Leaf optical properties and photosynthesis of fern species with a wide range of divergence time in relation to mesophyll anatomy

Abstract

Background and Aims

For a comprehensive understanding of the mechanisms of changing plant photosynthetic capacity during plant evolutionary history, knowledge of leaf gas exchange and optical properties are essential, both of which relate strongly to mesophyll anatomy. Although ferns are suitable for investigating the evolutionary history of photosynthetic capacity, comprehensive research of fern species has yet to be undertaken in this regard.

Methods

We investigated leaf optical properties, gas exchange and mesophyll anatomy of fern species with a wide range of divergence time, using 66 ferns from natural habitats and eight glasshouse-grown ferns. We used a spectroradiometer and an integrating sphere to measure light absorptance and reflectance by the leaves.

Key Results

The more newly divergent fern species had a thicker mesophyll, a larger surface area of chloroplasts facing the intercellular airspaces (S_c), thicker cell walls and large light absorptance. Although no trend with divergence time was obtained in leaf photosynthetic capacity on a leaf-area basis, when the traits were expressed on a mesophyll-thickness basis, trends in leaf photosynthetic capacity became apparent. On a mesophyll-thickness basis, the more newly divergent species had a low maximum photosynthesis rate, accompanied by a low S_c.

Conclusions

We found a strong link between light capture, mesophyll anatomy and photosynthesis rate in fern species for the first time. The thick mesophyll of the more newly divergent ferns does not necessarily relate to the high photosynthetic capacity on a leaf-area basis. Rather, the thick mesophyll accompanied by thick cell walls allowed the ferns to adapt to a wider range of environments through increasing leaf toughness, which would contribute to the diversification of fern species.

INTRODUCTION

Photosynthesis is a key function for plant growth and survival, and a wide range of variation in the rate of photosynthesis, more than 100-fold, has been obtained on a leaf-mass basis across C₃ plant species (Wright et al., 2004). In the evolutionary history of land plants, plant photosynthetic capacity overall has increased with evolution, namely, in order of the earliest plants such as mosses and bryophytes, to ferns and to angiosperms (Brodribb et al., 2009; Carriquí et al., 2015; McAdam and Brodribb, 2015). For a comprehensive understanding of the mechanisms of improving plant photosynthetic capacity during plant evolutionary history, knowledge of leaf gas exchange and leaf optical properties are essential, both of which relate strongly to leaf mesophyll anatomy. Effective leaf gas exchange is linked to a reduction in CO₂ diffusional limitations through the stomata and mesophyll, as well as to improved photosynthetic biochemistry (Flexas et al., 2012; Gago et al., 2020). Leaf optical properties that involve high radiation use efficiency of the leaves are linked to some anatomical and physiological mechanisms for efficient light absorption without photoinhibition, which depends highly on the light environment of their habitats. Namely, plants need to have a high light-harvesting efficiency under low light (Gotoh et al., 2018), while under excessive light, plants should decrease light-harvesting efficiency to avoid photoinhibition (Lambers et al., 2008; Ruban, 2015). In fact, an evolutionary decreasing trend in some carotenoid content has been reported from algae, mosses and ferns to basal angiosperms, related to improving photoprotection with evolution (Esteban et al., 2009).

Ferns, early vascular plants, are suitable for investigating the evolutionary history of photosynthetic capacity in relation to leaf anatomy. Ferns comprise more than 10 000 species that have a wide range of divergence times (Pryer et al., 2004; Schuettpelz and Pryer, 2009), in which the earliest ones, such as lycophytes, diversified in the Devonian, ~370 million years ago (Mya; Testo et al., 2018), whereas some latest fern species, such as the family Polypodiaceae, diversified in the Cenozoic era, about <10 Mya (Pryer et al., 2004). Many fern species have a similar leaf structure, serrated flat leaves, but with large variations in leaf anatomy among species. Some of the earliest ferns, lycophytes in the Selaginellaceae, have only one or two layers of mesophyll cells with a thick epidermis, and with a very low rate of photosynthesis (Carriquí et al., 2019). More recently diversified fern species, such as Dryopteris erythrosora in the family Dryopteridaceae, have more than three layers of mesophyll cells (Tosens et al., 2016), and their leaf anatomical structure is very similar to those of angiosperms and enables a high photosynthesis rate. Although the increase in photosynthetic capacity with evolution has been reported for the limited number of species of ferns (Veromann-Jürgenson et al., 2017), comprehensive research on the relationship between leaf anatomy and photosynthetic capacity in relation to the evolution of fern species is lacking.

For another important factor to improve photosynthetic capacity, namely high radiation use efficiency, it is important to improve the light-harvesting ability by photosynthesis, because many fern species grow under light-limited conditions (Ebihara, 2018). Indeed, fern species are an important component of the understorey layers of temperate and tropical forests (Royo and Carson, 2006). It is possible that during the evolutionary history of ferns, they evolved efficient light-harvesting ability of photosynthetic pigments such as chlorophylls. However, among fern species, few studies have reported on the increasing evolutionary trend in the amounts of photosynthetic pigments. On the other hand, many studies exist on the evolution of the light-sensing mechanisms of photoreceptors among ferns (Kawai et al., 2003; Li et al., 2014; Li and Mathews, 2016; Cai et al., 2021). The functional types and numbers of photoreceptors are different between fern clades and orders, in which the chimeric photoreceptor neochrome, which fuses red-sensing phytochrome and blue-sensing phototropin (Kawai et al., 2003), has been reported only in the orders Cyatheales and Polypodiales (Cai et al., 2021), which diversified 190 Mya (Schuettpelz and Pryer, 2009). Although not yet studied, it is possible that, similar to the light-sensing mechanisms, there exists some evolutionary trends in light-harvesting traits among fern species.

