Leaf Form and Photosynthetic Physiology of Dryopteris Species Distributed Along Light Gradients in Eastern North America

Functional Ecology 2014, 28, 108–123 doi: 10.1111/1365-2435.12150 Leaf form and photosynthetic physiology of Dryopteris species distributed along light gradients in eastern North America

Emily B. Sessa*,† and Thomas J. Givnish Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, WI 53706 USA

Summary 1. Despite the ubiquity of ferns and at least tacit recognition by botanists that their physiology is unique among land plants, most studies on fern physiology have focused on only a few, locally distributed, usually distantly related species. No previous study has attempted to examine physiological adaptations in a group of widespread taxa that are closely related and whose relationships are well understood. Here we report leaf form and physiological measures for such a group, the 11 eastern North American species of Dryopteris (Dryopteridaceae), and examine differences in these parameters for evidence of adaptation to light availability. 2. Economic theory predicts that species from sunnier habitats should have narrower, more steeply inclined leaves, lower specific leaf area, higher stomatal density, higher rates of maximum photosynthesis, respiration and stomatal conductance, and require more light to saturate photosynthesis. Species should show adaptive crossover in net carbon uptake per unit leaf mass, with a relative advantage by sun-associated species at high photon flux densities (PFDs) and by shade-associated species at low PFDs. 3. Field studies allow us to begin characterizing the range of native light environments occupied by members of this group, and to examine interspecific variation in several aspects of leaf form and photosynthetic light response for evidence of adaption to light availability. We also present a novel means for incorporating phylogeny in tests of correlated evolution in a reticulate lineage. 4. Synthesis. Observed trends in physiology and morphology generally agree with qualitative predictions but are often not statistically significant. We found no support for adaptive crossover in mass-based carbon uptake, and thus for light availability being the most important var- iable driving morphological and physiological adaptation in these ferns. We propose that hydraulic factors related to water balance may have played a larger role in determining their morphological and physiological variation. Allopolyploid hybrids did not show transgression in any physiological parameter that may have allowed them to coexist regionally with their parents. The results of our phylogenetically structured analyses highlight the importance of incorporating phylogeny into comparative studies, particularly when hybrid or polyploid taxa are present.

Key-words: comparative methods, ferns, gas exchange, hydraulics, light regimes, photosynthetic light response, polyploidy, transgressive trait expression

vascular plants. They are second only to angiosperms in Introduction species diversity among land plants (Smith et al. 2006) and Ferns bridge a critical functional gap in land plant evolu- inhabit a large range of climates, substrates and light tion between nonvascular bryophytes and seed-bearing regimes, both in habitats where flowering plants dominate and where few angiosperms can survive (Page 2002). *Correspondence author. E-mail emilysessa@ufl.edu Despite their broad ecological success and key position in †Present address. Department of Biology, University of Florida, land plant evolution, relatively little is known about the Gainesville, FL 32611, USA. physiological traits that have driven fern diversification or

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society Leaf form and photosynthesis in Dryopteris 109 allowed them to persist despite limiting constraints of their constraint on survival and competitive success under dif- biology. Ferns are characterized ecologically by an inde- ferent light regimes than variation in photosynthetic pendent, ephemeral gametophyte stage (Watkins, Mack & parameters. A comparative study of the evolution of pho- Mulkey 2007), low rates of photosynthesis, vascular tissue tosynthetic parameters among related fern species native development and leaf hydraulic conductance (Brodribb & to different light regimes should thus be illuminating, Holbrook 2004; Brodribb, Feild & Jordan 2007; Boyce regardless of whether the traditional patterns seen in flow- et al. 2009; Watkins, Holbrook & Zwieniecki 2010), rela- ering plants are observed in ferns or not. tively high photosynthetic capacity in low light (Page A better understanding of traits that drive adaptation to 2002), slow plant growth rates (Page 2002) and low stoma- different environments and lead to niche differentiation tal densities (Franks & Farquhar 2006; Kessler et al. may also allow us to evaluate possible mechanisms for the 2007). persistence of hybrid and polyploid taxa, which are extre- Biologists have traditionally viewed ferns as once-domi- mely common in ferns (Manton 1950; Wood et al. 2009). nant organisms of ancient ecosystems that were quickly Newly formed polyploids are subject to minority cytotype out-competed and relegated to the shadows following the exclusion, in which polyploid cytotypes experience a fre- evolution of seed plants in the Devonian, and especially quency-dependent mating disadvantage and waste repro- after the rise of flowering plants in the Cretaceous (Niklas, ductive effort in ineffective matings with the established Tiffney & Knoll 1983; Lupia, Lidgard & Crane 1999; Nag- parental cytotype (Levin 1975). Niche differentiation alingum et al. 2002). Recent research has challenged this between progenitors and offspring has been suggested as a view (Rothwell 1996), and studies based on fossil and potential mechanism allowing polyploids to persist region- molecular dating evidence have demonstrated that ferns ally with their parental species on evolutionary time-scales underwent their major radiation into modern lineages only (Rodriguez 1996). In particular, ploidy increases are likely after the appearance of the angiosperms in the Cretaceous to increase cell volume (Melaragno, Mehrota & Coleman (Schneider et al. 2004; Schuettpelz & Pryer 2009). Recent 1993; Comai 2005), potentially increasing values for traits research has identified several physiological features of such as specific leaf area (SLA, cm2 g1), as well as tra- ferns that may have contributed to their success in various cheid diameters and thus hydraulic conductance. Such terrestrial ecosystems, including cavitation-resistant, tra- increases may have important implications for light cheid-based xylem (Watkins, Holbrook & Zwieniecki 2010; capture and water loss, potentially making hybrids less Pittermann et al. 2011), a host of adaptations to xeric hab- susceptible to internal self-shading and drought. itats (Hietz 2010), and chimeric photoreceptors that The 11 eastern North American members of the wood- enhance plastid movement in response to light at low flu- fern genus, Dryopteris, are an ideal group for addressing ences (Kawai et al. 2003; Suetsugu et al. 2005; Kanegae these questions. These species vary extensively in leaf mor- et al. 2006). Chimeric photoreceptors combine phyto- phology and habitat, from species found only on brightly chrome and phototropin functionality to allow red light, lit, well-drained cliff faces and possessing narrow, short, which is typically enriched relative to blue light in forest erect fronds (e.g. D. fragrans), to species of heavily shaded, understoreys (Endler 1993), to trigger phototrophic mesic forest understoreys that often have relatively broad, responses and chloroplast relocation, which are typically tall, sprawling fronds (e.g. D. goldiana; Fig. 1). Some of initiated only by blue or UV light. Arens (1997) found that these species also occur in Europe and Asia, but eight are several leaf characters in tree ferns are strongly correlated endemic to eastern North America. Sessa, Zimmer & with light environment; together these data suggest that Givnish (2012a) recently demonstrated that the 11 North traits related to light response may have played an impor- American species are not each others’ closest relatives, and tant role in shaping fern evolution. However, no study has that each of the six diploids in the group is more closely yet attempted to test the extent to which adaptation to dif- related to Asian or European species than to other North ferent light environments may drive the evolution of mor- American diploid taxa. The remaining five eastern North phological and physiological characters within a fern American species are allopolyploids, formed over the last lineage, or to trace the evolution of such traits in a phylo- 13 million years via hybridization and genome doubling genetic framework. A high sensitivity of plastid movement involving three of the extant North American diploids and to light, for example, opens the possibility that tight corre- one extinct progenitor (Sessa, Zimmer & Givnish 2012b; lations among the photosynthetic parameters Amax, R and Table 1). Tani & Kudo (2003, 2005), and Tessier & Bornn k (maximum photosynthetic rate, dark respiration rate and (2007) have examined biomass allocation and the contribu- the amount of light required to half-saturate photosynthe- tions of overwintering leaves to carbon uptake in several sis, respectively) seen in many angiosperms (e.g. see Dryopteris species, and Runk,€ Zobel & Zobel (2010) and Bjorkman€ 1981; Givnish 1988; Walters & Reich 1996; Runk€ & Zobel (2007) have compared growth and survival of Givnish, Montgomery & Goldstein 2004; Chen et al. 2011) a few European species as a function of edaphic conditions might break down in ferns, with plastid motion affecting and light availability, but no rigorous study of photosyn-

