Ecophysiological Diversity

Ecophysiological Studies Overview

In this section of the proposal, we address Question 3 in Table 1.

Is functional diversity of desiccation tolerance evident among lineages and do these differences vary by life phase or sex, providing evidence for fitness tradeoffs over evolutionary time scales?

We expect to uncover a variety of physiological responses to desiccation that serve to identify patterns of diversity among the 15 different North American species of Syntrichia. This includes patterns specific to genotypes (ecotypes) across the region as well as the different life phases and sexes within the species.

Phenotypic plasticity and ecological strategies of DT

Phenotypic plasticity describes the ability of a genotype to express different phenotypes in different environments and can be an adaptive strategy and evolutionary driver to cope with variable environments (Gabriel 2005; Pigliucci 2005; Nicotra et al. 2012). Desiccation tolerance in bryophytes is fundamentally a plastic trait, and bryophytes present a wide range of phenotypes (both plastic and non-plastic) in response to desiccation. Plant responses to surviving desiccation can be described, using norms of reaction (the phenotypes produced by a given genotype in response to systematic environmental variation; Windig et al. 2004), along a continuum from highly plastic to approaching non-plastic (Stark & Brinda 2015). Recent studies demonstrate that bryophytes exhibit both reversible phenotypic acclimation to DT induced by an environmental stimulus (which reverts to the non-DT state when the stress disappears) and non-plastic DT (e.g., Stark et al. 2012; Cruz de Carvalho 2014). By tracing norms of reaction for each factor of DT, we can locate the population or species along this plasticity gradient, derive a quantitative measure of assessing DT and relate it, via qPCR-based “signature transcriptomic assays”, to gene expression states, thereby defining the role of gene expression in plasticity.

Factors of DT

The fitness advantage of reversible phenotypic plasticity has been modeled (e.g., Gabriel 2005) and found to depend upon the environmental cue, the intensity of the stress, the duration of the stress, and the time necessary to reverse the plastic response. The ecological factors of DT have only recently been conceptualized (Green et al. 2011; Stark 2014) or indicated (Pichrtová et al. 2014). These factors reflect the modeled dependent factors of reversible phenotypic plasticity above, and for DT are (1) the Rate of Drying (RoD), (2) equilibrating relative humidity or water content at equilibration (RHeq), (3) Duration Dry (Duration Dry), and (4) Dehardening Time (Deharden T). Our approach will incorporate two factors (RoD and RHeq) that have proven significant in previous experiments.

Ecotype detection via norms of reaction

We will look for ecotypes by testing for differences in reaction norms across the species of Syntrichia by exploring patterns in their responses for each factor of DT (Table 4).

Table 4. Ecophys TreatmentsEach of the specimens collected following our protocol will be subcultured to a single uncontaminated clonal line in the lab (Horsley et al. 2011), thus eliminating the physiological history of desiccation stress expected in field-collected material (Stark et al. 2014). Each of these 40 clones (20 per S. ruralis and 20 per S. caninervis lineage) will be subjected to a DT experiment (N=20) where adult shoots are exposed to each of these factors of drying: (1) RoD min = time required from full turgor to desiccation morphology (leaf curling, Schonbeck & Bewley 1981, which occurs at a RH ~86%, Dilks & Proctor 1979; Proctor 2001) that upon rehydration produces no or a minimum of discernable damage (as assessed using chlorophyll fluorescence) to the shoot or structure. We vary rate of drying by varying the amount of substrate water on filter paper in closed Petri dishes, which correlates well to time at subturgor (Stark et al. 2013), and (2) RHeq min = the lowest water potential tolerated without incurring damage (as assessed using chlorophyll fluorescence), equivalent to the protoplasmic DT, PDT, used in assessing the DT of vascular plants (Gaff & Oliver 2013).

We will create a series of saturated salt solutions equating to a range of descending relative humidities, from 80% to 1% above each solution. Plants are initially desiccated at the highest RH, and then equilibrated at each descending stop for 24 h (modified from Tetteroo et al. 1995; Oldenhof et al. 2006). Thus we have two objective metrics by which to compare the degree of ecological DT across and within species. The use of the qPCR-based “signature transcriptomic assays” will also illuminate the gene expression characteristics of each factor of DT, which in turn can refine the assays for more detailed analysis of the trait and inform each factor of DT. We expect to recover distinct ecotypes of S. caninervis and S. ruralis from throughout North America that exhibit a range of capacities for DT.

Life history phases and assessing degree of DT

An unanswered question relates to how the expression of DT varies with life history phase, i.e., is it possible that different phenophases exhibit different strategies of DT? In Syntrichia pagorum, juvenile shoots exhibit reversible phenotypic plasticity (an environmentally inducible DT phenotype), whereas adult shoots of the same species exhibit a nonplastic (phenotypically constitutive) response (Fig. 2), the first report in vegetative tissues for a bryophyte and only the fourth known instance of this phenomenon (one angiosperm, Vander Willigen et al. 2003, and two ferns, Farrant et al. 2009; Testo & Watkins 2012).

Further, sporophytes of a moss can transition in the same fashion from embryonic to postembryonic phenophases (Stark & Brinda 2015). We will focus on S. caninervis and S. ruralis, using clonal lines described above, and test cultured life phases of protonema, protonemal gemmae (if present), juvenile shoots, adult shoots (male and female), and sporophytes (embryonic and postembryonic, if present). Reaction norms will be determined utilizing the two aforementioned factors of drying on each life phase. The qPCR-based signature transcriptomic assays will also be utilized to assess gene expression as it relates to the plasticity of the DT response in both species at each life stage. We expect to elucidate how the physiological diversity in the trait DT varies with life phase and contributes to the survival and reproductive vigor of these species.

Characterizing the trait DT for 15 species of Syntrichia

North American Syntrichia consists of 15 species (Table 2). We have 10 of these species currently in single clone pure cultures, and will extend this number to cover all 15 species with the assistance of postdoctoral associate Brinda. For at least five single clone cultures of each species derived from a range of environments across North America, we will derive norms of reaction (Fig. 3) using adult shoots for RoD min and RHeq min as described above, and monitor recovery using chlorophyll fluorescence, gas exchange, regenerative growth rate, and gene expression where appropriate. Gas exchange measurements will be performed using a customized bryophyte chamber (Li-Cor 6400-24) attached to an infrared gas analyzer (Li-Cor 6400XT). The data generated by these measurements will be used to infer changes in carbon balance per wetting event following methods proposed by Mishler & Oliver (2009) and recently implemented by Coe et al. (2012a). These quantitative traits of DT will be added to our phylogenetic analysis by mapping them onto the phylogeny for each species and testing the associations among them as described in section D5 below.

Testing the productivity tradeoff hypothesis

This hypothesis predicts that plants incorporating greater degrees of DT will, because of resource limitations on allocation, either grow more slowly or reproduce with less vigor than plants incorporating lesser degrees of DT (Alpert 2006). Conversely, plants with higher inherent growth rates should exhibit greater phenotypic plasticity (less DT) than slower growing counterparts (Herms & Mattson 1992). We will measure degree of DT using the two DT factors described above and predict that, for each species, lower values of RoD min and RHeq will correlate with reduced vegetative growth and reproductive allocation. Assessments will be made by observations of protonemal and shoot production, gemmae production, sexual expression, and sporophyte production in culture (as in Horsley et al. 2011). We plan on testing all 15 North American species in this regard, as well as comparing males and females in their ability to grow and reproduce in the face of desiccation stress. In this fashion, we expect to show that productivity trades off with DT in ways specific to each species.