It is also possible that such light-harvesting traits, as well as photosynthetic traits, may be related to the evolutionary trend in mesophyll anatomy, because light-harvesting efficiency is greatly affected by mesophyll anatomical traits (Terashima et al., 2011). For efficient light harvesting, light needs to be captured by all the chloroplasts distributed along the cell surfaces throughout the leaf (Terashima et al., 2011). Well-developed palisade tissues that contain many chloroplasts, which are often observed in sun-grown leaves, allow plant leaves to direct light to penetrate into the lower leaf layer (Brodersen and Vogelmann, 2010), increasing the light-harvesting efficiency of leaves with a thick mesophyll. At the same time, the development of palisade tissue reduces the absorbed photons per chloroplast (Kume, 2017). Given that palisade and spongy tissue is not distinguishable in many fern species, to maximize light-harvesting efficiency for each chloroplast in fern species, reducing mesophyll thickness will be preferable under a low-light environment where many fern species grow. Photosynthetic efficiency is affected by light absorption, which is related to mesophyll anatomy; there is a vertical gradient in the chloroplast properties within a leaf, in which maximum photosynthetic efficiency will be realized if photosynthetic capacity is proportional to light absorption at each point in the leaf (Terashima et al., 2011). The importance of anatomical traits to light harvesting and the photosynthesis of leaves is also evident in the movements of chloroplasts. Chloroplast accumulation along the periclinal cell walls increases absorption over the range of wavelengths (350–750 nm) of visible light, which enhances the photosynthesis and plant biomass production of Arabidopsis thaliana (Gotoh et al., 2018). In contrast, avoidance response of chloroplast reduces photodamage in plants (Kasahara et al., 2002).

We performed two experiments to investigate the changes in light use and photosynthesis of fern species with different divergence times in relation to leaf anatomical traits. In the first, we collected 66 fern species from four natural habitats in Japan, to obtain comprehensive evolutional trends in their mesophyll anatomy. In the second experiment, we performed a growth experiment on eight fern species with different divergence times to investigate light absorption capacity, leaf and stomatal anatomy, and leaf photosynthetic capacity. We tested the following hypotheses:

• (1) Fern species have a distinct trend with divergence time in mesophyll anatomy: more recently diverged ferns have a larger chloroplast surface area facing intercellular airspaces (S_c) and thinner cell wall in the mesophyll cells.

• (2) Newer diversified ferns have a high light absorption capacity and high leaf photosynthetic capacity.

• (3) The high light absorption capacity and high leaf photosynthetic capacity in more recently diverged ferns are linked to mesophyll anatomical traits.

MATERIALS AND METHODS

Collection of ferns from natural habitats: field collection

During field collection, plant samples of ferns were collected from their natural habitats in mountainous regions such as Mt Matsuo, Kyoto City (35°00ʹN, 135°40ʹE, 130–190 m altitude), in June 2014; Gongen Valley, Shiga Prefecture (35°15ʹN, 136°22ʹE, 230–370 m altitude), in August 2013; Mt Makio, Osaka Prefecture (34°23ʹN, 135°30ʹE, 330–630 m altitude), in January 2014; and Yakushima Island, Kagoshima Prefecture (30°22ʹN, 130°32ʹE, 800–1300 m altitude), in September 2014 in Japan. The fully expanded fronds of mature healthy ferns were collected from the forest floor around trails. Most of the ferns were growing in shady habitats, along rivers valleys. The base of the petiole of the ferns was cut with scissors, placed in a plastic bag filled with water, sealed and transported to the laboratory.

From 1991 to 2020, mean annual temperature was 19.6 °C and mean annual precipitation was 4700 mm on Yakushima Island (Japan Meteorological Agency). For the other habitats, mean annual temperature was 15.0–16.5 °C and mean annual precipitation was 1200–1600 mm (Japan Meteorological Agency). In total, 66 species were, of which 17 were from Kyoto City, 26 from Shiga Prefecture, 18 from Osaka Prefecture and five from Yakushima Island.

Growth experiment for the eight fern species

The growth experiment was conducted from 2018 to 2021 for eight fern species, Selaginella uncinata (Selaginellaceae), Angiopteris lygodiifolia (Marattiaceae), Osmunda japonica (Osmundaceae), Lygodium japonicum (Lygodiaceae), Sphaeropteris lepifera (Cyatheaceae), and Coniogramme intermedia, Asplenium antiquum and Microsorum insigne (all Polypodiaceae). The ferns were purchased from commercial nurseries, and transplanted to larger plastic pots (0.5–1.5 L) filled with soil at a ratio of cultivated soil/red ball/perlite of 11 : 11 : 3. The size of the pots was altered depending on the size of the plants. The potted plants were grown in a temperature-controlled glasshouse, 25 °C, at the campus of the Kyoto Institute of Technology in Kyoto City (35°03ʹN, 135°47ʹE). The plants were watered about three times per week, and fertilized with 200 mL of 1/500 Hyponex solution (Hyponex Japan, Osaka, Japan) once a week. The ferns were grown for at least 1 year in the glasshouse before the experiments. The leaves that emerged after being transferring to the glasshouse were used for the following analyses.