Amax strongly and k and R more weakly, if at all. Alterna- thetic adaptations to light regime has been undertaken for tively, the low leaf hydraulic conductance of ferns may the North American group, despite their strikingly diﬀerent make diﬀerential modulation of hydraulic traits a greater habitats and leaf morphologies.

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k)

Fig. 1. Eleven species of eastern North American Dryopteris included in the current study. (a) D. campyloptera, (b) D. carthusiana, (c) D. celsa, (d) D. clintoniana, (e) D. cristata, (f) D. expansa, (g) D. fragrans, (h) D. goldiana, (i) D. intermedia, (j) D. ludoviciana, (k) D. marginalis.

Table 1. North American allopolyploid Dryopteris species and In this paper, we present gas exchange data from ﬁeld their putative progenitors, with ploidy indicated. The ploidy of the studies and tests for correlated evolution of several mor- extinct progenitor “D. semicristata” is unknown, but it is hypothe- sized to have been diploid (Montgomery & Wagner 1993) phological and physiological traits with light regime, which we predict based on the following three hypotheses: Allopolyploid Putative parents 1. Species from brighter environments should have narrower, more steeply inclined leaves, lower SLA D. campyloptera (49) D. expansa (29), D. intermedia (29) 2 1 9 9 (cm g ) and higher maximum photosynthetic rates D. carthusiana (4 ) D. intermedia (2 ), “D. semicristata” l 2 (29) (Amax, mol CO2 m per s), light compensation points D. celsa (49) D. goldiana (29), D. ludoviciana [the instantaneous light compensation point, ICP and (29) the leaf compensation point, LCP, the latter incorporat- D. clintoniana (69) D. cristata (49), D. goldiana (29) ing night-time respiration and leaf construction costs, 9 9 D. cristata (4 ) D. ludoviciana (2 ), “D. semicristata” both measured in photon ﬂux density (PFD, lmol pho- (29) tons m2 per s)], dark respiration rates (R, lmol

2 CO2 m per s), maximum stomatal conductances (gs, Goldstein 2004). In C3 plants, stomatal conductance (gs) – 2 2 mol H2Om per s) and stomatal densities (per mm ) determined by stomatal size, depth and density per unit compared to species from shadier environments area (Franks & Beerling 2009b; Franks, Drake & Beerling (Bjorkman,€ Ludlow & Morrow 1972; Bjorkman€ 1981; 2009; Taylor et al. 2012) – is expected to be greater in Givnish 1988; Givnish, Montgomery & Goldstein 2004); brighter, moister and/or more fertile microsites (Givnish & 2. Species should show adaptive crossover in net carbon Vermeij 1976; Cowan & Farquhar 1977; Wong, Cowan & uptake per unit leaf mass as a function of PFD, with Farquhar 1979; Farquhar & Sharkey 1982; Givnish 1986; sun-associated species outperforming others at high Franks & Beerling 2009a; Taylor et al. 2012) in order to PFDs and shade-associated species outperforming oth- maximize photosynthetic rate at a given rate or cost of ers at lower PFDs (Bjorkman,€ Ludlow & Morrow 1972; water loss, and lower in drier microsites in order to protect Givnish 1988, 1995; Givnish, Montgomery & Goldstein the hydraulic pathway leading from the roots to the leaves 2004); and from cavitation (Brodribb & Holbrook 2004, 2006). Across

