals. Hydrological regime: The hydrology of riparian ecosystems can be assessed in many different ways: days flooded in a year, date of first flood, date of last flood, flooding over the growing season only, and height above mean water level. These measurements tend to be correlated and a great deal of correlation can be found when many related variables are used to analyse the effects of flooding on plants (Toner and Keddy 1997). For this reason I only calculated the percentage of three years time during which a focal plant was flooded as an index of its hydrological regime (Appendix 2). To do this the elevation difference of each focal plant from the river was measured using a surveyor's level and stadia rod. I then used the nearest water gauging station (Figure 2, Table 3) to obtain the daily water elevations to estimate the plant's elevation relative to the average river level. In several cases, the gauging station was located more than a kilometer from a study site. To find the difference in water elevations between the study site and gauging station I measured the elevation of the water at the geodesic marker nearest to the study site several times and compared it with the water level for that day at the gauging station. After taking these field measurements I then could convert the gauging station's water elevation to the study site's elevation above the river by using an average

16 difference between the two points of reference. I used three years of daily water level data from gauging stations along the sampled rivers (Environment Canada 2006) to calculate the percentage of time each focal plant was flooded. Data of the three most recent years with complete daily measurements across all gauging stations (2003, 2004, and 2005) were used in these calculations. Fertility regime: After removing litter, I collected soil at the base of each focal plant from the soil surface to a depth of 15 cm using a 2 cm slotted soil probe in six random locations around the focal plant. Within two weeks of air drying at room temperature, soil was dried completely in a forced air oven at 55°C. The soil aggregates were then crushed and sieved through a 2 mm mesh screen (Appendix 3). The Macdonald Soil Test Lab (Ste. Anne de Bellevue, Qc) analysed the percent organic matter as loss on ignition (Bell 1964). The N/NH4+, N/NO3", P, K, Ca, and Mg of the soil were analysed by the Sol-For Lab at Laval University (Quebec City, Qc). Exchangeable ammonium and nitrate in soil samples were assayed in an extract of KCI (Kalra and Maynard 1991). Sol-For (Quebec, Qc) determined calcium, magnesium and potassium on a NH4OAc extract at pH 7 (Chapman 1965) and available phosphorus using a NH4F-

H2S04 extract assay (Bray and Kurtz 1945). I estimated the total soil nitrogen by combining only the nitrogen component amount in the nitrate and ammonium for each soil sample. Total nitrogen was estimated because nitrogen can change forms easily over time and during storage and processing of the soil. Sol-For also estimated the pH of the soil samples using a water suspension (Kalra 1995). Local topography: The nature of the terrain immediately around each focal plant was characterized in two ways: 1) local slope and aspect, and 2) micro-topographic categories. Local slope angle was measured between two points (1 M upslope and 1 M downslope from the focal plant) using a clinometer; aspect was determined with a compass. When there was no apparent slope, I recorded it as zero. Micro-topography was estimated for each focal plant by observation and classified as either slope, concave, convex, or flat. Miscellaneous habitat characteristics: Other variables measured included: percent vegetation cover immediately around the focal plot, tree basal area, and wave action. The percent cover of vegetation and non-vegetation within 0.5 M of the center of each focal plant was estimated by the same researcher throughout the summer. The basal area of trees in the vicinity of each focal plant was estimated using a prism with BAF = 3 using each focal plant as a sampling point (Bitterlich 1984). I also recorded the

17 degree of exposure of focal plant to wave action, determined by observation in the field, categorizing exposure as low, mid, high, or not applicable (for individuals away from the river channel).

Trait data: Diaspore buoyancy: Traits of roots and diaspores of each species were measured to compare with environmental preferences. The diaspore or dispersal unit of a Carex is considered the nutlet containing the seed along with the perigynium covering the nutlet. In the field, diaspores of each species were collected from 3 to 5 different populations in July and August, 2006, and were air-dried for storage. Mature diaspores with undamaged perigynia were separated from the field collection and stored in separate small paper bags for each population at room temperature. From this subset, diaspores were randomly selected and weighed in batches of 100 for each of the 3 to 5 sampled populations. One hundred diaspores each were selected from three random populations of diaspores for buoyancy analysis. Diaspores of a population were placed in a shallow bowl with 200 mL of tap water. The surface tension in natural settings where the diaspores are floating is broken occasionally by wave action. I simulated this wave action by stirring the diaspores with a gentle back and forth motion. After placing the diaspores in the vessel, the diaspores were stirred, the number of sunken diaspores recorded, and any sunken diaspores removed from the bowl. The vessels were then placed on the shelves of a refrigerator at 12°C in a completely randomized design. I stirred the diaspores every day for the first week of the experiment and recorded and removed the sunken diaspores. Sunken diaspores were counted every two days for the second week, every three days for the third week, and then every fourth day until the end of the experiment - 225 days. The diaspores were always stirred before counting. The bowls were stored in the same position in the refrigerator for the duration of the experiment. The amount of water (200 ml) was also kept constant in the bowls throughout the experiment. I calculated the average floating duration for each species at the end of the experiment (i.e., average time taken for 50 diaspores in a bowl to sink per species). The few populations where 50 diaspores did not sink by the end of the experiment were assigned an average floating duration of 225 days. Aerenchyma: I collected root sections from four randomly selected focal individuals of each species to observe aerenchyma formation, or lack thereof, within

18 species roots (Appendix 4). Half-centimeter root sections of each selected focal plant were collected from June through August 2006 and preserved in FAA solution. The FAA consisted of 5% Formaldehyde, 5% glacial acetic acid, 33% deionised water, and 57% ethanol (Ruzin 1999). The first section was taken where the root connected with the stem (high), the second at 1cm from the root tip (lower), and the third starting 2.5 cm from the tip (mid). If a was present in the plant, a 0.5 cm section was cut from it where the rhizome met the shoot. Lower, mid, high root and rhizome cross-sections of each species were used to produce slides by means of wax histology (Ruzin 1999). I viewed slides for presence or absence of aerenchyma and photographed the slides at 90X magnification using an Olympus SZX12 dissecting scope and Ql CAM Fast Color 12-bit camera.

Statistical analyses: The goal of the statistical analyses of environmental data was to describe variation between species and to assess which of the measured environmental variables were important in the partitioning of niche space by Carex species. I first determined the mean, median, standard error, minimum and maximum values of each variable to visualize the distribution of the data. Secondly, I tested for covariance between data types and removed variables to reduce redundancies that would complicate interpretation of the niche axes. For example, nitrate, ammonium and total nitrogen were all highly correlated, so I only included total nitrogen in the multivariate analyses. Similarly, I only included the total understory radiation as a measure of the insolation regime because gap fraction, diffuse, direct and total understory radiation were redundant. Calcium and magnesium were also highly correlated and were both removed from the final analysis because the soils from some sites contained many small shells that confounded the soil fertility assay. These shells were dissolved in the acid bath that was part of the protocol for obtaining the soil calcium value. In nature the calcium within the shells would not be readily available to focal plants, making this a misleading measurement. The magnesium measurement was similarly misleading. Slope, aspect, micro-topography, and wave action were all removed from the final multivariate analysis. Slope and aspect were removed because most of the slopes were between 0 and 2%, which shows that there was a very small difference in local drainage at each focal site. Micro-topography was removed for the same reasons, and

19 too few species had a wave action value to include this variable in the multivariate analyses. I used transformations to normalize the remaining data (Table 4).

Table 4: Environmental data collected, its use in the final analysis, and transformations to data for final analysis. PPFD-direct per day is the direct understory radiation on the plant, PPFD-diffuse per day is the diffuse understory radiation, and PPFD-total per day is the total understory radiation for an average day of the growing season. Environmental Included Variable in CVA? Transformation Gap fraction no PPFD-direct per day no PPFD-diffuse per day no

PPFD-total per day yes None % time flooded yes Arcsin Soil pH yes None SoilP yes Log Soil K yes Log Soil Ca no Soil Mg no Soil N/NlV no Soil N/NO/ no Soil Total N yes Log Soil % OM yes Arcsin Slope no Aspect no Microtopography no Wave action no Vegetative cover yes Arcsin Tree basal area yes None

To define the environmental niches of the Carex species, I used a multivariate technique called Canonical Variates Analysis (CVA). A CVA derives canonical variables, a linear combination of the measured environmental variables, that summarize among- class (in this case, species) variation. The CVA analysis was done using PROC CANDISC in SAS version 9.1 (SAS Institute, Cary, North Carolina, USA). PROC CANDISC also creates a mean value in canonical space for each species and calculates the squared Mahalanobis distance of means between pairs, which I use as a measure of niche difference (Table 7). For the CVA analyses, I included 9

20 environmental variables, most with transformations (Table 4) total understory radiation, % time flooded, soil P, soil K, total soil N, % organic matter, soil pH, % vegetation cover, and tree basal area. I conducted a Mantel test for relationship between niche distances (Mahalanobis distances, Table 7) and species relatedness (patristic distances, Table 1). A Mantel test circumvents the problem of partial correlations within data matrices, which prevents us from using a simple regression. I used PC-ORD (MjM Software, Gleneden Beach, Oregon, USA) to run this test using Mantel's asymptotic approximation method.