Classification of ferns and their divergence times

The nomenclature and classification of the fern species followed Ebihara (2018). The 66 species from natural habitats included four species of lycophytes, one species of Osmundales, three species of Hymenophyllales, two species of Cyatheales and 56 species of Polypodiales (Fig. 1). These 66 species were classified into six classes according to genus divergence times (Schuettpelz and Pryer, 2009): which are 5–30 Mya (class 6), 30–40 Mya (class 5), 40–50 Mya (class 4), 50–60 Mya (class 3), 70–160 Mya (class 2) and 290–320 Mya (class 1, Supplementary Data Table S1). Each class includes 5–18 species. For the eight glasshouse-grown ferns (Fig. 1), the mean divergence time of each genus was calculated using two references for each species, which had a wide range of divergence times, from 22 to 317 Mya (Pryer et al., 2004; Li and Lu, 2007; Schuettpelz and Pryer, 2009; Arrigo et al., 2013; Brandt et al., 2016; Wei et al., 2017; Testo et al., 2018).

Fig. 1.

Phylogenetic tree of the fern species used in the present study. The phylogenetic structure follows previous studies (Pryer et al., 2004; Ebihara, 2018).

Leaf anatomical properties and stomatal traits

For all the fern species, leaf sections of 1 × 3 mm were obtained from the central part of the lamina, avoiding the main veins, fixed in 2.5 % glutaraldehyde in 0.2 m sodium phosphate buffer (pH 7.4), and then stored at 4 °C. For the species collected from natural habitats, fixation in 2.5 % glutaraldehyde was performed within 6 h of plant collection. Leaf sections were post-fixed in 1 % OsO₄ solution at 4 °C for 3 h in darkness, dehydrated in a graded ethanol series, and then embedded in Spurr’s resin (Low Viscosity Resin kit, TAAB, Aldermaston, UK). For the eight glasshouse-grown fern species, leaf sections were obtained from four leaves for each species in September 2021, fixed in 2.5 % glutaraldehyde, dehydrated in a graded ethanol series after fixation in glutaraldehyde, and then embedded in Technovit 7100 (Technovit 7100 set, Kulzer Technique, Wehrheim, Germany). Technovit 7100 was used because of its low toxicity. We found no difference between the images of leaf sections embedded in Spurr’s resin and those in Technovit 7100. Transverse sections of leaves of 700 nm thickness were stained with 1 % toluidine blue solution, and then digitized images were obtained using a light microscope (BX51-33, Olympus, Tokyo, Japan) with a CCD camera (DP22, Olympus) at 200× magnification. Three images of the ferns from the natural habitats, and eight images (i.e. two images from four leaf sections) for the glasshouse-grown ferns were used for analysis. The images were analysed using ImageJ software (Schneider et al., 2012). Leaf anatomical traits, such as numbers of mesophyll cell layers, mesophyll thickness and chloroplast surface area exposed to intercellular airspaces per unit leaf area (S_c) in the mesophyll tissues and epidermis, were measured using digitized images, following Hanba et al. (2002). For the estimation of S_c, the curvature cell correction factor was measured and calculated for each species following Thain (1983), giving an average length–width ratio of five cells from two images. For the measurement of chloroplast size and cell wall thickness, mesophyll cells just below the upper epidermis were analysed, using three leaf sections for each species. We manually circled five randomly selected chloroplasts in the mesophyll cells (n = 15 for each species), and then analysed their area. We measured the thickness of the cell walls where the chloroplasts were attached for the five mesophyll cells (n = 15 for each species). These analyses were performed using ImageJ software (Schneider et al., 2012).

Stomatal density and stomatal size, which are related to photosynthetic capacity, were estimated for the eight glasshouse-grown fern species using four fully expanded mature leaves for each species, in October 2019. A mixture of a silicone rubber monomer (KE-10) and a catalyst (CAT-RA, both from Shinetsu Chemical, Tokyo, Japan) was poured on the lower side of the leaves, and a secondary replica was then taken by applying nail varnish. Light micrographs of the stomata were taken using a microscope (BX51-33, Olympus), digitally recorded with a CCD camera (DP22, Olympus) and analysed using ImageJ software (Schneider et al., 2012). Stomatal density was measured with two different fields of view of the images from the four leaves. The length and width of the stomata were measured for five closed stomata from the four images of each leaf.

Leaf gas exchange measurements and chlorophyll content

Photosynthesis measurements were performed for healthy fully expanded mature leaves of the glasshouse-grown plants in October–November 2019, with four attached leaves used for each species (n = 4). Leaf photosynthesis was measured using a Li-6400XT (Li-Cor, Lincoln, NE, USA). Photosynthetic biochemical parameters, such as maximum carboxylation rate (V_cmax) and maximum electron transport rate (J), were obtained from A/C_i curves with 12–15 different CO₂ levels (Ethier and Livingston, 2004), in which CO₂ was decreased stepwise from 400 to 50 µmol mol^–1, and then returned to 400 µmol mol^–1 and finally increased stepwise to 1900 µmol mol^–1. Measurements were conducted under 1500 µmol mol^–1 photosynthetic photon flux density (PPFD). The light-saturated CO₂ assimilation rate (A_max) was obtained using light-response curves measured at 400 µmol mol^–1 of ambient CO₂ with 14–17 different values of PPFD, in which PPFD was increased stepwise from 400 to 2000 µmol m^–2 s^–1, and then returned to 400 µmol m^–2 s^–1 and finally decreased stepwise to 0 µmol m^–2 s^–1. In these photosynthesis measurements, mean (s.d.) leaf temperature and vapour pressure deficit (VPD), were 25.7 (0.8) °C and 1.4 (0.4) kPa, respectively. Stomatal and non-stomatal limitations were calculated using A/C_c curves obtained from the A/C_i curves, following previous studies (Grassi and Magnani, 2005; Matsumoto et al., 2022).