3. Allopolyploid species should exhibit physiological, C3 plants, stomatal density and individual size are nega- morphological or ecological traits that fall outside the tively correlated, and high stomatal conductance is posi- range of variation exhibited by their parental taxa [i.e. tively correlated with stomatal density and photosynthetic they should be transgressive relative to their progenitors capacity (Franks & Beerling 2009a,b; Franks, Drake & (Rieseberg, Archer & Wayne 1999)], and doing so may Beerling 2009; Taylor et al. 2012). In general, we might enable them to coexist regionally with those progenitors expect photosynthesis per unit leaf mass to be higher in in the long term. species native to sunnier microsites in comparisons made at higher PFDs, and higher in species from shadier The rationale for these predictions is as follows: in microsites in comparisons made at lower PFDs (Bjorkman,€ brightly lit sites with high heat loads and/or vapour pres- Ludlow & Morrow 1972; Givnish 1988, 1995; Givnish, sure deficits, plants must balance increased rates of photo- Montgomery & Goldstein 2004). synthetic carbon gain due to increased light and For hybrid allopolyploid taxa, it is possible that the temperature with greater water loss and low leaf water expression of certain morphological, physiological or eco- potentials, which can lead to desiccation. Narrower leaves logical traits extends significantly beyond that seen in one are expected in such environments in order to maintain or both parents. Successful polyploids are often those that high rates of photosynthesis while reducing water loss and inhabit an ecological niche different from either parent high leaf temperatures (Givnish & Vermeij 1976; Givnish (Schwarzbach, Donovan & Rieseberg 2001; Maherali, 1979), as are more steeply inclined leaves, which reduce Walden & Husband 2009), or that display phenotypic traits heat load and transpiration by decreasing the angle of inci- which are outside the range exhibited by the parents [i.e. dent radiation on the leaf (Givnish 1979). For deeply transgressive character expression (Rieseberg et al. 2003)], divided foliage, however, such as most Dryopteris share, especially if their distributions overlap. The conspicuous the main effect of shorter, narrower fronds may be to success of allopolyploid taxa in the Dryopteris complex, reduce the drop in local leaf water potential across the several of which occupy ranges that are transgressive rela- length and breadth of the frond, favouring narrow, short tive to one or both parents (Sessa, Zimmer & Givnish fronds in sunny microsites with high evaporative loads, 2012b), may be due to such transgressive expression, which and broader, taller fronds in shadier microsites. Greater would have allowed them to avoid minority cytotype exclu- stature should also increase competitive ability, especially sion following polyploidization (Abbott & Lowe 2004; in sites with dense coverage by competitors (Givnish 1982, Quintanilla & Escudero 2006; Jimenez et al. 2009). 1995). SLA is a measure of leaf area per unit dry mass; Here we present data on native light regime, leaf form thicker leaves or leaves with denser tissue have more dry and photosynthetic light response from field studies of 11 biomass per unit area and thus lower levels of SLA (Evans Dryopteris species in eastern North America. These data & Poorter 2001; Wright et al. 2004; Shipley et al. 2005). allow us to test the hypotheses described above, in order SLA is positively correlated with high photosynthetic rates to determine whether leaf traits and photosynthetic param- per unit leaf mass and high rates of whole-plant growth eters show the expected patterns of correlated evolution (Shipley 1995; Kruger & Volin 2006; Shipley et al. 2006) with photosynthetic light availability in these species’ and is expected to decline with increasing light and native habitats. decreasing moisture availability (Givnish 1978, 1988; Ack- erly et al. 2002). According to classical economic theory (Bjorkman€ 1981; Givnish 1988), maximum photosynthetic Materials and methods rates (Amax) are expected to be higher in species from brighter environments, as are dark respiration rates (R), SPECIES, STUDY SITES AND LIGHT REGIMES the amount of light required to half-saturate photosynthe- For 10 species of eastern North American Dryopteris – D. campy- sis (k) and the amount of light needed to balance leaf loptera*, D. carthusiana*, D. celsa*, D. clintoniana*, D. cristata*, respiration on an instantaneous basis (ICP, instantaneous D. expansa, D. fragrans, D. goldiana, D. ludoviciana, and D. mar- light compensation point; Givnish, Montgomery & ginalis – we located one field population and measured 5–10 plants

Table 2. Locations of ﬁeld populations of Dryopteris measured in this study

Species Location of study site GPS coordinates

D. campyloptera Bear Pen Gap, Blue Ridge Parkway, North Carolina 35°19′16″N, 82°53′25″W D. carthusiana Huron Mountains, Big Bay, Michigan 46°53′22″N, 87°53′56″W D. celsa Private property, Hertford, North Carolina 36°13′46″N, 76°33′53″W D. clintoniana Thurber Preserve, Dryden, New York 42°33′17″N, 76°″W D. cristata Huron Mountains, Big Bay, Michigan 46°53′22″N, 87°53′56″W D. expansa Chequamegon-Nicolet National Forest, Wisconsin 46°17′46″N, 90°49′40″W D. fragrans Huron Mountains, Big Bay, Michigan 46°53′08″N, 87°54′22″W D. goldiana Fernwood Gardens, Niles, Michigan 41°51′55″N, 86°20′52″W D. intermedia Huron Mountains, Big Bay, Michigan 46°53′22″N, 87°53′56″W D. ludoviciana Rocky Hock Swamp Preserve, Hertford, North Carolina 36°11′19″N, 76°40′37″W D. marginalis Private property, Madison, New York 42°52′07″N, 75°30′57″W

in each population (Table 2). For an eleventh species, D. interme- MORPHOLOGICAL AND FUNCTIONAL TRAIT dia, we made measurements on 10 individuals at two separate populations in the Huron Mountains in Michigan. Allotetraploid MEASUREMENTS species are indicated by asterisks. Parental taxa are listed in We measured the length and width at the widest section of each Table 1. fully expanded, mature leaf and analysed these as the ratio frond We assessed light regimes in the field using digital hemispheric narrowness (length/width), with larger values describing relatively photographs taken with a Nikon Coolpix 5400 digital camera longer, narrower fronds. We used a clinometer (Suunto, Vantaa, (Melville, NY, USA) with a fisheye lens attachment. Photographs Finland) to measure the angle of inclination away from vertical of were taken immediately above each plant and were analysed using each leaf tip, with vertical = 0° and horizontal = 90°. Values >90° Gap Light Analyzer (Frazer et al. 1999) to estimate the percentage describe leaves whose tips drooped below horizontal. The total of total (direct plus indirect) light transmittance through the width and height of each plant were also measured and analysed canopy. as the ratio plant narrowness, similar to frond narrowness, which effectively integrates frond angles and lengths over a whole plant. Individuals with higher plant narrowness values tend to be taller GAS EXCHANGE MEASUREMENTS than wide and have leaves held at steeper angles on average, while We measured photosynthetic light response on attached, fully plants with lower values tend to be broader than tall and have expanded leaves using an LI-6400 portable photosynthesis system leaves that lie at an angle approaching parallel with the ground. (Li-Cor, Lincoln, NE, USA) equipped with a red/blue LED light We used negative epidermal impressions taken from one pinna source and a CO mixing system. All measurements were made (leaf division) per plant with clear nail polish to calculate stomatal 2 2 under ambient relative humidity (c.55–65%), leaf temperature (c. densities. The numbers of stomata per mm were counted at five 20–26 °C) and CO (400 lmol per mol) between 07.00 and locations on each epidermal impression and averaged. We mea- 2 2 1 13.00 h. Leaves were clamped into the measurement cuvette and sured SLA (cm g ) by removing and photographing against a allowed to equilibrate at a moderate light intensity (200 lmol m2 cm scale a single pinna from each plant. Pinnae were then dried in per s). We then measured photosynthesis at the following PFD silica gel and weighed using a Sartorius electronic balance = values: 450, 750, 1000, 500, 260, 150, 90, 60, 40, 20, 10 and (precision 0 00001 g, Goettingen, Germany). Areas of pinnae 0 lmol m2 per s and fit these data to the Michaelis–Menten were calculated from photographs using IMAGEJ software (Abram- eqn (1) following Givnish, Montgomery & Goldstein (2004). off, Magelaes & Ram 2004). Data are archived in DRYAD (doi: 10.5061/dryad.38h06).