21 Chapter 3: Results

Univariate analysis of environmental variables: I was able to sample a fairly wide range of variables representing environmental heterogeneity in riparian habitats in the Saint Lawrence Valley (Table 5). Some of the largest differences between species were found in insolation and flooding time. Species found most often in forested habitats (Carex intumescens, C. grayi, C. typhina, C. tuckermanii) tended to grow under low light levels whereas marsh species (C. comosa, C. lacustris, C. pseudocyperus, C. vesicaria) grew in the highest light levels. All but four species had individuals that were not subjected to flooding in the years 2003-2005. All individuals of the marsh species were flooded at some point within this three-year span. Carex intumescens, C. grayi, and C. typhina all grew at higher elevations in the riparian zone (Figure 3) and were flooded the least often. Carex lupulina, C. retrorsa, C. tuckermanii, and C. crinita grew at microsites with a broad range of flooding time and in general had intermediate elevation values from the river compared with marsh species and upper swamp species. For ease of writing, I will refer to these groups as marsh, upper swamp and lower swamp species, respectively. There were other environmental variables that showed similarities in each of the species groups. Tree basal area was the least for marsh species sites and greatest in two of the three upper swamp species (C. grayi and C. typhina). Sites where marsh species grew, excluding C. vesicaria, had the highest values of soil potassium, soil total nitrogen, and soil organic matter. Sister-species had different ranges and averages for several environmental variables. Of all the species, C. grayi had the highest values of soil pH while C. intumescens sites had a much lower average pH, although both species were found in a wide range of pH. These two sister-species also differed from one another in soil phosphorus and total soil nitrogen at their focal sites. Carex lupulina and C. retrorsa, were not sister species in the phylogeny including other wetland Carex, but they are only one speciation event away. Carex lupulina and C. retrorsa sites differed in their soil organic matter, soil pH, and tree basal area (C. retrorsa being higher for these). Carex comosa and C. pseudocyperus sites differed from one another, on average, in flooding (C. comosa higher), amount of vegetation cover (C. comosa higher) and tree basal area

22 (C. pseudocyperus higher). Carex tuckermanii and C. vesicaria sites differed from one another along most environmental gradients but were similar in soil pH.

Multivariate analysis of the environment: The plotted results of the canonical variates analysis (Figure 4) were consistent with the habitat groupings of species that were evident in my interpretation of the univariate analyses (Figure 3). Canonical axes 1 through 4 accounted for 63%, 17%, 6%, and 7% of the variance in the data, respectively (Table 6). All four axes were significant in describing the species pattern in canonical space (Table 6), but the first two axes captured he large part of the variation in environment among the study species. The environmental variables with the highest standardized canonical coefficients were the total understory radiation and percent time flooded (Table 6). The second axis was mainly correlated with total understory radiation, soil phosphorus, and soil organic matter; the third axis with tree basal area, total soil nitrogen, and percent ground cover of vegetation; and the fourth axis with soil pH, total soil nitrogen, and soil phosphorus (Table 6). Most species pairs were different in their overall environmental affinities, which are indicated by the significant difference when comparing the mean canonical scores of pairs using Mahalanobis distances (Table 7). Only the sites for the species pairs C. lupulina and C. tuckermanii, C. lupulina and C. crinita, C. intumescens and C. typhina, and C. crinita and C. tuckermanii did not differ significantly in their average environment. Species environmental partitioning varies with habitat type. Upper swamp species segregate along a mixture of light, flooding, pH, nitrogen and phosphorus gradients (Figure 5). The lower swamp species segregate along gradients in tree density, vegetation density, and nitrogen as well as insolation and flooding gradients. Marsh species segregate along flooding, insolation, phosphorus and organic matter gradients. These individual environmental gradients are correlates of the canonical axes that best visually partition each habitat group (Table 6).

Trait analysis: Diaspore traits (Table 8) had some relationship with environment regime. There is a positive trend between diaspore floating duration and the average percent time a species was flooded (Figure 6). Related species tended to have similar diaspore floating time values (Figure 7). There was also a negative linear relationship between diaspore

23 weight and floating duration (Figure 8) and total understory radiation (Figure 9). For two species (C. crinita and C. lacustris) very few diaspores sank. All species in this study displayed aerenchyma formation in the first half centimeter of the root closest to the stem (Figure 10) (sample size per species: n=4). The mid- and low-sections sampled from each root were often immature tissue although mature mid and low sections also did display aerenchyma (sample size 1-2 per species). Carex crinita and C. typhina exhibited the least amount of aerenchyma in the sections closest to the stem.

Phylogenetic analysis of the niche: The correlation between niche distances and patristic distances was not different from random by Mantel's asymptotic approximation test (P=0.309, r = 0.18). Although there appeared to be a positive correlation of niche differences and patristic distances and low niche differences between pairs C. lupulina - C. retrorsa, C. pseudocyperus - C. comosa, and C. crinita - C. typhina, there was a much larger niche difference between C. tuckermanii and C. vesicaria (Figure 11). There also appears to be a general trend where species pairs have more variation in their niche distances when they are more distantly related (Figure 11).

24 Table 5: Univariate statistics for environmental variables at each focal plot (Total n=229, 17-25 per species). Species grouped by elevation on floodplain, from the upper floodplain to the river shore.

Species (ordered by environmental riparian std. variable habitat type) mean median error min max

Average C. intumescens 32.25 10.41 0.53 4.29 14.3

Total Understory C. grayi 11.11 11.65 0.67 5.38 16.16

Radiation in C. typhina 11.33 9.61 1.13 5.77 23.6

Growing Season C. lupulina 13.31 12.26 1.62 1.37 33.48

[Mol/m2day] C. retrorsa 16.74 15.34 1.43 8.23 32.36

C. crinita 14.99 14.09 1.24 5.49 25.81

C. tuckermanii 12.06 11.16 1.05 3.02 21.87

C. vesicaria 34.00 35.99 1.88 13.55 43.39 C. pseudocyperus 21.86 20.25 1.97 7.21 40.35 C. comosa 32.25 35.16 1.94 17.26 40.72

C. lacustris 28.55 31.01 2.46 10.60 43.01

Percent Time C. intumescens 13 1 3 0 61

Flooded C. grayi 8 0 3 0 56

2003-2005 C. typhina 28 20. 5 0 83

C. lupulina 50 41 10 0 100

C. retrorsa 58 59 6 0 100 C. crinita 50 46 7 0 100

C. tuckermanii 50 43 10 0 100

C. vesicaria 58 62 5 18 93 C. pseudocyperus 54 56 6 0 100 C. comosa 76 89 5 47 100

C. lacustris 99 100 0 98 100

25 environmental std. variable Species mean median error min max Soil pH C. intumescens

C. grayi 5.61 5.64 0.15 4.33 7.2

C. typhina 4.91 4.80 0.10 4.22 5.71

C. lupulina 5.24 5.20 0.13 4.46 6.28

C. retrorsa 5.74 5.82 0.09 5.10 6.68

C. crinita 5.33 5.38 0.09 4.39 6.16

C. tuckermanii 5.14 5.18 0.10 4.25 6.20 C. vesicaria 5.34 5.27 0.12 5.02 7.19 C. pseudocyperus 5.68 5.69 0.09 5.08 6.83 C. comosa 5.49 5.32 0.14 4.66 6.43

C. lacustris 5.37 5.47 0.10 4.44 6.14

SoilP C. intumescens 24.7 25.9 2.2 8.9 44.1

(ppm) C. grayi 35.3 23.9 10.6 4.8 300.6

C. typhina 19.4 13.5 3.6 5.1 61.8

C. lupulina 29.0 30.6 2.8 10.5 59.3

C. retrorsa 28.8 • 26.6 2.5 11.1 49.3

C. crinita 45.1 35.8 6.4 9.1 149.8

C. tuckermanii 37.1 34.1 3.7 13.5 75.0

C. vesicaria 21.1 17.5 3.6 5.3 57.4 C. pseudocyperus 46.2 46.1 3.2 26.0 87.5 C. comosa 45.1 39.6 8.1 7.1 130.4

C. lacustris 5.1.9 43.4 5.3 19.5 122.4

26 environmental std. variable Species mean median error min max Soil K C. intumescens 122.2 114.4 13.0 49.5 250.2