Leaf chlorophyll concentration was measured using the fully expanded mature leaves of the ferns in October 2021. Fresh leaf discs of 1.5 cm² or three–four leaf pinnules were taken and then ground with 1.5 mL of 80 % acetone. It was then centrifuged at 3000 r.p.m. for 5 min. The absorptance of the supernatant was measured using a spectrophotometer (U1800, Hitachi, Tokyo, Japan). The concentrations of chlorophyll a, chlorophyll b and total chlorophyll were calculated according to the equation given by Porra et al. (1989).

Leaf spectroscopy

Leaf optical properties (spectral reflectance, transmittance and absorptance of radiation) were measured using the fully expanded mature leaves in October–November 2019 (Supplementary Data Fig. S1A). Two to four leaf replicates were used for each species, except for Lygodium japonicum which was measured only once because of poor growth.

Generally, accurate leaf optical properties (spectral reflectance and transmittance of radiation) can be measured using a spectroradiometer attached via an optical fibre to an integrating sphere (Knapp and Carter, 1998; Carter and Knapp, 2001; Asner et al., 2009). In this study, we used a FieldSpec 3 spectroradiometer (Analytical Spectral Devices, Boulder, CO, USA) and an LI-1800 integrating sphere (Li-Cor), the inside of which is coated with BaSO₄ with perfectly diffuse reflectance in the target wavelength range (Supplementary Data Fig. S1B).

When the entire sample port of the integrating sphere was covered with the leaf, we measured leaf optical properties using a standard protocol: (1) the leaf was set on the sample port to face the leaf adaxial side inward, (2) the white reference was measured, (3) the reflectance of the leaf adaxial side was measured, and (4) the leaf was reversed; steps 2–4 were then repeated for the opposite side. When the leaf was too narrow to cover the sample port (i.e. for Selaginella uncinata, L. japonicum, S. lepifera), we used the protocol for narrow leaves proposed by Noda et al. (2013). Following this protocol, we made a leaf array (see Supplementary Data Fig. S1), and then the gap fractions at 400 nm in reflectance mode (G_r) and transmittance mode (G_t) were calculated as:

$${\displaystyle \begin{array}{l}{\displaystyle G_{\mathrm{r}}=(\frac{F_{\mathrm{l}\mathrm{r}}-F_{\mathrm{v}\mathrm{r}}}{F_{\mathrm{l}\mathrm{w}}-F_{\mathrm{v}\mathrm{r}}}-\frac{F_{\mathrm{a}\mathrm{r}}-F_{\mathrm{v}\mathrm{r}}}{F_{\mathrm{a}\mathrm{w}}-F_{\mathrm{v}\mathrm{r}}})\frac{{\rho }_{\mathrm{w}}}{{\rho }_{\mathrm{p}}}}\end{array}}$$ (1)

$${\displaystyle \begin{array}{l}{\displaystyle G_{\mathrm{t}}=\frac{F_{\mathrm{a}\mathrm{t}}}{(F_{\mathrm{a}\mathrm{w}}-F_{\mathrm{v}\mathrm{r}})}\frac{{\rho }_{\mathrm{w}}}{{\rho }_{\mathrm{c}}}}\end{array}}$$ (2)

where ρ_w, ρ_p,and ρ_c are the reflectance of the BaSO₄ reference surface, from the paper surface and of the cavity wall, respectively, and F_lr, F_vr, F_ar, F_at, F_aw, and F_lw are the flux of the overlapping leaf array and paper, of stray light measured in the reflectance mode with a vacant sample port, from the leaf array in the reflectance mode and from the overlapping leaf array and paper measured in white reference mode, from the leaf array measured in the white reference mode, and from the leaf array in the transmittance mode, respectively.

After obtaining the gap fractions using eqns (1) and (2), we calculated the reflectance from the leaf array (ρ_a) and transmittance of the leaf array from the illuminated side (τ_a) as follows:

$${\displaystyle \begin{array}{l}{\displaystyle {\rho }_{\mathrm{a}}=\frac{F_{\mathrm{a}\mathrm{r}}-F_{\mathrm{v}\mathrm{r}}}{F_{\mathrm{a}\mathrm{w}}-F_{\mathrm{v}\mathrm{r}}}{\rho }_{\mathrm{w}}\frac{1}{1-G_{\mathrm{r}}},}\end{array}}$$ (3)

$${\displaystyle \begin{array}{l}{\displaystyle {\tau }_{\mathrm{a}}=(\frac{F_{\mathrm{a}\mathrm{t}}}{F_{\mathrm{a}\mathrm{w}}-F_{\mathrm{v}\mathrm{r}}}{\rho }_{\mathrm{w}}-G_t{\rho }_c)\frac{1}{1-G_{\mathrm{t}}}}\end{array}}$$ (4)

We calculated leaf absorptance using the following equation:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}=1-\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}-\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}.}\end{array}}$$ (5)

We classified the absorption spectrum from 350 to 699 nm according to the absorption spectrum of the main pigments considering the blue shift of the absorbance spectrum measured in vitro (Solovchenko, 2010; Kume et al., 2018): 350–449 nm (absorbed mainly by chlorophyll a and flavonols), 450–549 nm (absorbed mainly by chlorophyll b and carotenoids) and 550–699 nm (absorbed mainly by chlorophyll a and b). Furthermore, the absorption spectrum from 700 to 1099 nm was divided into 700–799 and 800–1099 nm. For analysis of mesophyll thickness traits in latter part of the study, the data for 450–549 and 550–699 nm were pooled, because the trends in absorptance were similar between these wavelength classes.