AðPFDÞ¼AmaxPFD=ðk þ PFDÞR eqn 1 STATISTICAL ANALYSES where Amax – R is the asymptotic maximum rate of net photosynthesis per unit leaf area, R is the rate of dark respiration, k is the We performed all statistical analyses in R (R Development Core amount of light required to half-saturate photosynthesis, and all Team 2008). We first conducted standard linear regressions for the 2 parameters have units of lmol m per s. Amax, R and k were fit means of each morphological and physiological trait per species as to the light response data using a three-parameter Newton–Raph- a function of per cent canopy openness, and for all traits against son approach (Givnish, Montgomery & Goldstein 2004). R values each other; significance at P = 005, 001 and 0001 indicated by *, were normalized to 25 °C using a standard Q10 value of 2. We **, and ***. Data for the two populations of D. intermedia were calculated the ICP (lmol PAR m2 per s) by setting A(PFD) = 0 not statistically significantly different and so were combined prior and solving for PFD in eqn (1). Leaf compensation points (LCPs, to regression analyses, as were data for the dimorphic sterile and lmol PAR m2 per s) were calculated using the same equation, fertile leaves of D. cristata. following (Givnish, Montgomery & Goldstein 2004) to adjust the When making interspecies comparisons, phylogenetic noninde- value of R to account for night-time leaf respiration and leaf con- pendence can lead to an overestimation of degrees of freedom in struction costs. We obtained mass-based values of Amax (Amax, statistical analyses when species are treated incorrectly as fully 2 2 mass, nmol CO2 g per s) and R (Rmass nmol CO2 g per s) by independent units (Felsenstein 1985). We therefore employed the multiplying the area-based photosynthetic values by SLA phylogenetically independent contrast (PIC) method (Felsenstein 2 1 (cm g ). We also recorded maximum stomatal conductance (gs, 1985) to determine whether correlations remained significant when 2 mol H2Om per s) for each individual. Data are archived in phylogeny was taken into account. The PIC method incorporates DRYAD (doi: 10.5061/dryad.38h06). branch lengths from a phylogenetic tree to account for relation-

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 28, 108–123 Leaf form and photosynthesis in Dryopteris 113 ships, which we derived from the multilabelled phylogeny pro- from 129 14% for D. goldiana to 356 19% for duced by Sessa, Zimmer & Givnish (2012b) based on the biparen- D. clintoniana (Table 3). Of the 13 traits measured, most tally inherited nuclear marker gapCp (Fig. 2). In order to were positively correlated with canopy transmittance standardize branch lengths, we added a small value (00001) to edges with negligible (≤000000001) branch lengths, so that across species in nonphylogenetically corrected analyses, unequal weight would not be given to contrasts involving shorter but the correlation was significant only for Amax branches (Garland, Harvey & Ives 1992). Tetraploid and hexa- (r = 052*) with gs (r = 058*; Fig. 4, Table 4). Several ploid taxa possess two and three copies, respectively, of nuclear other traits were significantly correlated with each other, loci due to whole-genome duplication and are thus represented by including A with k (r = 081**), frond narrowness multiple tips on the gapCp tree. If left uncorrected, this multila- max = * = *** belled nature would lead to each tetraploid taxon being inter- (r 0 69 ) with gs (r 0 94 ); Rmass with Amax, mass preted as two separate species and each hexaploid as three (r = 044*); R and Rmass with each other (r = 088***) and separate species during calculation of contrasts. To avoid this, we each with ICP (R, r = 089***; Rmass r = 074**) with LCP used a novel repetitive process to perform contrast analyses, con- (R, r = 083**; Rmass r = 072*); SLA with Amax, mass sidering each possible combination of polyploid positions on the (r = 072**), R (r = 066*), frond narrowness tree. In the first step, tips were dropped from the tree at random mass = ** = * such that polyploids were represented by only one tip each (r 0 72 ), gs (r 0 54 ) with average stomatal den- (Fig. 3). Contrasts were then calculated for per cent canopy open- sity (r = 076**); ICP with LCP (r = 098***); plant nar- ness and each of the dependent trait variables using this random rowness with leaf tip angle (r = 064*); and gs and k each tree, and a standard linear regression carried out on the contrasts with frond narrowness (gs, r = 075**; k, r = 066*), and and forced through the origin (Garland, Harvey & Ives 1992). with each other (r = 086***; Table 4). There was no evi- P-values were stored at each step and the process repeated 10 000 times to ensure that all possible binary tree topologies (48) were dence of adaptive crossover in static light response curves sampled in the contrast analyses. We produced histograms of on either a mass or area basis; species from the brightest P-values for contrasts of each trait against contrasts of per cent environments consistently outperformed species from the canopy openness in order to assess the frequency of regression shadiest environments (Fig. 5). Average SLA did not differ analyses that produced significant vs. nonsignificant results. Val- significantly between polyploid (382 39 cm2 per g) and ues of the correlation coefficient (r) for each of the 10 000 repeti- 2 = tions were also recorded, in order to determine the percentage of diploid (298 49 cm per g) taxa (P 0 23). correlations for each set of contrasts that were positive or negative. In the phylogenetically structured analyses, we found Finally, we performed a one-tailed t-test to determine a significant correlation (P ≤ 005) between trait and whether SLA differed significantly between polyploid and diploid canopy transmittance contrasts in a majority of the species. 10 000 regression analyses on binary trees for Amax (58% with P ≤ 005), SLA (61% with P ≤ 005) and k (61% with P ≤ 005; Table 5, Appendix S1 in Support- Results ing Information). Contrasts for each of the other 10 In the field, Dryopteris occupies a range of light regimes, traits were not significantly correlated with per cent can- with canopy transmittance varying 28-fold across species, opy transmittance contrasts for a majority of repetitions.

D. cristata B D. clintoniana A D. celsa B D. ludoviciana D. celsa A D. goldiana D. clintoniana B D. expansa D. campyloptera B D. carthusiana A Fig. 2. Initial phylogenetic tree used in the phylogenetically independent contrast D. campyloptera A (PIC) analyses, based on a maximum likeli- D. intermedia hood topology from Sessa, Zimmer & Givnish (2012a). A, B, C following taxon D. carthusiana B names denote multiple genomes present in D. cristata A polyploid species. Tips were dropped ran- domly in the regression analyses so that D. clintoniana C each polyploid was represented by only one tip at a time (see Fig. 3). Branches are pro- D. marginalis portional to expected number of nucleotide D. fragrans substitutions per site. Bar indicates 001 changes. 0·01

D. ludoviciana D. celsa A D. goldiana D. clintoniana B D. expansa D. campyloptera B D. carthusiana A D. intermedia Fig. 3. One random binary tree (out of 48 D. cristata A possible trees) used in the phylogenetically D. marginalis independent contrast (PIC) calculations for D. fragrans the ﬁeld studies. Branches are proportional to expected number of nucleotide substitu- 0·01 tions per site. Bar indicates 001 changes.