(ppm) C. grayi 90.2 81.3 8.9 44.0 226.4

C. typhina 94.9 89.0 8.9 34.2 160.9

C. lupulina 101.2 97.9 7.9 43.2 170.7

C. retrorsa 106.2 93.4 10.8 59.5 275.5

C. crinita 146.8 117.7 24.0 31.1 519.3

C. tuckermanii 104.9 93.4 10.2 39.6 218.0

C. vesicaria 86.6 90.7 14.6 18.5 235.6 C. pseudocyperus 137.3 134.8 7.7 70.34 207.6 C. comosa 128.9 110.1 11.9 64.6 246.8

C. lacustris 125.6 99.0 14.3 55.9 280.1

Total Soil N C. intumescens 28.7 25.7 3.5 10.0 57.6

(ppm) C. grayi 15.4 13.8 1.1 7.1 30.5

C. typhina 20.9 16.1 2.7 5.7 40.3

C. lupulina 16.3 14.5 1.3 9.5 28.6

C. retrorsa 14.8 14.5 1.2 6.9 26.4

C. crinita 17.5 14.8 2.1 4.6 54.5

C. tuckermanii 17.8 15.1 1.9 5.6 38.6

C. vesicaria 15.7 11.8 2.3 6.3 33.6 C. pseudocyperus 29.4 22.6 4.2 12.2 95.6 C. comosa 29.9 26.3 4.3 8.2 72.1

C. lacustris 34.8 24.1 6.7 12.1 138.8

27 environmental std. variable Species mean median error min max Percent Soil C. inturnescens 15 12 2 4 54

Organic Matter C. grayi 10 g 0 2 20

C. typhina 15 12 3 3 57

C. lupulina 15 16 2 6 31

C. retrorsa 28 20 5 4 63

C. crinita 23 20 3 5 64

C. tuckermanii 14 10 2 4 40 C. vesicaria 10 8 2 2 22 C. pseudocvperus 40 44 4 5 66 C. comosa 40 42 5 5 68

C. lacustris 47 45 6 10 88

Percent C. inturnescens 55.1 62.5 6.7 6.0 92.0

Vegetation C. grayi 55.0 50.0 4.1 10.0 98.0

Cover around C. typhina 57.9 60.0 4.2 30.0 87.0

Focal Plant C. lupulina 56.4 50.0 5.4 14.5 95.0

C. retrorsa 54.7 55.0 4.4 15.0 92.0

C. crinita 62.8 63.5 4.7 19.0 98.0

C. tuckermanii 38.4 33.5 4.8 7.0 80.0

C. vesicaria 81.7 90.0 4.5 42.0 100.0 C. pseudocvperus 73.4 77.0 4.8 20.0 98.0 C. comosa 83.4 90.0 3.7 55.0 100.0

C. lacustris 68.0 70.0 5.1 15.0 99.0

28 environmental std. variable Species mean median error min max Tree Basal Area C. intumescens 7.59 24.00 1.75 12.00 42.00

in vicinity of C. grayi 29.61 28.50 1.34 12.00 45.00

Focal Plant C. typhina 29.21 30.00 1.96 10.50 45.00

m2/ha C. lupulina 17.29 18.00 1.97 4.50 34.50

C. retrorsa 25.43 28.50 3.32 3.00 54.00

C. crinita 18.44 18.00 1.92 0.00 37.50

C. tuckermanii 18.48 18.00 1.72 3.00 34.50

C. vesicaria 2.74 0.00 1.01 0.00 13.50 C. pseudocvperus 17.41 16.50 3.13 0.00 45.00 C. comosa 7.59 0.00 2.94 0.00 39.00

C. lacustris 4.43 0.00 2.22 0.00 33.00

29 Upper Swamp Lower Swamp Marsh 300

250 -

200 - 150 - I 100 - ± 50 -

• =* 0

-50

-100 INTU GRAY TYPH LUPL RETR CRIN TUCK VESI PSEU COMO LACU Species Figure 3: Boxplots of species elevation from the average river level (in cm) displaying habitat groupings used in the discussion of ecological differences among the study species. Dots are outliers, whiskers are the 10th and 90th percentiles. The box is defined by the 25th and 75th percentiles. The bar within the box is the median. In this compilation and comparison of focal plant data sampled across sites, the average water level from 2003-2005 is standardized at zero elevation for each site.

30 C mUtmescens C. gray) C. typhina

3 3

• / "» i 3 c ^ -j •» ~4™l i—i I l_-A- - -3 -i - c i : s » , ** «s *z >i c a *

C. Itipuiina C. retrorsa C. crinita

C. vesicaria C. tuckermam

C pseudocyperus C. comosa C. iscusiris * l 3 .: I i : /TT \ • * m n v I '• : ('"•P -J -2 -.X i * ,1 t ( , ( .(,.,. » -S -I -"5 C t IS*' i -i -i -* ; * i 3 * =!•

Figure 4: Biplots of species distribution on the first two axes of a Canonical Variates Analysis based on 9 environmental variables for 11 riparian Carex species. The first and second canonical axes explain 63 and 17% of the variation in this data set, respectively. Black dots represent individual focal plots. Inner Ellipses are the 95% confidence interval of the mean; Outer ellipses are the 95% confidence interval of the species distribution. Sets of biplots grouped within thick bold lines contain species of the same riparian habitat type; from top to bottom of the figure these are upper swamp, lower swamp, and marsh. Boxes around two biplots denote pairs of sister species.

31 Table 6: Total-sample standardized canonical coefficients of environmental variables, P values (P>0) and percent variance explained for each axis.

Can1 Can2 Can3 Can4 P>F O.0001 O.0001 <0.0001 <0.0001 % variance explained 63 17 8 7 Total Understory 0.81 -0.94 0.1 0.35 Radiation % Organic 0.61 0.42 0.38 -0.4 Matter 0.56 0.61 -0.13 -0.14 % Time Flooded

0.32 0.49 -0.3 0.43 Tree Basal Area

% Vegetation 0.21 0.3 0.26 0.71 Cover 0.01 -0.38 0.5 0.13 Soil pH

-0.21 -0.18 0.15 0.26 SoilP

-0.29 -0.01 0.57 -0.6 SoilK

-0.49 -0.04 0.76 0.34 Total Soil N

32 ^

Table 7: Niche distances. Squared Mahalanobis distance among species are given above the diagonal, with associated p-values below the diagonal. Bold numbers indicate the few species pairings that are not significantly different (p>0.01). C. c. c. C. C. C. C. C. c. C. C. intume­ grayi typhina lupulina retrorsa tucker­ vesi­ crinita comosa pseudo- lacustris scens manii caria cyperus 1.3 4.6 9.1 6 18.8 6.7 19.1 12.1 26.9 c. • intumescens C. grayi IHIIKlX ! 3.1 3.4 4.7 4.4 15.7 4.4 16.6 9.1 24.1 .

C. typhina (1.2212 1 I.I !• If 1 [ 4.5 7.4 6.4 16.7 6.9 18.1 12.1 25.5

C. lupulina O.0001 O.Oi'i'l 11.111M11 2.4 0.9 10.6 1 10.1 4.9 13.1

C. retrorsa 0.0001 0.0001 O.0001 0.0U7 3.4 11.3 2 7.7 2.6 10.1

C. <0.0001 0.0001 O.0001 0.4713 O.0001 13.7 2 12.9 6.6 . 13.5 tuckermanii C. vesicaria 0.0001 0.0001 O.0001 O.0001 O.0001 "O.060T 10.4 4.8 10.2 12.9

C. crinita 0.0001 0.0001 O.0001 0.3637 0.0112 0.4713 "0.0001 "." 3.2 11.4

C. comosa 0.0001 0.0001 O.0001 O.0001 O.0001 O.0001 O.0001 11111111I 2.9 4.3

C. pseudo- 0.0001 0.0001 O.0001 O.0001 0.0022 O.0001 O.0001 O.UUOl U.OUIS 5.4 cyperus C. lacustris 0.0001 0.0001 O.0001 O.0001 O.0001 O.0001 . O.0001 0.0001 O.0001 O.0001 Upper Swamp

o

Lower Swamp Marsh

o o

Figure 5: Biplots of species by habitat groups in canonical space. Canonical axes in each biplot are those that best visually separate the species for that particular group. Crosses (+) are species means, ellipses are 95% bivariate confidence intervals of focal plots. IN: Carex intumescens, GR: C. grayi, TY: C. typhina, CR.C, crinita, RE: C. retrorsa, LU: C. lupulina, TU: C. tuckermanii, VE: C. vesicaria, CO: C. comosa, LA: C. lacustris, PS: C. pseudocyperus.