Data analysis

Anatomical data for the ferns collected from the natural habitats are shown for the six divergence time-based classes using box plots, in which the minimum, maximum, median, first quartile and third quartile of each class are shown with the plots of mean data for each species. For the eight glasshouse-grown fern species, the anatomical traits are shown as means with standard errors. The trends of these anatomical variables against divergence time were tested using the Jonckheere–Terpstra trend test. For the gas exchange and stomatal traits for the eight glasshouse-grown fern species, means with standard errors are shown with the result of Jonckheere–Terpstra trend test. Correlations among these gas exchange and stomatal traits were analysed using Pearson’s product-moment correlation coefficient. For the light absorptance and chlorophyll concentration of the glasshouse-grown eight fern species, means with standard errors are shown with the result of Jonckheere–Terpstra trend test. The relationships between two traits were analysed using linear regression. These statistical analyses were performed using EZR (Kanda, 2013) and Rcmdr (ver. 2.7–2) for R software, R ver. 4.2.1 (R Core Team, 2022).

RESULTS

Changes in mesophyll anatomy with divergence time

We analysed how the change in mesophyll anatomy relates to the six divergence time classes, using the 66 fern species from natural habitats. Significant variations in the leaf anatomy were observed among the fern species in the present study (Supplementary Data Fig. S2, Table S2). The two lycophyte species, Selaginella remotifolia in the natural habitats (Fig. S2A) and Selaginella uncinata grown in the glasshouse (Fig. S2B), which had the oldest divergence time of 310–330 Mya, had less developed mesophyll tissues and large cells of the upper epidermis with large chloroplasts. All fern species in the present study had chloroplasts in the upper and lower epidermis cells, except for the three species of the family Hymenphyllaceae, Crepidomanes birmanicum, Hymenophyllum polyanthos and Hymenophyllum barbatum, which had only one layer of leaf cells (Fig. S2A). For most of the species, palisade tissue and spongy tissue in the leaf mesophyll were not distinguishable.

Significant trends with divergence time were obtained for some mesophyll anatomical traits, which were similar overall between natural-habitat species and glasshouse-grown species, except for chloroplast size in the lower epidermis (Fig. 2A, B). Mesophyll thickness was greater in more recently diverged species, related to the increased S_c. Cell wall thickness of mesophyll cells was low in species with an older divergence time. The increases in mesophyll thickness and S_c in the more recently diverged species corresponds to the increased numbers of mesophyll cell layers (Tables 1 and 2, Fig. 2B). On the other hand, no significant trend with divergence time was obtained for mesophyll porosity. For chloroplast size, reductions with divergence time were obtained for chloroplasts in the upper and lower epidermis, while no significant trend with divergence time was obtained for chloroplasts in the mesophyll. The ratio of epidermis S_c to mesophyll S_c was decreased in more recently diverged species.

Fig. 2.

(A) Divergence time of the genus of the species, and anatomical traits such as mesophyll thickness, surface area of the chloroplasts facing intercellular airspaces (S_c), and cell wall thickness of the leaves of the ferns. Box plots are shown for the six divergence time classes of the 66 ferns collected from natural habitats, with means of each species shown as circles. Means are shown for the glasshouse-grown plants, in which bars indicate standard errors. Divergence time was obtained following previous studies (Li and Lu, 2007; Pryer et al., 2004; Schuettpelz and Pryer, 2009; Arrigo et al., 2013; Brandt et al., 2016). (B) Results of statistical analysis for the anatomical traits, tested by the Jonckheere–Terpstra trend test. P-values for the increasing or decreasing trends are shown using colours.

Table 1.

Mean anatomical traits of the 66 fern species collected from natural habitats

Evolutional age class	Trait
	Number of mesophyll cell layers	Mesophyll porosity (m3 m–3)	Chloroplast size in the mesophyll (μm2)	Chloroplast size in the upper epidermis (μm2)	Chloroplast size in the lower epidermis (μm2)	Ratio of chloroplast surface area* in epidermis to that in mesophyll cells
1	2.4 (0.7)	0.29 (0.05)	17.0 (3.6)	51.3 (17.2)	19.7 (3.7)	0.91 (0.29)
2	3.1 (0.6)	0.52 (0.05)	17.1 (2.4)	34.0 (5.8)	25.3 (5.7)	0.47 (0.12)
3	3.3 (0.4)	0.55 (0.03)	16.2 (1.8)	30.3 (4.4)	23.4 (3.2)	0.48 (0.08)
4	4.8 (0.7)	0.46 (0.04)	22.8 (1.9)	26.9 (5.8)	15.3 (2.0)	0.50 (0.09)
5	7.0 (0.3)	0.41 (0.03)	20.1 (1.9)	11.7 (1.3)	10.7 (1.9)	0.23 (0.02)
6	6.3 (0.7)	0.76 (0.23)	20 (3.8)	21.5 (4.0)	12.7 (2.8)	0.27 (0.06)

Values are means (s.e.) for 5–18 species per each evolutionary class.

*Chloroplast surface area: chloroplast surface area facing the intercellular airspaces per leaf area.

Table 2.