Table 3. Environmental, morphological, photosynthetic and functional traits measured in the ﬁeld. Means standard errors are given. No stomatal density data were collected for D. fragrans due to density of abaxial trichomes

LCP (lmol Amax, mass 2 % Canopy Amax (lmol CO2 R (lmol CO2 k (lmol PAR ICP (lmol PAR PAR m (nmol CO2 Species transmittance m2 per s) m2 per s) m2 per s) m2 per s) per s) g2 per s)

D. campyloptera 280 13400 04030 004 294 48235 0543 093 190 27 D. carthusiana 205 20527 05033 009 444 58266 055 49 110 223 21 D. celsa 249 34245 03045 024 278 38763 506 2208 1726 84 6 D. clintoniana 356 19669 05046 005 413 38312 043 569 082 160 14 D. cristata 253 12596 03039 005 499 43348 051 634 097 259 15 D. expansa 174 14411 04103 023 298 461012 273 2575 847 202 16 D. fragrans 292 25713 04047 006 643 63441 06799 11 115 31 D. goldiana 129 14353 04032 007 284 43296 079 57 172 123 15 D. intermedia 212 16494 02067 011 464 30830 186 1843 489 155 11 D. ludoviciana 229 27486 03044 007 324 29311 033 577 07 100 7 D. marginalis 326 14499 02016 002 283 18094 011 166 02 126 5

Average Plant Frond

Rmass (nmol CO2 stomatal narrowness narrowness Leaf tip gs (mol H2O g2 per s) SLA (cm2 g1) density (mm2) (height:width) (length:width) angle (0–90°) m2 per s)

D. campyloptera 141 26 452 18 327 18062 006 201 003 876 10006 001 D. carthusiana 146 41 433 33 273 13081 003 271 004 650 17007 001 D. celsa 132 51 341 23 369 20098 006 325 007 623 38003 00 D. clintoniana 113 17 246 14 398 14127 011 401 008 450 4101 001 D. cristata 172 23 439 15 349 18120 008 430 009 553 22009 001 D. expansa 515 116 497 27 347 19091 006 223 004 560 22004 001 D. fragrans 73 18 155 35 – 064 005 477 010 852 36014 001 D. goldiana 114 27 356 17 282 14077 003 272 005 700 29004 00 D. intermedia 182 22 316 20 442 17076 003 303 003 638 12006 00 D. ludoviciana 89 13 211 6521 29129 018 399 010 749 35007 00 D. marginalis 40 04 256 12 544 38065 004 333 003 720 19007 001

ICP, instantaneous compensation point; LCP, leaf compensation point; SLA, speciﬁc leaf area.

For all traits, regressions on binary trees produced both contrasts, while the unstructured analysis produced a positive and negative correlations between trait contrasts positive correlation (Tables 4, 5). and native per cent canopy transmittance contrasts (Table 5). For most traits, the direction of the correla- Discussion tion (either positive or negative) for the majority of repetitions was the same as in the phylogenetically CORRELATED EVOLUTION OF PHOTOSYNTHETIC unstructured analyses. For three traits – A , LCP and max PARAMETERS AND LEAF TRAITS WITH LIGHT REGIME ICP – exactly half of the correlations were positive and half negative, and for one trait – k – the majority of Our ﬁeld measurements indicate that trends in photosyn- repetitions in the phylogenetically structured analyses thetic, morphological and ecological traits in eastern produced negative correlations with light availability North American Dryopteris generally accord with

(a) (b) D. campyloptera D. carthusiana D. celsa D. clintoniana

) D. cristata 1

− D. expansa s

D. fragrans ) 2 1 − D. goldiana − m s

D. intermedia 2 2

D. ludoviciana −

D. marginalis m

mol mol (µ (µ R max A 0·2 0·4 0·6 0·8 1·0 1·2 234567

10 15 20 25 30 35 40 10 15 20 25 30 35 40 % Canopy transmittance % Canopy transmittance

2 s

− 2 − m

mol mol (µ (µ k ICP 30 40 50 60 70 2 4 6 8 10 12

10 15 20 25 30 35 40 10 15 20 25 30 35 40 % Canopy transmittance % Canopy transmittance

(e) (f) ) 1 − s

2 ) − 1 g −

s 2

2 − CO

mol nmol ( (µ LCP mass

max A 100 150 200 250 010203040

10 15 20 25 30 35 40 10 15 20 25 30 35 40 % Canopy transmittance % Canopy transmittance

Fig. 4. Field measurements of gas exchange, morphological and functional traits plotted against per cent canopy transmittance. Species means SE are shown, and standard linear regression lines are plotted for those traits that were significantly correlated with per cent canopy transmittance. (a) Amax; (b) R; (c) k; (d) ICP; (e) LCP; (f) Amax, mass (g) Rmass; (h) specific leaf area (SLA); (i) average stomatal density; (j) plant narrowness; (k) frond narrowness; (l) leaf tip angle; (m) gs. Data points are coloured by species according to the legend in (a); polyploids are shown as squares, diploids circles (For interpretation of the references to colour in this figure legend, the reader is referred to the online version of this article.). ICP, instantaneous compensation point; LCP, leaf compensation point. theoretical predictions based on classical economic theory longer than broad; Fig. 4). We did not find significant (Bjorkman,€ Ludlow & Morrow 1972; Bjorkman€ 1981; statistical support for most of these results, however, as

Givnish 1988; Givnish, Montgomery & Goldstein 2004). only Amax and gs are significantly correlated with light Species from sunnier habitats tend to have higher photo- availability in the phylogenetically unstructured analyses, synthetic and stomatal conductance rates per unit area, the remaining correlations being in the expected direc- require more light to saturate photosynthesis, have lower tions but not achieving statistical significance at the 5% SLAs, higher stomatal densities and increased frond nar- level (Table 4). In the phylogenetically structured analy- rowness (the latter indicating that leaves were generally ses, Amax is again significantly correlated with per cent

(g) (h) ) 1 − ) s

1 2 − − g

2 cm (

nmol (

SLA mass R 200 300 400 500 10 20 30 40 50 60

10 15 20 25 30 35 40 10 15 20 25 30 35 40 % Canopy transmittance % Canopy transmittance

(i) (j) 2 − mm

stomata

number

Plant narrowness (height:width) Average 30 35 40 45 50 55 0·6 0·8 1·0 1·2 1·4

10 15 20 25 30 35 40 10 15 20 25 30 35 40 % Canopy transmittance % Canopy transmittance

(k) (l) Leaf tip angle Frond narrowness (length:width) 40 50 60 70 80 90 2·0 2·5 3·0 3·5 4·0 4·5 5·0

10 15 20 25 30 35 40 10 15 20 25 30 35 40 % Canopy transmittance % Canopy transmittance (m) ) 1 − s

2 − 0·10 0·12 0·14 m

mol (

s g 0·06 0·08 0·04

10 15 20 25 30 35 40 % Canopy transmittance

Fig. 4. Continued.