34 Table 8: Univariate statistics for trait data of each species.

trait standard variable species mean median deviation min max n 100 C. intumescens 1.3 1.2 0.4 1 1.8 4 Diaspore C. grayi 1.3 1.3 0.2 1.2 1.5 3 Weight C. typhina 0.2 0.2 0 0.2 0.3 4 (g) C. lupulina 0.7 0.7 0.1 0.6 0.7 4 C. retrorsa 0.2 0.2 0 0.2 0.3 6 C. crinita . 0.1 0.1 0 0.1 0.1 6 C. tuckermanii 0.5 0.5 0.2 0.3 0.7 4 C. vesicaria 0.2 0.2 0 0.2 0.2 3 C. pseudocyperus 0.1 0 0 0 0.1 3 C. comosa 0.1 0.1 0 0.1 0.1 3 C. lacustris 0.1 0.1 0 0.1 0.1 3 Floating C. intumescens 33 18 46 1 224 300 Duration C. grayi 15 14 16 1 148 299 (days) C. typhina 79 71 51 3 224 294 C. lupulina 71 51 64 1 224 299 C. retrorsa 159 188 71 2 224 290 C. crinita 205 224 47 9 224 293 C. tuckermanii 136 144 76 1 224 293 C. vesicaria 101 79 72 3 224 288 C. pseudocyperus 132 140 89 4 224 293 C. comosa 104 75 82 3 224 290 C. lacustris 217 224 38 3 224 279

35 250

A LA c o 200 , • CR

3 •o * RE o^ 150 •£ >. • TU A PS "Si «S o 2- s= 100 AVE A CO 0) O) • TY 2 • LU o > re 50

|GR

0.5 1 1.5 median time flooded (log(%))

Figure 6: Scatterplot of species average location on flooding gradient in relation to floating duration. Species acronyms are as in Figure 5. Triangles indicate marsh species, squares: lower swamp, and diamonds: upper swamp.

250 i

>. re Q 200 - • • *

;nc e C. tuckermanii/ C. vesicaria • • E 150 * pair

Dif f • • • § 100- • • • • P • • • • • • • • • • • # e n • • • Floatin g •

o i • i • (D 50. 100 150 200 250 300 Patristic Distance

Figure 7: Scatterplot of patristic distance (Table 1) vs. difference in floating time.

36 250

A^ 200 *CR O to 3 • RE •o 150 APS »TU re S^•o ro 100 CO 2 • TY • LU S CO 50 • IN • GR I 0.2 0.4 0.6 0.8 1 1.2 1.4 average 100 diaspore mass (9)

Figure 8: Scatterplot displaying negative correlation between diaspore mass and diaspore floating duration (r2=0.60). Species acronyms are as in Figure 5. Triangles indicate marsh species, squares: lower swamp, and diamonds: upper swamp.

40

AVE 35 COA

ALA 30 c .2 25 15 c Ej20 A PS c o .2 E "S 15 • RE "CR E LU IN TU • TY 10 GR

5

0 0.2 0.4 0.6 0.8 1 1.2 1.4 average 100 diaspore mass (g)

Figure 9: Scatterplot of diaspore mass vs. insolation. Species acronyms are as in Figure 5. Triangles indicate marsh species, squares: lower swamp, and diamonds: upper swamp.

37 a) b)

•d|:S

0 5 mm

9) h)

Figure 10: Representative images of upper root cross-sections of 11 riparian Carex species arranged the same as in Figure 4. a) C. intumescens, b) C. grayi, c) C. typhina, d) C. lupulina, e) C. retrorsa, f) C. vesicaria, g) C. tuckermanii, h) C. crinita, i) C. pseudocyperus, j) C. comosa, k) C. lacustris. ex, exodermis; ae, aerenchyma; en, endodermis; co, cortex. All species display at least some aerenchyma formation.

38 30.0

25.0

20.0

• • «t 15.0 •u C. tuckermanii/ C. vesicaria pair 10.0 • • • •

• •

5.0 H

0.0 50 100 150 200 250 300 Patristic distance

Figure 11: Scatterplot of distance matrices. Patristic distance between pairs of species based on a parsimony analysis (Table 1) Vs. Niche difference or Mahalanobis distance between pairs from CVA analysis (Table 7) Chapter 4: Discussion According to niche theory, coexistence is limited by environmental filtering and interspecific competition (Chase and Leibold 2003). The goal of this thesis is to define the niches of Carex in riparian habitats and explore how this relates to their coexistence. Firstly, species segregation along environmental gradients in the floodplain will be addressed, and then the role of the environment in species distributions within the riparian zone. To highlight environmental filters acting in the riparian habitat, I consider how several adaptive traits contribute to the ability of species to grow in riparian environments. Lastly, I will discuss how this research supports the hypothesis that the niches of coexisting congeners are not conserved over evolutionary time.

Filtering by flood tolerance and segregation along multiple environmental gradients: Defining the niche as the environmental limits within which a species can exist (Hutchinson 1961) implies there can be influence from both spatial and temporal factors. Although riparian zones experience much variation in flooding episodes within a year, the average duration of flooding over the years is fairly equivalent (Junk et al. 1989, Environment Canada 2006) and spatial variation within the floodplain may be more important in determining plant distributions. Flooding tolerance can be a filter for admittance into the riparian zone that also acts on a finer scale to allow a subset of riparian Carex to grow only at certain segments of the flooding gradient that characterizes the zone as a whole. This appears to be the case for the distribution of Carex species in floodplains of the St. Lawrence Valley (Table 5), which falls into three zones: upper swamp, lower swamp, and marsh (Figure 3). This is not the case for all Carex communities. Height above water table was found to be of secondary importance in explaining sedge distributions in peatlands across Canada (Gignac et al. 2004). More generally, the dominance of water regime as a factor in niche differentiation is not unusual in plant communities (Grace 1981, Menges and Waller 1983, Bendix 1994, Sultan 1998, Silvertown et al. 1999, Cavender-Bares et al. 2004a, Sluis and Tandarich 2004). Not all adaptive traits in wetland Carices appear to be conserved and important to growth and survival at all spatial and temporal scales. Within a given portion of the riparian zone (upper swamp, lower swamp, marsh) species possibly have various trait differences that let them grow at different points along gradients other than just flooding

40 time (pH, insolation, etc.). Indeed the majority of the Carex species in this riparian sedge community are significantly different from one another in a multidimensional environmental space that is defined by many different environmental factors (Table 7), which supports the notion that species coexist by partitioning the environment. In the following paragraphs, I describe in the order of flooding groups the environmental niche of each species based on the environmental gradients that I measured. Upper swamp: Carex grayi grew under tree canopy at the highest elevations in the riparian zone, well above the average level of the river water. Individuals often grew in association with C. intumescens and C. typhina, but commonly in micro-sites with a higher pH than the micro-sites in which the other two species occurred nearby. All three of these Carex species were most often found in Acer-Fraxinus forests with little understory vegetation, except Toxicodendron radicans and Lysimachia nummularia. A study of riparian vegetation in Wisconsin (Menges and Waller 1983) only found C. grayi in the same elevation class as C. lupulina and C. retrorsa, not with C. typhina. Although we infrequently found C. grayi growing near C. lupulina, our study showed that C. grayi in Quebec grows along the drier, high elevation end of the flooding gradient. Carex intumescens occurred in the upper riparian zone under forest canopy and in canopy gaps. In nearby Vermont, Carex intumescens was present in the seedbank at 5, 25 and 50 M from the shore (Hughes and Cass 1997) but was only found growing 100 meters from the shore of a wide, slow-flowing stream under an Acer saccharinum canopy (Hughes and Cass 1997). This indicates that conditions for germination were only suitable beyond 50 M from the water's edge. At my Quebec study sites, C. typhina individuals grew in locations on environmental gradients very similar to C. intumescens, but also across a larger range in flooding time and insolation. This species appears to be more of a riparian generalist than C. intumescens. While C. intumescens had a smaller niche breath than C. typhina, its distribution reachs beyond riparian zones and into wet and mesic forest where competition from C. typhina is absent (Table 2). Lower swamp: Carex lupulina appears to be broadly tolerant to flooding variation in the riparian zone. Carex lupulina can be found from rocky shorelines to shaded swamps with clay soil, but the species distribution is narrow in terms total nitrogen and organic matter gradients (Table 5). On these gradients C. lupulina tolerates lower levels than either C. crinita or C. retrorsa, species that share similar ranges with C. lupulina on other

