An investigation into trait differentiation within and between two closely related Silene species. Daniel Connaghan Degree project in biology, Master of science (2 years), 2017 Examensarbete i biologi 45 hp till masterexamen, 2017 Biology Education Centre and Department of Ecology and Genetics, Uppsala University Supervisor: Dr. Sophie Karrenberg External opponent: Per Torang Table Of Contents Abstract 3 Introduction 4 Materials and Methods 9 Collection sites and plant material 9 Trait Measurements 10 Statistical Analysis 13 Results 14 Climate Results 14 Between Species and Populations within Species Results 20 Climate Relationship Results 26 Discussion 27 Acknowledgements 32 References 33 Abstract Ecological differentiation and adaptation are processes that can drive divergence and speciation. Measuring ecologically revenant traits can help to identify possible targets of natural selection that may have mediated ecological differentiation. This study looked for evidence of within and between species differentiation in seven ecologically relevant traits in two closely related species sampled across their range, and whether any of these traits were related to climate differences among site of origin. We measured seven traits under common garden conditions in seedlings of Silene dioica (11 populations, n=528) and Silene latifolia (14 populations, n=672) grown in the botanical garden in Uppsala in a randomised block design. Three traits related to leaf morphology were measured, and four related to water usage of the plant were measured. These traits were analysed for differences between the species as well as for differences within each species between populations using a linear mixed model. The traits’ relationship to a climate variable, derived from the axes of a principal components analysis of climate data for each site, was investigated using a linear model. A number of drought avoidance (e.g. succulence, max turgid weight) and morphological traits (e.g. leaf ratio) differed between the two species. Water use efficiency has been flagged before as possibly driving ecological differentiation between the two species and the results identify possible candidate traits for quantifying this separation. Differentiation between populations within each species was also present in two traits within S. latifolia and in four traits within S. dioica significantly varying between the populations. This could reflect either local adaptation or genetic drift acting on populations across the range. One trait related to the amount of water taken up by the leaf (wgain) was found to be significantly associated with the climate variable, which was extracted from the principal components analysis, in S. latifolia. The results identified a number of candidate traits which could reflect ecological differences between the species especially with respect to water relations. These traits may allow the species to respond differently during periods of water stress, which in turn could result in ecological separation of the species and determine their geographical ranges. 2 Introduction Looking for evidence of trait divergence is an important step to understanding how speciation may occur, and at what stage in this process natural populations are. Extrinsic (environmentally dependent) postzygotic isolation owing to differential adaptation and differences in ecogeographic distributions (Butlin et al., 2012) can reflect divergent ecological selection. The motivating idea behind the search for trait divergence between species is that gene flow between incipient species may be reduced due to differential adaptions to the environment (Favre & Karrenberg, 2011). This may then in turn lead to the evolution of intrinsic reproductive barriers between the two species, such as alteration in reproductive organs, or season in which they breed (Seehausen, 2014; Rahme, 2009 which act independently of the environment. Traits that may contribute to ecological differentiation between species can vary largely within one or each species. It is important to also consider trait divergence among populations of each species throughout their range. Trait variations within and between species measured in a common environment are generally interpreted as genetically-based and can be explained either by natural selection driving phenotypic differentiation across climate conditions, by local selective pressures, or by genetic drift (Hall et al., 2007; Kawakami et al., 2011). Measuring traits important to plants’ survival across their range can provide an insight into which selective pressures are important for the plants and how these pressures vary in importance across space. In order to select relevant traits a number of important areas for plant ecology needed to be considered. Leaves are the main organs of the plant for carbon assimilation and energy balance and thus a leaf’s functional significance in managing a whole host of stressors is obvious. Water use efficiency and specifically how plants deal with even moderate water stress is known as one of the most important forces governing range distribution (Taiz, 2014) and as such is worthy of investigation. In North America, vegetation analysis has shown that the distribution of plants is more correlated to traits related to the water relations of a plant, than with other commonly used metrics of climate (Stephenson, 1990), as changes in these water use traits more aptly reflects the effects of climate as sensed by plants (Stephenson, 1990). As well as water use traits, comparative investigations of traits related to leaf shape have also been indicated as useful in investigating adaptive significance of morphological 3 variation (Givnish, 1984). The shape of the leaf has been found to have relevance to the boundary layer resistance due to dead non-moving air surrounding the leaf (Gates, 2011) as well as follow on effects in terms of rate of transpiration (Chiariello, 1984). Two-dimensional leaf shape has been shown to be strongly correlated to the structure of their veins, and broader leaves can potentially create stronger water gradients and higher photosynthetic function than found in narrower or more elongate leaves due to leaf tissue being positioned farther away from major veins (Leigh et al., 2014). Utilizing lower photosynthetic function to avoid water lost to transpiration exceeding the water uptake is a well known drought strategy of plants (Roberts, 1986; Fitter, 1995), and thick leaves are characteristic of drought resistant xerophytic plants, as it gives a higher ratio of photosynthetic mesophyll to transpiring leaf area (Abrams et al., 1994). Studies investigating changes in leaf traits in relation to environmental gradients such as specific leaf area or leaf dimensions (Ackerly et al., 2002; Ehleringer et al. 1981; Givnish, 1984) are relatively rare, but have found significant associations between species distribution and these leaf traits and have flagged the importance of further investigation of the changes in leaf traits across climate gradients (Ackerly et al., 2002). In terms of candidate traits that were worthy of investigation, I looked for ones which had been shown in the past to be of biological relevance. With regards to traits related to water relations in plants, leaf succulence has been indicated as associated with a progressive inhibition of photosynthetic cellular machinery, as well as an inhibition of the key enzyme rubisco (Griffiths et al., 2007) and so plays a key role in how the plant photosynthesizes under variable light conditions. Succulent leaves usually possess large cells with thin walls and a large vacuole which makes them function well as water storage tissue (Taiz, 2014), and so this was identified as a possibly ecologically relevant trait keeping in mind the different life histories regarding drought and shade tolerance of the two species discussed below. Maximum possible leaf turgidity as well as rate of water uptake has been shown to predict turgidity-dependent petiole flexibility which has knock on effects on the ability of a plant to resist wilting (Gonzalez-Rodriguez et al., 2016), and has previously been indicated as a candidate trait for drought tolerance in sugar beet (Ober et al., 2005). Specific leaf area, as a measure of variation in leaf structure, was related to patterns of variation in net photosynthetic capacity in an analysis of field data of 107 species from six different biomes, as well as based on a literature review of an additional 162 4 species (Reich et al., 1998), and so was of interest to the plants which have been known to inhabit different light environments (see below). These traits provided a useful starting point from which to hone in on the study of potential ecological differences between the species. This study focused on a number of aspects of trait divergence in traits in two closely related species of the plant family Caryophyllaceae. I looked at whether evidence for trait divergence could be detected in a collection of ecologically relevant traits including those related to water usage (eg. wgain, succulence, turgidw) and also leaf morphology (eg. SLA, leafratio) were investigated. The two study species, Silene dioica (L.) Clairv and Silene latifolia Poiret, have been model organisms for many past study questions in ecology and evolutionary biology and this wide breadth of literature made them an attractive model species pair for the study. There is existing research ranging from descriptions of life history and ecological observations for