Table 4. Correlations among all traits measured in the ﬁeld

Average stomatal Plant Frond Leaf tip

Amax RkICP LCP Amax, mass Rmass SLA density narrowness narrowness angle gs

Canopy 052 037 022 040 038 009 046 047 048 010 047 001 058

Amax – 007 081 032 044 027 024 046 022 015 069 008 094 R –– 006 089 083 016 088 028 009 017 018 038 014 k –– – 000 013 024 018 034 013 003 066 004 086 ICP –– – –098 002 074 027 012 004 022 033 031 LCP –– – – –008 072 030 011 004 028 034 041

Amax, mass –– – – –– 044 072 049 009 021 033 011 Rmass –– – – –– – 066 032 007 047 038 035 SLA –– – – –– – – 076 008 072 024 054 Average ––– –––––– 017 047 010 019 stomatal density Plant ––– –––––– – 043 064 003 narrowness Frond ––– –––––– – – 008 075 narrowness Leaf tip ––– –––––– – – – 008 angle gs ––– –––––– – – – –

ICP, instantaneous compensation point; LCP, leaf compensation point; SLA, speciﬁc leaf area. See text and Table 3 for data and units. Signiﬁcant correlations (P ≤ 005) are shown in bold. canopy transmittance in a majority of analyses, as are eqn (1) (Farquhar & Sharkey 1982): when maximum pho-

SLA and k. Amax is thus the only trait to be strongly tosynthetic rate increases, so too should the PFD required correlated with light availability in both the phylogeneti- to half-saturate photosynthesis. R and Rmass are also both cally structured and unstructured analyses. Our measures strongly positively correlated with ICP and LCP, and the of plant stature (plant narrowness and leaf tip angle) PFDs at which carbon consumed by respiration and pro- show very little or no correlation with PFD (Fig. 4, duced by photosynthesis are balanced. As respiration Table 4). increases (i.e. more carbon is lost to respiration), the

The values of Amax and other photosynthetic parameters amount of light required to balance carbon consumption that we report here for Dryopteris are similar to those that must increase. ICPs and LCPs range from 001% to 02% have been found in ferns previously. Amax in our species full sunlight and are each well below the PFDs necessary 2 ranged from 25 03to71 04 lmol CO2 m per s, to partly saturate photosynthesis. Both are negatively cor- which corresponds roughly to the middle of the range of related with PFD, which is contrary to expectations, as the

Amax values that have been reported for other fern genera amount of light required to balance respiration should (Ludlow & Wolf 1975; Nobel, Calkin & Gibson 1984; increase with daily PFD to which a species is adapted Hollinger 1987; Brodribb, Feild & Jordan 2007). Previous (Givnish, Montgomery & Goldstein 2004). 2 estimates for Dryopteris range from 18 lmol CO2 m Several photosynthetic parameters show greater integra- per s in Asian D. crassirhizoma (Maeda, 1970) to 40 lmol tion with each other and with other traits when considered 2 CO2 m per s in D. arguta (Pittermann et al. 2011) and on a mass rather than an area basis (Table 4), although 2 90 lmol CO2 m per s in D. filix-mas (Bauer, Gallmetzer only area-based Amax is significantly correlated with native & Sato 1991). Previous researchers have reported instanta- PFD. Because competition should favour plants that neous light compensation points in ferns infrequently, but maximize returns on energy investment (Givnish 1988, our values (Table 3) are somewhat higher than those docu- 1995), and mass-based estimates incorporate energy invest- mented previously for Dryopteris [which ranged from ment in leaf construction, photosynthesis on a mass basis 05 lmol m2 per s in the shade to 17 lmol m2 per s in should be more closely tied to relative performance and the sun (Bannister & Wildish 1982)], and are more similar competition between species than photosynthesis on an to values reported by Hollinger (1987) for two understorey area basis. It is therefore surprising that Amax but not 2 species of Blechnum and Pteridium (73 and 116 lmol m Amax, mass is significantly correlated with native PFD; in per s, respectively). fact, Amax, mass shows almost no relationship to native In addition to the relationships with PFD, several traits PFD (Fig. 4f). However, ferns are known to respond con- are also correlated with each other as expected based on servatively to desiccation (Brodribb & Holbrook 2004), theoretical predictions. Amax is significantly positively cor- and this sensitivity is driven at least in part by low rates of related with k, as expected based on the Michaelis–Menten area-based leaf hydraulic conductance (Woodhouse &

(a) Table 5. Results of independent contrast analyses on binary trees for traits measured in the ﬁeld against native per cent canopy transmittance

P ≤ 005 (%) % Positive

Amax 58 50 R 45 42 k 61 33 ICP 17 50 LCP 17 50

Amax, mass 49 33 Rmass 39 25 SLA 61 8 Average stomatal density 34 81 Plant narrowness 42 55 Frond narrowness 39 59 Leaf tip angle 42 33

gs 42 83 (b) ICP, instantaneous compensation point; LCP, leaf compensation point; SLA, speciﬁc leaf area. Percentages indicate the frequency of binary trees (out of 48 possible topologies) for which P-values were below 005 and r was positive.