41 environmental gradients. The affinities of C. tuckermanii are very similar to C. lupulina in flooding, total nitrogen, and organic matter gradients. Carex lupulina diaspores were present in the seedbank of an Acer saccharinum floodplain forest at 5, 25, and 50 M from the shore, yet no established plants were found at the site (Hughes and Cass 1997). However, in fall 2006 I scouted the shores of their study site and did find C. lupulina present within 5 meters of the shore. Their transect sampling may not have detected the presence of C. lupulina because the species has more patchy distribution than C. intumescens, a species that they did find in their study. From personal observation, I know that C. tuckermanii never grew on the shore edge like individuals of C. lupulina frequently do. Often C. tuckermanii grew along with Acer seedlings in sparsely vegetated areas near edges of small pools higher in elevation than the river. In the literature, this species has been known to grow in marshes along with swamps (Table 2). My results also are contrary to findings in Wisconsin floodplains (Menges and Waller 1983) that reported C. tuckermanii as a high light specialist growing in less flooded areas alongside C. typhina. Perhaps their large plot size for vegetation sampling was too general for assessing the finer scale habitat of C. tuckermanii. Carex crinita commonly grows in the lower swamp along the edge of the shore, which may or may not have organic soil. In another study along the Ottawa River, ramets of C. crinita were planted at positions -40, -20, 0, 20 or 40 cm higher (or lower) than the average water level, which is a wider range than where C. crinita is actually found along the river (Shipley and Keddy 1987, Shipley et al. 1991). There was no difference in the survival rate of these ramets (Shipley et al. 1991), indicating that the fundamental niche of C. crinita is larger than its realized niche along the Ottawa. At my study sites this species appeared to be a lower swamp generalist (Figure 5) that shares much of its niche space with C. lupulina, C. tuckermanii and C. retrorsa. The range of the species elevation in relation to the average water level was broader than the breadth of elevations in the ramet experiment (Shipley et al. 1991) although the majority of individuals did grow 40 cm above and below the mean water level at my study sites (Figure 3). The distribution of C. crinita may depend more on dispersal and establishment more than on niche requirements. Carex retrorsa was restricted to shorelines and marsh borders, often occurring in the same position relative to the water level as marsh species, yet this species also grows under tree canopy. The species was considered a high light specialist in Wisconsin floodplains (Menges and Waller 1983). Compared with forest understory

42 species, Carex retrorsa grows in higher light conditions. Its insolation is much lower than that of marsh species and the species can occur in the same light conditions as some lower swamp species. Marsh: In the marsh, Carex lacustris formed large mats of sedge meadow with its rhizomatous growth making it a superior competitor in such situations. The roots of this species were often constantly in the water. Carex lacustris was also dominant in several shrub wetlands and was found in the understory in forested wetlands. The species was most often found in marshes at the wettest end of the flooding gradient and in the highest insolation areas. The majority of the individuals were collected along Lake St. Francois (Huntingdon Marsh) because: a) there were many different habitat types along the lake where C. lacustris was common and b) there were very few other sites where this species was found in the St. Lawrence Valley. Carex /acusfr/s-dominated marshes along Lake St. Francois had an average pH of 5.4 and soil was mainly organic (66%) (Jean and Bouchard 1993), which was similar to the pH observed in this study, but, because I collected focal data outside of the Carex /aci/sfrv's-dominated marshes, the average soil organic matter for this species was much less (Table 5). Carex pseudocyperus and C. comosa grew along the edges of C. lacustris marshes and also grew on top of logs in marshes and in pits formed by uprooted trees in the swamp. In general, the roots of both species were in anaerobic conditions, which is consistent with a European study that also observed C. pseudocyperus in anaerobic conditions (Moog and Janiesch 1990). Carex pseudocyperus is also an infrequent aquatic species in cutoff channels of the Rhone River in France in soils at pH 7.5 - 7.7 that have very little phosphorus (0.00-0.02 ppm). The pH at C. pseudocyperus sites in my study was much lower (pH 5.0 - 6.8) and soil phosphorus was much higher (Table 5) but I nonetheless did find this species growing in cut-off channels at Pointe-Riviere-a-la- Raquette. Carex pseudocyperus has a smaller niche breadth than C. comosa (Figure 5) and the two species differed along the insolation gradient. Carex pseudocyperus appears to have better shade tolerance. Carex vesicaria has been considered a swamp species in the literature (Table 2), yet in this study it was only found in marshy river shores with little organic matter. At Otter Creek, Vermont, diaspores of this species were found in the seedbank at 5, 25, and 50 M from the shore in an Acer saccharinum floodplain forest but there were no individuals found in the understory (Hughes and Cass 1997). This could be because C.

43 vesicaria tends to grow only in riparian marshes, as found in the current study. It also is possible that this taxon is actually a composite of several species that have not yet been recognized as distinct (Ford et al. 1993; Waterway personal communication). Perhaps Carex vesicaria in riparian zones is a different species from what has been described in the qualitative studies of Table 2.

Role of traits: The effects of anoxic conditions on plant roots are very stressful to both upland and wetland plants (Armstrong 1979). In riparian habitats, plants undergo anoxic conditions (Table 5) at varying times throughout the year. Many wetland plants have trait adaptations to allow them to tolerate anoxia, including Carex species (reviewed by Armstrong 1979). To better understand how species segregate along environmental gradients, like flooding time, an understanding of their adaptive traits is necessary. Lysogenous aerenchyma formation in roots and leaves has been observed in several wetland and upland Carices in Europe (Visser et al. 2000). This network of air spaces provides a pathway to allow oxygen to reach cells in anoxic conditions, so that tissue may survive and even grow (Armstrong 1979). Although there has been some research on how aerenchyma helps C. pseudocyperus (Moog 1998), C. lacustris (Busch 2001), and also C. retrorsa and C. vesicaria (Visser et al. 2000) tolerate anoxic conditions, to my knowledge no studies have reported other Carex species in riparian habitats that form root aerenchyma. I observed lysogenous aerenchyma formation in all 11 species, in all three sections of the root (high, mid, low). Carex typhina displayed very little aerenchyma compared to the other species, which in part may explain why this species grew on the dry end of the moisture gradient. Interestingly, Carex crinita also displayed very little aerenchyma although it was found over a broad range of flooding regimes. Carex typhina and C. crinita are closer relatives with each other then with any other species in this study (Tablel). The presence of aerenchyma in all my study species leads me to conclude that aerenchyma is a conserved trait in the wetland Carex clade that underlies the ability of species to inhabit saturated, sometimes anoxic, floodplain soils. Another plant stressor in riparian habitats is shade. Diaspore mass has been correlated with adaptation to various environmental stresses, most often shading (Westoby et al. 2002). In the case of riparian Carex, I found a negative trend between diaspore mass and site insolation (Figure 9). It has been shown repeatedly that seed

44 mass is correlated negatively with seed production (Westoby et al. 2002), which is likely to be true for our species (personal observation). A larger diaspore mass provides more resources to the germinating plant than smaller diaspores, whereas less resource devoted to each diaspore allows more seeds to be produced from a given amount of resource (Westoby et al. 2002). I also found diaspore weight was negatively correlated with floating time (Figure 8), which may play a role in dispersal to habitats with different environmental regimes. It has often been postulated that the inflated perigynia of species in the Lupulinae section, are an adaptation to hydrochory (Reznicek and Ball 1974). My experimental results showed that, to the contrary, species of the Lupulinae section floated for the shortest duration of time out of all riparian species. Floating time of diaspores for Carex species in general varied greatly, which is consistent with what van den Broek and others (2005) found with wetland Carex from different communities in Europe. They found floating duration increased in species from rich fens, to wet meadows and then to reed beds (van den Broek et al. 2005). My buoyancy experiment yielded results for C, pseudocyperus that were within the 109 to 220 day range they observed (Table 5; (van den Broek et al. 2005). My study also showed species diaspore floating duration increased with occurrence at flooded sites (Figure 6). Finally, Carex crinita and C. lacustris diaspores floated the longest of my study species, up to the 225 day limit of my study. Carex crinita is flooded less often than many other species exhibiting shorter diaspore floating duration. The floating duration of C. crinita allows it to disperse farther along the river and may increase the likelihood of being deposited on a patch of land rather than sinking to the bottom of the river. From these various examples, it is clear that some particular traits can be important in determining the ability of a species to grow at certain points along the flooding and insolation gradients in riparian systems. While some traits may be conserved across my study species to allow them to persist in flooded conditions, other traits are different enough among the species to support their segregation along environmental gradients within different riparian zones. These results are an illustration of how ancestral traits can be conserved or evolved as new species evolve in a lineage, allowing a new species to either stay in its ancestral habitat or colonise another (Cavender-Bares and Wilczek 2003).