Mean anatomical traits of the eight ferns grown in the glasshouse

Species	Evergreen or summer-green	Trait
		Numbers of mesophyll cell layers	Mesophyll porosity (m3 m–3)	Chloroplast size in the mesophyll (μm2)	Chloroplast size in the upper epidermis (μm2)	Chloroplast size in the lower epidermis (μm2)	Ratio of chloroplast surface area* in epidermis to that in mesophyll cells
Selaginella uncinata	E	1.4 (0.3)	0.81 (0.04)	63.9 (4.7)	138.8 (9.6)	56.0 (3.4)	3.00 (0.80)
Osmunda japonica	S	4.6 (0.4)	0.59 (0.02)	29.9 (1.8)	12.4 (0.9)	13.6 (1.6)	0.49 (0.07)
Lygodium japonicum	E/S	4.9 (0.6)	0.52 (0.03)	30.6 (2.0)	20.7 (1.5)	16.7 (1.4)	0.32 (0.09)
Sphaeropteris lepifera	E	4.5 (0.3)	0.39 (0.03)	35.3 (2.6)	12.4 (1.0)	13.2 (0.8)	0.58 (0.06)
Coniogramme intermedia	E	5.0 (0.3)	0.60 (0.02)	45.9 (2.4)	43.6 (4.7)	37.6 (2.2)	0.57 (0.06)
Asplenium antiquum	E	8.8 (0.2)	0.32 (0.02)	52.7 (6.3)	14.0 (1.2)	8.9 (1.1)	0.06 (0.01)
Angiopteris lygodiifolia	E	7.5 (0.3)	0.47 (0.02)	106.9 (7.2)	31.1 (2.4)	30.9 (3.3)	0.21 (0.02)
Microsorum insigne	E	5.3 (0.5)	0.53 (0.02)	32.8 (3.8)	20.3 (1.7)	13.9 (1.2)	0.32 (0.02)

Values are means (s.e.) for the mesophyll and S_c ratio traits (n = 8) and chloroplast size traits (n = 20).

*Chloroplast surface area: chloroplast surface area facing the intercellular air paces per leaf area.

Photosynthetic and stomatal traits of the glasshouse-grown ferns with different divergence times

We analysed how the photosynthetic and stomatal traits of ferns depend on divergence time. Although the photosynthetic traits, such as A_max,V_cmax, g_m and g_s, were significantly different among the eight species grown in the glasshouse (P < 0.001, one-way ANOVA), the trend of these traits against divergence time was not clear (Fig. 3A). Correlation analysis revealed that photosynthetic traits, such as A_max, V_cmax, g_m and g_s, were strongly correlated with each other (Fig. 3B). In contrast, the stomatal traits showed a significant trend against divergence time: stomatal density was decreased while the stomatal size was increased with more recent divergence times (Fig. 3A). However, neither stomatal trait was correlated with stomatal conductance and the other photosynthetic traits (Fig. 3B).

Fig. 3.

(A) Leaf gas exchange and stomatal traits for the eight glasshouse-grown fern species. Leaf gas exchange for each species was measured for the attached leaves (n = 8), and stomatal density (n = 4) and stomatal size (n = 20) were measured using secondary replica of the lower sides of the leaves of each species. (B) Correlation coefficients (r) between the mean values of the traits for the eight species (n = 8), with values from –0.8 to 0.8 shown using colours. Significance levels were such that †P < 0.1, *P < 0.05, **P < 0.01 and ***P < 0.001.

Absorptance spectra for the glasshouse-grown ferns with different divergence times

The shapes of the absorptance spectra were similar for all of the eight species (Fig. 4A), in which we found significant and almost constant increasing trends with newer divergence time in the mean absorptance at 450–549 nm (blue–green) and 550–699 nm (green–red, Fig. 4B). The leaf-area-based chlorophyll a + b concentration showed overall an increasing trend against newer divergence time (Fig. 4B). Similarly, the reflectance of near-infrared, 700–799 nm and 800–1099 nm had an increasing trend with newer divergence time (Fig. 4B), in which mesophyll porosity correlated negatively with the reflectance of these wavelengths (Fig. 4C). The regression coefficient between light absorptance and divergence time was high, 0.6–0.9, at wavelength of 450–710 nm (Fig. 4D). The regression coefficient between light absorptance and chlorophyll a + b concentration had two peaks, at 520–560 and 700–740 nm, respectively (see the right-hand figure in Fig. 4D).

Fig. 4.

(A) Absorptance spectra of the leaves of the eight glasshouse-grown fern species. The data for two–four leaves were pooled except for Lygodium japonicum and then mean values were calculated. For L. japonicum, data were obtained for a single leaf. (B) Mean absorptance for two wavelength regions of 450–549 and 550–699 nm, sum of the content of chlorophyll a and chlorophyll b for each species, and mean reflectance of the two wavelength regions of 700–799 and 800–1099 nm. The results of the Jonckheere–Terpstra trend test are shown. (C) Correlation between reflectance of 700–799 or 800–1099 nm light and mesophyll porosity. Each point represents the mean value of each species, with bars showing standard errors. Lines represent regression lines, with regression coefficients (r²) and their significance levels, †P < 0.1 and *P < 0.05, are shown. (D) Left: the regression coefficients (r²) between absorptance and divergence time at a certain wavelength for the eight fern species. Right: the regression coefficients (r²) between absorptance and chlorophyll a + b content at a certain wavelength for the eight fern species. A higher regression coefficient indicates that differences in the divergence time or that in the chlorophyll a + b content explain the difference in the absorptance of the wavelength more strongly.