photosynthetic parameter but was signiﬁcantly positively

correlated with area-based Amax (Table 4). The lack of broad statistical support for the observed trends in trait correlation with native PFD may reflect several phenomena. First, individual species do not appear to demonstrate the differential adaptation to light regimes we had expected. We saw no evidence of adaptive crossover (Givnish, Montgomery & Goldstein 2004) in static light Fig. 5. Static light response curves of species from the two bright- responses, in which species from sunnier microsites outper- est habitats (D. marginalis and D. clintoniana) and the two shadi- form species from shady habitats at high PFDs, with the est habitats (D. goldiana and D. expansa). (a) area-based curves 2 1 reverse at low PFDs. In contrast, species from sunnier sites (lmol CO2 m per s); (b) mass-based curves (nmol CO2 g per s). Points are mean SE. Curves do not show adaptive crossover, outperformed species from shadier sites at all PFDs, in with species from shady habitats outperforming sun species at low both area-based and mass-based curves (Fig. 5). As noted photon flux densities (PFDs) and the reverse at high PFDs (For above, the absence of the expected mass-based relationship interpretation of the references to colour in this figure legend, the is particularly striking, as these estimates incorporate leaf reader is referred to the online version of this article.). construction costs via SLA and are therefore expected to be more tightly tied to species’ relative competitive abili- Nobel 1982; Brodribb & Holbrook 2004). Our measure- ties. Species from the brightest environments, including ments of maximum stomatal conductance (Table 3) are at D. clintoniana, D. fragrans and D. marginalis, rarely the low end but well within the range of values previously occupy the extremes of any given trait, usually being out- reported for Dryopteris by Pittermann et al. (2011), and performed by species from intermediate habitats (Fig. 4, for other ferns by Hollinger (1987) and Brodribb & Table 3). Such species rarely co-occur with other Dryopter- Holbrook (2004); these estimates range from 001 to is in the field, but apparently this is not due to any advan- 2 023 mol H2Om per s. gs was significantly positively tage such ‘sun’ species have over ‘shade’ species in carbon correlated with native PFD, Amax, k and frond narrow- uptake at high PFDs per se. A likely explanation for the ness, and negatively with SLA, indicating that longer, nar- somewhat lower photosynthetic rates in these species is the rower and thinner leaves tended to have higher maximum much greater evaporative demand in their high-PFD envi- stomatal conductance rates. Low conductance rates will ronments. In ferns – as opposed to angiosperms or gymno- directly influence transpiration and photosynthetic rates, sperms – low hydraulic conductance may strongly restrict which suggests that area rather than mass-based estimates photosynthesis, as well as growth and survival, interfering of these parameters may be more meaningful for ferns, in with the usual rise in photosynthesis with PFD. A second that they incorporate the surface area from which evapora- explanation for the lack of significant patterns across spe- tion proceeds. Maximum stomatal conductance in Dryop- cies related to PFD may be the fact that overall, there was teris is not significantly correlated with either mass-based relatively little variation among species in PFD, with

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 28, 108–123 Leaf form and photosynthesis in Dryopteris 119 canopy openness varying only 28-fold across species. A for our general observation of expected but nonsignificant narrow range of PFD decreases by itself the likelihood of correlations between traits and PFD, and for the lack of significant relationships of individual traits to PFD across expected pairwise performance comparisons at the species species, even if such relationships can be seen for species level between taxa from sun and shade environments. It is inhabiting a broader gradient. We note that many of the important to recognize that several traits expected to show expected positive relationships among plant traits and positive relationships to PFD based on classic economic PFD were observed but were simply not significant; per- theory (i.e. based solely on the putative effects of PFD) haps a greater number of species, or occupancy of a might also show such relationships based on hydraulic broader range of light regimes, might have made these considerations. As we suggested in the Introduction, a patterns significant. greater sensitivity of plastid movement to PFD in ferns

Finally, and perhaps most importantly, many of the might preserve a positive relationship of Amax to PFD traits we examined are likely influenced not just by light while reducing or eliminating that of k and R, by reducing availability but by the correlation of PFD with tempera- self-shading and maintaining a constant plastid mass. ture, vapour pressure deficit, wind speed and other envi- Thicker leaves or leaves with higher mass per unit area ronmental variables. Several previous studies on ferns have may be an adaptation to greater evaporative demand in documented one or more of the expected relationships brighter sites, reducing leaf and evaporative surfaces in among photosynthetic parameters that we see in Dryopter- order to reduce tissue desiccation and preserve xylem func- is. In a study of three species from different genera (Adian- tion (Creese, Lee & Sack 2011). Our gs values were signifi- tum, Thelypteris and Woodwardia) growing in different cantly correlated with PFD (Table 4), but this correlation light regimes, Hill (1972) found that species from sunnier could also have been driven by differences in water avail- habitats had higher maximum photosynthetic rates, light ability between the sites, if brighter habitats happened also compensation points and light saturation points compared to be drier. Conductance in several fern species has been with species from a shadier habitat. Lower compensation reported to be more strongly influenced by soil water sta- points and SLAs in species from shady habitats have been tus than by other variables (Nobel 1978; Hollinger 1987), documented in single species from several fern genera and several recent studies have revealed that fern stomata (Ludlow & Wolf 1975; Bannister & Wildish 1982; Brach, lack many of the active control mechanisms found in an- McNaughton & Raynal 1993), as have higher stomatal giosperms (Doi, Wada & Shimazaki 2006; Doi & Shima- densities (Ludlow & Wolf 1975) in species from sunny zaki 2008; Brodribb et al. 2009; Brodribb & McAdam habitats. Higher photosynthetic capacity with increasing 2011), instead opening and closing passively in response to light availability has also been documented in Blechnum hydraulic changes and leaf water status (Brodribb & (Saldana~ et al. 2009), Adiantum (Yeh & Wang 2000) and McAdam 2011; McAdam & Brodribb 2012). This further in D. intermedia (Brach, McNaughton & Raynal 1993), suggests that hydraulic constraints, rather than responses among others. However, in additional studies examining to PFD per se, may be primarily responsible for shaping fern traits and habitat preference, soil moisture was found morphological and physiological diversity in these ferns. to be a primary factor influencing fern distributions in Indeed, decreased conductance in brighter environments Ohio (Greer, Lloyd & McCarthy 1997) and Quebec (Karst, could countervail the general tendency towards higher