45 Absence of phylogenetic signal: There are several different perspectives on how traits might shift as speciation proceeds in a lineage. If interspecific competition between congeners is strong, we expect closely related species to have less similar niches than random species pairs (Cavender-Bares et al. 2004b). If environmental filtering only allows species with similar traits to occupy a given area, then we would expect related species, with their similar traits, to occupy more similar positions along environmental gradients than expected between random pairs (Webb et al. 2002). Neither alternative trend in fact was found in our group of species (Mantel test p<0.05). Losos et al. (2003) came to the same result using a Mantel test to compare species relatedness with position in multivariate ecological space in Anolis lizard communities. They concluded that when strong interspecific interactions cause niche divergence, species may be diverging from near relatives to an extent where they interact with distantly related species just as strongly as closely related species. Using a different but complementary approach to these questions, (Cavender-Bares et al. 2004b) supported the hypothesis that the convergent evolution of traits and the dominance of filtering over interactions among related species in the community may have caused phylogenetic overdispersion in oak species within different habitats. The apparent environmental filtering of Carex along the flooding gradient and lack of niche conservatism also supports this hypothesis. More focus on trait evolution and the role of competition between species is needed to further explore this idea. Silvertown et al. (2006) hypothesised that the relationship of niche differences and species relatedness will not be the same at different phylogenetic and niche scales. They reasoned that related species living in the same area must be different enough to relax competitive exclusion and allow coexistence, so that traits that define the alpha niche need to be at the terminals of phylogenetic trees for coexistence of related species, and these traits therefore should be evolutionary labile (Silvertown et al. 2006b). On the other hand, if there is no phylogenetic trend in the traits, then the differences in environmental niche between species pairs should have no evolutionary trend either. This is the case in my study where there was a lack of phylogenetic signal in the ecological structure of riparian Carex species. Furthermore, the environmental niches of sister-species did differ, supporting the idea of evolutionary lability in at least some of the traits that determine the ability to grow at different points along environmental gradients in the riparian system. For example, the sister pair C. grayi and

46 C. intumescens apparently shared traits that allowed them to grow in the shady and drier upper swamp, but differentiated in that zone on a pH gradient (Table 6). Other sister pairs that were similar in site location were the C comosa and C. pseudocyperus pair and the C. lupulina and C. retrorsa pair in the marsh and lower swamp, respectively. Within these riparian zones, these sister pairs differentiated on different gradients. Carex comosa grew in open marsh more often than C. pseudocyperus, which appears to tolerate shading better. Carex retrorsa grew in areas with more organic matter than found at C. lupulina sites. On the other hand, the sister pair C. tuckermanii and C. vesicaria is an example of habitat differentiation at the terminal branches of a phylogenetic tree. Carex tuckermanii grows in swamps while its sister, C. vesicaria, was only found in marshes in the riparian zone. This sister pair has the least amount of genetic difference of all the sister pairs in this study (Table 1), yet surprisingly this sister pair showed the largest difference in environmental affinities. Another example of habitat differentiation of close sister species is between C. crinita and C. gynandra. Carex gynandra was not included in this study because it was entirely absent from my study sites and indeed from all locations along the floodplain that I scouted for potential field sites. A thorough literature review conducted by the St. Lawrence Centre (Environment Canada; Montreal, Qc) on the flora of the riparian zone along the entire length of the St. Lawrence River also did not ever find C. gynandra present. Conversely, C. gynandra is common at wet, shaded upland forest sites in the Gault Nature Reserve at Mont-Saint-Hilaire, Qc, where C. crinita is uncommon (K. Flinn, Biology Dept., McGill University, personal communication). A between-habitat study of Cyperaceae species niches in peatlands across Canada used a TWINSPAN analysis to separate species into groups (Gignac et al. 2004). In their study, 25 of the species were Carex, and 5 sections in the genus had more than one species represented. Species of the same section were sometimes found in the same habitat and sometimes across habitats implying that there may not be beta niche conservatism in peatland Carex. In contrast both the present study and a phylogenetic analysis in the subgenus Carex by (Waterway et al. in press) found beta niche conservatism along moisture gradients. Why would there be moisture gradient conservatism of species between- habitats? Prizing and others (2001) have hypothesised that this is a result of ancestral opportunities for speciation. Gradients dominant in a landscape with large amounts of

47 heterogeneity in the past increase the likelihood of species having been able to colonize extreme ends of environmental gradients, triggering speciation (Prinzing 2001). Most of today's plant lineages diversified during the Cretaceous epoch that had a strong contrast in soil moisture regimes from place to place (Prinzing 2001). The presence of beta niche conservatism on moisture gradients today supports the notion that niche position in extant species may have been determined by these types of opportunities in the past.

Summary and conclusion: The results obtained from this field study have updated and significantly expanded the ecological information on riparian Carex species by gathering quantitative data that describes their environmental preferences. This research also has increased our knowledge of traits that allow species to occur in temporarily flooded environments. I have shown that species appear to first be organized into groups along the flooding gradient in riparian systems and secondly differentiated on other environmental gradients within different riparian zones. The most notable gradients for this secondary differentiation are: soil organic matter, insolation, and soil pH. The high degree of spatial environmental heterogeneity in the riparian zone contributes to the coexistence of riparian Carex species. Although the riparian Carex community is structured ecologically, the ecology is not phylogenetically structured. This supports the notion that rapidly evolving traits that are different between and among species allow congeners to coexist at the within-habitat scale (Silvertown et al. 2006b) and suggests that competition between closely related species may not be any stronger than that between random species pairs (Losos et al. 2003). A study focusing more on the adaptive traits of wetland Caricies would be needed to further explore these hypotheses, and a study of Carex niches at larger spatial scales would be particularly interesting for exploring the possible hierarchical organization of the niche (Silvertown et al. 2006b).

48 References

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56 Appendix I: Canopy Analysis

Photograph Collection: Canopy photographs were taken using a Cannon EOS 30D digital camera with a Costal Optics Systems, Inc. (West Palm Beach, FL, USA) 185° angle view Fisheye lens mounted in a Regent Instruments (Quebec City, Quebec, Canada) Universal O-Mount with NorthFinder, all in turn mounted on a sturdy tripod. Photos at the different sites were taken from August to the very beginning of September, 2006. The tripod was placed 50-100 cm above the soil surface directly above each focal plant; the tripod was in the higher height range in situations where the water level was above the soil surface. Photographs were taken at dawn and dusk or under cloud cover as much as possible. Photographs taken under sunny skies were about 10% of the total number of canopy photos taken. Photographs were never taken when the sun was high in the sky (10am - 2pm). The photographs were downloaded onto a computer, labelled, and backed up on a CD. All analyses on the photographs were done using the WinSCANOPY (Regent Instruments Inc. 2006) program. Pixel Classification: Although an attempt was made to avoid having sun reflections in the images, some pictures did have tree trunks and leaves that appear shiny. This can be a problem because using an automated grey scale pixel classification yields light parts of the canopy interpreted as sky on the WINSCANOPY program. The cut off point for grey- scale sky versus canopy can be adjusted manually, but sometimes there are parts of the sky that are darker than the canopy, making an accurate adjustment impossible. Manual adjustment also risk introducing analyst bias. Another alternative I tried was using the WinsCANOPY "edition" working mode to modify the original image content. Parts of the canopy that were too light were colored with a darker shade so the program analyzed the region as canopy instead of sky. Manual coloring is time consuming and, when there is a large amount of shiny or washed out canopy in the image, it can become difficult to predict what colors are at the cut-off point for sky. Coloring the photograph is thus also a poor way to fix the problem of photographs with bright lighting.