Mesophyll-thickness-based traits of leaf anatomy, leaf gas exchange and light absorptance

Although the trends with divergence time for leaf gas exchange traits on an area basis were not clear (Fig. 3A), the trend became apparent when these traits were expressed on a mesophyll-thickness basis; maximum photosynthesis (A_max) decreased in the species with a more recent divergence time (Fig. 5A). Similarly, mean S_c was decreased in species with a more recent divergence time. On the other hand, a strong negative correlation was obtained between mesophyll thickness-based S_c and mesophyll thickness (Fig. 5B). A strong negative correlation was also obtained between light absorptance of 450–699 nm (green—red) and the inverse of mesophyll thickness. A decreasing trend of mesophyll-thickness-based S_c for more recently diverged species was obtained for the 66 fern species in the natural habitats from divergence-time classes 2–6, although the decreasing trend was less distinct (Fig. 5C). A strong positive relationship between A_max and S_c was obtained when our data for glasshouse-grown ferns were plotted with data from as previous study (Fig. 6A; Tosens et al., 2016). A negative correlation was obtained between S_c/mesophyll thickness and mesophyll thickness (Fig. 6B) when our data for glasshouse-grown ferns and ferns collected from natural habitats were plotted together with data from a previous study (Tosens et al., 2016).

Fig. 5.

Leaf gas exchange, anatomical traits and light absorptance related to mesophyll thickness. (A) Mesophyll-thickness-based maximum photosynthetic rate (A_max) and S_c for the eight glasshouse-grown species. (B) Relationship between mesophyll-thickness-based S_c and mesophyll thickness, between absorptance of 450–699 nm light and the inverse of mesophyll thickness for the eight glasshouse-grown species. Lines indicate linear regression lines, with r² values with significant levels shown: *P < 0.05 and ***P < 0.001. (C) Box plot of mesophyll-thickness-based S_c for the six divergence time classes for 66 species from natural habitat. Means of each species are shown as circles. The results of the Jonckheere–Terpstra trend test except for divergence-time class 1 are shown.

Fig. 6.

Correlations between mesophyll-thickness-based gas exchange and mesophyll-thickness-based S_c (A), and between mesophyll-thickness-based S_c and mesophyll thickness (B). Data from 35 fern species were redrawn from a previous study (Tosens et al., 2016). The data were fitted using linear regression, with the regression coefficient, r², shown. The solid and dashed lines in B show the regression lines for all data and that for data eliminating three species circled with the dashed line, Equisetum telmateia (Tosens et al., 2016), Lemmaphyllum microphyllum and Neocheiropteris ensata, respectively.

DISCUSSION

A distinct effect of divergence time on mesophyll anatomy was obtained for the 66 species collected from natural habitats as well as for the eight species grown in the greenhouse, in which increasing numbers of cell layers are related to an increase in mesophyll thickness and involves a increase in S_c (Fig. 2A, B). This increasing trend in the anatomical trait S_c potentially enhances CO₂ diffusion in the leaf mesophyll, while the increasing trend of the other anatomical trait, namely cell wall thickness (Fig. 2A, B), potentially imposes the opposite effect on CO₂ diffusion in the leaf mesophyll, inhibiting CO₂ diffusion (Terashima et al., 2011; Tosens et al., 2016; Carriquí et al., 2020). We found no significant effect of divergence time on the photosynthetic capacity of the glasshouse-grown ferns (Fig. 3A), although some previous studies reported increasing trends of photosynthetic capacity with a more recent evolutionary age (Carriquí et al., 2015; Veromann-Jürgenson et al., 2017). The less distinct effect of divergence time on photosynthetic capacity in the present study can be partly explained by the fact that the increase in cell wall thickness offsets the enhancement in mesophyll diffusion caused by the increase in S_c. The unclear trend of photosynthetic traits against divergence time is not surprising because adaptation to environmental conditions, such as light and moisture levels, which do not necessarily change constantly along with divergence time, can induce more significant evolutionary changes in photosynthetic traits compare with their divergence time (Tosens et al., 2016; Veromann-Jürgenson et al., 2017).

The strong interrelationship among the photosynthetic traits, A_max, V_cmax, g_m and g_s, supports a previous study on 35 fern species (Tosens et al., 2016), which indicates that both diffusional limitations (g_m, and g_s) and biochemical limitation (V_cmax) regulate the photosynthetic capacity of these eight fern species, irrespective of their divergence time. Although the two stomatal traits (i.e. stomatal size and stomatal density) showed significant trends with divergence time (Fig. 3A), neither are correlated with stomatal conductance and the other photosynthetic traits (Fig. 3B). This may be because the negative correlation between stomatal density and stomatal size offsets their effects on stomatal conductance (Ohsumi et al., 2007).

Significant increasing trends with divergence time are obtained at mean absorptance of 450–549 nm (blue–green) and 550–699 nm (green–red, Fig. 4B), which correspond to the increasing trend in leaf-area-based chlorophyll a + b concentration (Fig. 4B) because light of 450–549 and 550–699 nm is absorbed largely by chlorophyll a and b (Campillo et al., 2012). Note that light of 450–549 nm is also absorbed by carotenoids (Solovchenko, 2010). The large regression coefficients between light absorptance and chlorophyll a + b concentration at 520–560 and 700–740 nm (Fig. 4D), which is well in line with a previous study (Carter and Knapp, 2001), also confirm the strong relationship between chlorophyll concentration and absorptance of blue–green and green–red light. These results suggest that fern species with a more recent divergence time are capable of efficient absorption of light that is used for photosynthesis by increasing the chlorophyll a + b concentration, in agreement with a previous study that reported an evolutionary increasing trend in chlorophyll concentration across different plant taxa (Karst and Lechowicz, 2007).