Gilbert & Lechowicz 2005), and Nobel, Calkin & Gibson Amax, k and R and lower SLAs in higher-PFD environ- (1984), in a study of gas exchange in 14 species of ferns in ments. a greenhouse, found that water vapour and CO2 conductance were better predictors of maximum photosynthetic TRANSGRESSIVE TRAIT EXPRESSION IN POLYPLOIDS rates than was PFD. Reudink et al. (2005) noted that D. intermedia growing in a damp, but open site appeared The eastern North American Dryopteris complex includes to experience greater water stress, as indicated by negative five allopolyploids – tetraploids D. carthusiana, D. cristata, water potential, than other species growing in the same D. campyloptera, D. celsa and hexaploid D. clintoniana – environment, and concluded that D. intermedia’s ability to the latter three of which have both parental species extant acclimate to high PFDs was limited, with moisture playing (Sessa, Zimmer & Givnish 2012b). We expected to see evi- a greater role in determining its distribution. Runk,€ Zobel dence of transgressive expression in these species relative & Zobel (2010), in a study of three Estonian species of to their parents in at least some of the traits measured. Dryopteris (including D. carthusiana and D. expansa), Novel or extreme trait expression can allow hybrids to determined that species’ distributions and abundance were escape competition and the effects of minority cytotype most influenced either by edaphic factors or by ‘locale exclusion and will tend to favour their establishment and effects’ that were not specifically related to any one vari- long-term success (Abbott 1992; Rieseberg 1997; Riese- able. Taken together, these results suggest that fern distri- berg, Archer & Wayne 1999; Buerkle et al. 2000; Rieseberg butions and adaptation of specific traits are likely shaped et al. 2003; Quintanilla & Escudero 2006). Two recent by the interaction of a number of environmental factors. studies have also shown that polyploids tend to occupy That light availability is probably joined in its effects by larger geographic ranges than their progenitor taxa moisture availability and evaporative demand may account (Parisod, Holderegger & Brochmann 2010; Parisod 2012).

This is true of D. campyloptera, D. celsa and D. clintoni- which traits can be compared among species grown within ana (Sessa, Zimmer & Givnish 2012b) and suggests that in the same PFD and related to the native PFDs of each some traits they must be transgressive relative to their species in the field (Givnish, Montgomery & Goldstein parents. Sessa, Zimmer & Givnish (2012b) also found 2004), and ‘hard’ tests, which will allow us to ask whether that the allotetraploid hybrids have resulted from crosses species showing the highest realized rates of photosynthesis between diploid parents that display intermediate levels shift with light treatment, and if they are the species of genetic divergence relative to all possible progenitor that occupy habitats with similar native light regimes. Our pairs. Such intermediate levels of parental divergence results also indicate that hydraulic demands may have have been found to be linked to transgressive trait played a key role in the physiological and morphological expression in hybrid angiosperms (Stelkens & Seehausen evolution of these ferns, supporting results from several 2009). recent studies that have found significant differences Contrary to our expectations, we found little evidence between ferns and other land plants in traits related to for transgressive trait expression in any of the three allopo- water balance, including hydraulic conductance, vein lyploids we examined. In almost all traits, measurements density and the nature of fern stomatal responses in the polyploids were intermediate relative to their parents (Brodribb & Holbrook 2004; Brodribb, Feild & Jordan and frequently overlapped significantly with one or both 2007; Boyce et al. 2009; Watkins, Holbrook & Zwieniecki parents (Fig. 4). SLA was also not significantly different 2010; Brodribb & McAdam 2011; McAdam & Brodribb between polyploids and diploids. Transgressive expression 2012). of photosynthetic parameters is therefore evidently not the This study is the first to propose a statistical framework means by which these allopolyploid taxa are being main- for incorporating phylogeny into comparative analyses tained. However, the native PFDs of two of the three when the subjects include species with reticulate evolution- allopolyploids (D. campyloptera and D. clintoniana) were ary histories. Phylogenetic relationships must be consid- transgressive relative to their parental species (Fig. 4), with ered in comparative analyses due to the danger of inflating each allopolyploid occupying a much brighter habitat type I error rates when species are erroneously considered than its parents. Coupled with the transgressive ranges of to be independent samples (Felsenstein 1985). The method these species relative to their parents, this points to the we present allows for the incorporation of phylogeny even existence of physiological differences that provided the in the case of hybridization or polyploidy, by accounting allopolyploid taxa an advantage in geographic range that for all possible sets of relationships (trees) for a given set apparently allowed them to coexist with their progenitors of taxa. The results of the phylogenetically structured and at a regional scale. As discussed above, water balance unstructured analyses presented here are largely congruent mediated by hydraulics is the most likely driving factor. with each other, and this agreement increases our confi- Although we found no significant difference in SLA dence in the results. However, the distribution of negative between polyploids and diploids, this does not rule out the and positive r values (Table 5) obtained from repetitive possibility of an increase in tracheid size due to polyploidy PIC analyses utilizing all possible trees indicates that the that could affect species’ hydraulic conductance and thus placement of hybrid species in the tree can greatly their ability to tolerate brighter habitats. Further studies influence the results of such analyses, and speaks to the of the allopolyploids and their parents, including sampling importance of incorporating phylogeny in comparative from multiple populations of each in the field, will be studies and considering carefully the placement of reticu- needed in order to determine the relative roles of light and late taxa. If we had selected only a single tree, for example, water availability in influencing these species’ evolution. our results for any given trait would have been either 100% positive or negative, when in fact there is a distribution between negative and positive correlations depending Conclusions on which tree is utilized in a given repetition of the analy- The current study is the first to document physiological sis. Many researchers now incorporate phylogeny into and morphological traits in a widespread, closely related their comparative studies of ecological and physiological group of ferns whose phylogeny is well understood, and to traits, and we hope that the method presented here will be attempt to determine whether photosynthetic PFD has useful in such studies involving hybrid and/or polyploid been a primary factor driving adaptation in fern leaf form organisms. and physiology. Although we found little evidence to support PFD as the main determinant of diversification in the traits measured, our results suggest several avenues for Acknowledgements future studies. Common garden studies, in which species The authors thank A. Jandl, T. Goforth, P. Gonsiska, C. Line, J. Perry are grown together and environmental variables can be and K. Woods for help in the field, and the Finger Lakes Land Trust, Nat- carefully controlled, will be particularly important, and ure Conservancy of North Carolina, Huron Mountain Club, Blue Ridge will allow us to more rigorously examine the extent to Parkway, Fernwood Botanical Gardens, and J.E. Watkins, Jr. and J. Perry for allowing us access to field populations. Many thanks to E. Kruger for which species may be adapted to their native light regimes. advice on this manuscript, and to C. Ane for assistance with developing the Such studies will allow both ‘soft’ tests of adaptation, in statistical tools used in this study, particularly for the phylogenetically

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 28, 108–123 Leaf form and photosynthesis in Dryopteris 121 structured analyses of polyploids. Financial support for this research was Creese, C., Lee, A. & Sack, L. (2011) Drivers of morphological diversity and provided by the Huron Mountain Wildlife Foundation (EBS, TJG), a mi- distribution in the Hawaiian fern ﬂora: trait associations with size, growth croMORPH award from the National Science Foundation (EBS), and form, and environment. American Journal of Botany, 98, 956–966. graduate research awards from the Botanical Society of America, the Doi, M. & Shimazaki, K.-I. (2008) The stomata of the fern Adiantum capil-

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