Fortunately, the pixels can also be classified by color automatically using the WINSCANOPY software. The researcher needs to program which colors belong to the canopy group, and which ones belong to the sky group. By using a photograph with a

57 large range of colors and shades, I identified color groups that became the standard for assessing all my images. The color groups are archived in a calibration file in the electronic appendix associated with this thesis (Appendix V). I defined several color classes in each color group (canopy or sky). The sky group consisted of white, light blue and a darker blue shades; the canopy group consisted of various tree trunk colors (dark, medium and light) and four leaf colors (dark, medium, light and grey). The program defined pixels with the closest color class. By toggling between the actual image and the computer's color pixel classification, one can check that the sky and canopy is not misrepresented. Before each analysis was completed, I checked the program's interpretation of sky and canopy and found them satisfactory.

To be sure my technique was working well, I also compared the color analysis with the default grey scale analysis (with the sky-canopy cut off determined by an algorithm) in images in which the default grey scale analysis determined canopy from sky accurately (accuracy determined visually). The results showed that both color and grey analyses had a very similar percent openness (±<1% difference) for most images. Since the color analysis could distinguish the light colored canopy from sky pixels, and the analyses of the pictures taken in low light are about the same as with the default grey scale setting, all of the images were analyzed using the color classes I made for my 229 images of floodplain canopies. In very few cases, the sun was so bright on leaves that they appeared white, a color inevitably analyzed as sky by any automated algorithm. In only these exceptional instances, I used the "edition" working mode to color in the white canopy so it was properly interpreted as canopy.

WinSCANOPY Data: Each photograph was analysed in a random order using the WinSCANOPY program. Because the camera was not correctly aligned with the Lens.cal folder delivered with the program of this new instrument, I had to be define the hemisphere (hemisphere - camera lens - click interactive - copy from "Lens.cal" - ok) and make one other modification to the default (analysis - preferences - check off resize image when

58 hemisphere goes outside of it)1 . The location of magnetic North was defined for each image along with the latitude, longitude, slope, aspect, and sky condition. The date and time of each image was part of the image file. Once the program analysed the image, the estimated canopy openness and radiation data under canopy (direct and diffuse average from May 1st to August 31st) were recorded. Note that the radiation regime was analyzed for each image using the default 5-day interval in WinSCANOPY; thus the daily averages reported in my thesis are based on the daily radiation regime assessed every 5th day throughout the growing season, not every single day.

1 N.B. Since the summer of 2006, the canopy camera has been realigned. Future researchers using our WinSCANOPY system can use the Lens.cal folder default for the program, but must still use the keyboard's side arrows to define north.

59 Appendix II: Estimation of percent time flooded

During field work in July and August 2007, I used a stadia rod and surveyor's level to measure the elevation of focal plants from the water level of the river channel on the day a site was visited (Figure 12). One focal plant (or a "benchmark") in a patch was measured twice so we could check the consistency of our survey measurements. The largest difference between two measurements from the focal plant to the river water was 10 cm; most of the time the difference between the two measurements were within a centimeter.

taking more than one measurement at the water because waves or statis sinking can reduce accuracy. When it was difficult to get the water level measurements right at water level, we placed the statia rod in the water and made a note on how tar it was in so we could add that amont to the elevation.

$/ ;=W- for focal plants in the channel, we used a ruler to measure the back measurement to check out "benchmark" / height of the water torn the t accuracy. soil at the focal plant.

i I focal plants

Figure 12: Example of surveying lines to estimate the elevation difference between a focal plant and the river water level.

When we visited the Oka site, the water level was above the base of every plant in both the swamp and the marsh. Because the marsh was so vast, it was impossible to take a measurement at the water channel. For the Oka site we used a ruler to measure the water depth above the base of the focal plant.

60 At the Huntingdon marsh site, there were some plots where focal plants were rooted in mats of floating peat. Since they were floating, changes in water level would change the elevation of the plant base so that it was always at the height of the water. For these plants, the elevation from the average water level was considered zero, because the plants were always floating on top of the water.

For the majority of the sites, water gauging stations were too far away (upstream or downstream) to directly measure the water elevation at the site on a given day. By exception Oka's Grande Baie was right next to a gauging station, and Cap-St-Jacques was across the lake from the same station (Pointe Calumet) so at these two sites I made no benchmark measurements. For the other sites, I measured the actual elevation of the water using geodesic benchmarks as close to our sites as possible. For Plaisance, I used a benchmark near the provincial park that was verified by a professional surveyor; there were no other benchmarks near the park. I made one to six water elevation measurements for each site between July 2006 and May 2007 and used the average day elevation at the gauging station to compare water elevations between the site and station. I used this average difference between the site and station to calculate the elevation of the water at the site for the dates the focal plants were actually measured (gauging station daily average + or - the average difference from site and station). By estimating the water level for the day a focal plant was measured, we could estimate the elevation of the focal plant above the water level in a way that was consistent across sites and sampling times.

Finally, to obtain the percent time flooded for each plant, I collected data on the average daily water elevation for each gauging station (Environment Canada 2006) using the three most recent years with full data sets, which were the years 2003, 2004, and 2005. Using the average difference from site and station, I calculated the three year daily water levels for each site. I then calculated the number of days each focal plant's soil at or below the river level. From this I calculated the percent time flooded over three years.

61 Appendix III: Soil collection and preparation

Soil collection: Soil was collected at the base of each focal plant from the soil surface to a depth of 15 cm. I observed many roots of the focal plants in this depth range. For the majority of the collections, soil was collected within 0.5 m of the plant 6 times using a 2 cm diameter soil probe. All soil collected by the probe was then placed in a plastic Ziploc bag labelled with the focal plant's code. Every evening the bags of soil collected that day were opened to start the drying process. In some cases collecting soil with a soil probe was not possible. When the soil was extremely wet, the soil would slip out of the probe before it could be collected. In these cases a trowel was used for soil collection. On return from the field, I placed the samples in 5 lb Kraft paper bags, labelled them, and placed them in a forced draft oven at 55°C until dry (1-3 days). Soil was oven dried within two weeks after collection in the field. The oven-dry soil was then placed in Rubbermaid bins to be stored until processing.

Considerations on sampling and soil preparation: Each soil sample needed to be homogeneous for analyses. The floodplain soils had high clay content and dried in hard aggregates, which were not suitable for soil analysis without prior processing. The soils had to be broken up to pass through a sieve, split up into subsamples, and then shipped off for analysis. This preliminary process mixed up heterogeneous soil aggregates to make one homogenous soil sample, and removed all non-soil (i.e. rocks, twigs) from the sample.

Aggregates can be broken up by chemical or physical means. Chemical pre-treatments can result in destruction and dissolution of some soil minerals. Physical pre-treatments also have their drawbacks (chance of altering particle size), but they are fairly simple and more easily dealt with than chemical pre-treatments. For our soils we physically crushed soil aggregates, which is the most common treatment used.

Sieving the soil after crushing has its own set of considerations that need to be acknowledged. Soils are considered aggregates equal to or smaller than 2 mm particle size(1986). Any particle slightly larger is considered a pebble. Therefore it is common practice for soils to be sieved through 2 mm mesh screens. The abrasion of the steel

62 screen on the soil sample may cause some contamination (Soil and Plant Analysis Council Inc. 2000). Fortunately, our chemical analysis does not include heavy metals.

Soil crushing: Dust from dry soil can seriously irritate the lungs. The following procedure was done while wearing a proper dust mask in a ventilated area. Each soil sample was poured out of its paper bag onto a large 40 X 70 brown sheet of packaging paper. The soil was then spread out and a rolling pin was rolled over the soil to break the aggregates. If the rolling pin was not sufficient, the aggregates were placed in an agate mortar and crushed with a pestle until the clumps were small enough to be crushed by the rolling pin. After sufficient crushing, the soil was sieved through a 2mm mesh screen. Soil aggregates that were not entirely crushed were crushed and sieved again.

Quartering the crushed and sifted soil sample:

Figure 13: Quartering

1 2 It was necessary to separate the soil remove samples into several sub-samples to be .mix sent for different analyses. The quartering method was used for taking sub-samples for soil analysis. This 3 /P1 4 method was used so that every particle mix remove would have an equal chance of being sub- sampled; making each sub-sample is a representative of the larger soil sample. With the soil on the brown paper, the soil was divided into four equal portions using a ruler (Figure 13). Portions two and three were mixed together to make the subsample. Portions one and four were placed in a plastic Ziploc bag labelled "Storage". The remaining portion of the sample was quartered again. One half was placed in a plastic Ziploc bag labelled "Chem" and the other in a bag labelled "Particle". The "Chem" and "Particle" samples were shipped off to the Sol- For Lab at Laval University, Qc, for analysis. The "Storage" bags were left in the basement locker used by the Lechowicz Lab at McGill, and are available for re-assay or use in other experiments.