The increasing trends with divergence time in the reflectance of near-infrared light, 700–799 and 800–1099 nm (Fig. 4B), and their negative correlations with mesophyll porosity (Fig. 4C), contradicts a previous study that reported an increase in reflectance of near-infrared light (750–1250 nm) with increasing mesophyll porosity in Vitis vinifera leaves (Rapaport et al., 2014). This discrepancy is probably due to the much larger mesophyll porosity in the present study, 30–80 %, than those in the previous study, 26–41 %.

In contrast to the trends in the absorptance of 450–549 and 550–699 nm light, no clear trends in the absorptance of 350–449 nm light is obtained (UV–blue, data not shown), which might be attributed to the fact that ferns with an older divergence time, Selaginella species that diversified 300–420 Mya, had already acquired the photoprotection mechanism, as had more recently diversified ferns, by accumulating photoprotective pigments in their leaves. Some important antioxidant pigments, such as phenolic acids, flavonoids, xanthophylls and carotenoids, have high absorption in wavelength range 350–449 nm (Solovchenko, 2010). The biosynthetic pathway of one of these antioxidants, flavonoids, arose during the period of land colonization of plants around 470–550 Mya (Davies et al., 2020), which is before the divergence time of the oldest fern, Selaginella species.

The mesophyll-thickness-based A_max and S_c (Figs 5 and 6) reflects photosynthesis rate per unit leaf volume and chloroplast surface area facing the intercellular airspaces per unit leaf volume, respectively. The decreasing trends with more recent divergence time in the mesophyll-thickness-based S_c (Fig. 5A, C) and the negative relationship between the mesophyll-thickness-based S_c and mesophyll thickness (Fig. 5B, 6B), together with the increasing trend in mesophyll thickness with more recent divergence time (Fig. 2A), suggest that fern species have developed the mesophyll anatomy with decreasing chloroplast surface area: decreasing chloroplast numbers per mesophyll volume with an increase in mesophyll thickness. This decreased number of chloroplasts per mesophyll volume may enhance the light absorption by each chloroplast by facilitating light diffusion within the mesophyll (Terashima et al., 2009). Such a trend contrasts with that in sun-expised leaves, which increase S_c per mesophyll volume with an increase in mesophyll thickness (Oguchi et al., 2005). Our results support previous studies, which reported that less development of palisade tissue (i.e. less vertical overlapping of the chloroplasts) will increase the absorbed photons per chloroplast (Kume, 2017). Our results also suggest the importance of chloroplast positioning inside the leaves for light absorbance and photosynthesis, which supports a previous study that reported enhancement of photosynthesis by chloroplast accumulation in Arabidopsis (Gotoh et al., 2018). Maximizing light absorption by each chloroplast is advantageous in the light-limited conditions where many fern species are found (Royo and Carson, 2006).

The strong relationships among mesophyll thickness, 450–699 nm light absorption, mesophyll-thickness-based A_max and mesophyll-thickness-based S_c (Figs 5B and 6A, B) shows the strong coordination among light capture, mesophyll anatomy and photosynthesis rate in fern species for the first time. Fern species with thin leaves are often distributed on the forest floor with low irradiance levels, so the ability to capture light could directly improve photosynthesis. The observed enhancement in light absorption with more recent divergence time (Fig. 4B) is largely due to the increase in mesophyll thickness. It is likely that more recently diversified fern species optimize the area-based leaf photosynthesis rate by developing mesophyll cell layers.

In conclusion, we have found significant trends in leaf mesophyll anatomy and light absorptance with divergence time in fern species, using 66 species in natural habitats and eight species grown in the glasshouse, which supports our hypotheses. Mesophyll thickness increased in species with a more recent divergence time, matching with the increase in absorptance of green and red light. The increasing trends in S_c with more recent divergence time supports our hypothesis, while the increasing trend in mesophyll cell wall thickness contradicts our hypothesis. Gas exchange traits, such as maximum photosynthesis rate, had no distinct trends with divergence time, which also contradicts our hypothesis. Meanwhile, strong correlations were obtained among mesophyll anatomy, light absorptance and photosynthesis on a mesophyll-thickness basis, which supports our third hypothesis. Those species with a more recent divergence time had low mesophyll-thickness-based values for maximum photosynthesis rate and S_c, possibly because of the decreased numbers of chloroplasts to maintain the light absorbance of each chloroplast. We conclude that leaf-area-based photosynthesis efficiency is not necessarily high for more recently diverged ferns, particularly for the species of large mesophyll thickness. This weak relationship between leaf-area-based photosynthesis and mesophyll thickness reflects that the fern species of large mesophyll thickness have high leaf construction costs (Wright et al., 2004). Species of large mesophyll thickness have thick cell walls. This large cell wall thickness imposes higher resistance for CO₂ diffusion, which decreases area-based photosynthesis, and concurrently, increases their construction cost (Mizokami et al., 2022). Rather than optimizing area-based carbon gain, fern species with more recent divergence time improve the toughness of their leaves accompanied the thick cell walls, allowing the ferns to adapt to a wider range of environments including low water availability, which would contribute to the diversification of fern species.

SUPPLEMENTARY DATA

Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: description of the 66 fern species collected from natural habitats. Table S2: anatomical traits of the 66 fern species collected from natural habitats. Figure S1: appearances of the leaves of eight fern species used for measurements of leaf optical properties and the instrument for measuring leaf optical properties. Figure S2: light micrographs of the leaf sections of the fern species.

CONFLICT OF INTEREST

None declared.

FUNDING

This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI, 18H02511).