63 Appendix IV: Aerenchyma Estimation

Killing and preservation agent: The roots needed to be preserved so that the structure stayed similar to that of a fresh root. An FAA solution was used to both rapidly kill and preserve the roots. The FAA consisted of 5% Formaldehyde, 5% glacial acetic acid, 5% glacial acetic acid, and 90% seventy percent ethanol. 10 ml of FAA was placed in 30 ml Sarstedt screw top bottles for use in the field. Batches of FAA were made for two weeks worth of root collection so that the solution was fresh for optimum results. Bottles were stored in a cooler and placed in the middle of a backpack in the field to keep the chemicals away from the heat.

Field root collection, killing, and preserving: For each focal plant, a root was selected to collect sections from. This root was known to come from the focal plant, and the root tip was still intact. Each section cut was 0.5 cm in length so that the killing agent could penetrate all of the tissue. Roots were cut using a razor blade or knife. In retrospect, all roots should have been cut with a razor blade or a surgical scissors to prevent structural damage to the root. Each root had three sections taken for analyses. The first section was taken where the root connected with the stem, the second at 1cm from the tip, and the third starting 2.5cm from the tip. If a rhizome was present in the plant, a 0.5cm section was cut from it where the rhizome meets the shoot.

The root sections were immediately placed in a 30 ml Sarstedt screw top bottle containing ~10ml of FAA solution. A piece of paper with the focal plant label, date and section of root (written in pencil) was also placed in each bottle. Roots were left in FAA until made into slides the following winter/spring.

Making Slides: The roots of the Carex under study are fragile and easily deformed by slicing. Wax histology was used to support the tissue to prevent the deformation and tearing of root sections in the sectioning process (Figure 14). Due to time constraints only three individuals of each species were randomly selected to have their root samples analyzed. The following bullet points are the general methods used to make the slides (Ruzin 1999).

64 Figure 14: Wax Histology

1. Preliminary work

a. a. Coating Slides

• Add albumin and glycerin mix (Meyer's adhesive) and spread thinly to slide • Dry on slide warmer 40-42°C at least 24hrs before wax ribbons are placed onto them

b. b. Preparing Stain

• 2 g Safranin in 200 ml of distilled water • Place on stir platform for 5 min • Filter through 2 layers of fluted filter paper • Place in a screw top glass jar for storage. • 0.25g fast green in 50 ml absolute alcohol/coplin jar • Place fast green in reagent bottle for storage

2. Dehydration Day 1 • Move tissue into fresh 70% ethanol 3 times over the course of one work day

65 Day 2 • 80% ethanol 2 times (15min each) • 95% ethanol 2 times (15min each) • 100% ethanol 4 times (15min each)

3. Infiltration

Day 2 continued • CAUTION: xylene should be used only in the fume hood with proper thick gloves and eye protection. User should read MSDS of each chemical before proceeding. • Replace half of the 100% ethanol with xylene (30 min) • 100% xylene

Day 3 • 100% xylene 3 times (15 min each) • Pour contents of vial into glass vial that has some wax residue (10 min) • Melt wax in 60°C oven • Replace half of 100% xylene with wax (30 min in 60°C oven) • 100% wax 2-3 changes in 60°C oven • Store in oven overnight

Day 4 • 100% wax 2-3 changes in oven (can be stored indefinitely)

4. Embedding tissues in Paraffin wax

• Make sure specimens are warming up in oven for tissue removal • Turn on wax dispenser to melt paraplas flakes in dispensing bowl • Turn on hot and cold platforms • When wax melts (<1 hr) turn on cold platform switch • Remove metal embedding cups from xylene, drain on paper towels in fume hood, wipe dry with kimwipes • Warm metal cup on front platform of wax dispenser • Fill metal cup with hot wax using foot pedal • Orient specimen vertically in metal cup • Slide metal cup onto rear platform of wax dispenser to solidify • Fit a plastic holder on metal cup (projections at right angles) • Fill plastic holder to top • Slide onto rear platform • When skin forms on top of wax add a little more wax • After 2 or more hrs of cooling, remove metal cup after running under cool water for 3 s

66 5. Sectioning

• Trim wax to a trapezoid shape • Insert plastic specimen holder into microtome specimen holder and tighten in place • Remove blade from safety box and slide into blade holder • Place blade as far to the right as possible so it can be moved to the left as it becomes dull • Set sectioning thickness between 10-15 urn • Unlock rotary handle on right • Wax ribbons will be formed by cranking the right rotary handle. Discard the first few cuts. Retrim block face if block diverts from trapezoid shape throughout the cutting. • To form long ribbons, gently lift ribbon initials onto brush or forceps • Transfer ribbon to paper towel w/ labels. Can be stored between paper towels

6. Mounting

a. • Cut ribbon so that it will fit on slide • Orient shiny side down • Float ribbon on drop of distilled water to help orient ribbon section. Ribbons should start 0.5cm from bottom of slide (side away from frosted end) and should not overlap edges • Excess water blotted off with kimwipes • Transfer slides to warm plate (40-42°C). Ribbons are gradually stretched on warm plate. Leave to dry overnight on warm plate. Slides can be left indefinitely at room temp. b. • Place slides in slide holder ail facing the same direction • Wax removal: 100% SafeClear II, 3X 20min each • Rehydration: 100% ethanol 2X 10min each 95% ethanol 10min 70% ethanol 10min 50% ethanol 10min 25% ethanol 10min distilled water

7. Staining

• Place slide holder in safranin mix 30-40min • Take one slide out of mix at a time to process • Distilled water - 3 rinses w/ back and forth action in 3 consecutive containers. • Check background w/ microscope • Wipe back and blot edges of front slide • Pour rapid green into coplin jar • Dip slide holder once to several times in rapid green • Use rapid dips in 100% ethanol to remove excess. Wipe back of slide • Check with microscope - parenchyma green, collenchyma cell walls red • Several dips in clove oil to clean background (optional). Wipe back of slide • Store in 100% SafeClear until ready to mount(<20min)

67 8. Adding cover slips • Use paper lined tray to drain slides of SafeClear • Place slides on warming tray - add 2-3 drops of mounting medium (Permount or Canadian Balsam). Cover with cover slip. • Dilute drops with SafeClear if too thick or bubbly • Leave specimens on hot plate for several days to remove bubbles (add extra mounting medium on edges of cover slip to fill air spaces if necessary) • Clean up!

Photography: Photographs were taken of each section on a slide using the Image-Pro Discovery (Media Cybernetics Inc. 2005) program with a Olympus SZX12 dissecting scope and Ql CAM Fast color 12-bit camera. The magnification was noted so the diameter of each section could be recorded. Afterwards the photographs were converted into JPEG for viewing on other programs.

68 Appendix V. Electronic archive of thesis material

The following paragraphs provide a description of the content of a DVD provided to my co-supervisors Martin Lechowicz and Marcia Waterway with their copies of this thesis. Included in the DVD is data matrices, analyses, images, and relevant writings. The DVD can be used to verify methods and results and to access a digital copy of the entire thesis. Main folders: Data Analysis output Text Thesis Images

Data This file includes sub-folders titled "environmental", "trait", "genetic" that includes their respective data. The environment file has a master spreadsheet of all environmental data both in raw and final form. This file may be referred to in case of discrepancies in later analyses.

Analysis output This folder is organized similarly to the data folder with final outputs of analyses.

Text Content of this folder includes protocols, site descriptions, and PowerPoint presentations directly relevant to this thesis. Also included is a literature review of habitat preferences of over 57 Carex potentially found in the riparian zone of the St. Lawrence Valley. I have included my Community Ecology course papers.

Images Field images This folder contains close up images of focal plants (labelled with their focal plant code) along with images of their habitat. Other images of the site in general along with images of scouted sites not included in the study are also included in this folder.

69 WinSCANOPY Hemispheric images of canopy above focal plants are included in this folder (labelled with their focal plant code) organized by site. ImagePro Images Photographs taken with ImagePro Plus program of root cross sections and diaspore images are included in this folder first organized by part of plant then by species. ImagePro images converted to JEPEG are also in these folders.

Thesis An electronic copy of the entire thesis in final form is provided in this folder.

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