DOI: 10.5061/dryad.c59zw3r3z
title: A combination of morphological and photosynthetic functional traits maintains the vertical distribution of bryophytes in a subtropical cloud forest

description: The distribution and performance of bryophyte species are known to vary
 with vertical gradients, due to changes in environmental factors,
 especially light conditions. However, the morphological and physiological
 drivers of bryophyte distribution along forest vertical gradients are
 poorly understood. Methods: We conducted a comparative analysis of 28
 morphological and photosynthetic functional traits in 18 species of mosses
 and liverworts distributed among three vertical microhabitats (ground,
 tree trunk, and branch) to analyze trait variance among the microhabitats
 and bryophyte life–forms in a subtropical cloud forest in Ailao Mountain,
 Yunnan, southwest China. Principle component analysis (PCA) was used to
 summarize trait differences among bryophyte species. Key
 Results: In contrast to trunk and ground dwellers, branch
 dwellers tend to reduce light interception (smaller leaf and cell sizes,
 lower chlorophyll content); protect against damage from intense
 irradiation (higher ratios of carotenoids to chlorophyll); raise light
 energy utilization (higher photosynthetic capacity) and cope with lower
 environmental moisture conditions (pendant life-forms, thicker cell wall).
 Principal component analysis showed that ecological strategies of
 bryophytes in response to levels of irradiation were specialized in
 branch–dwellers, although those in ground and trunk–dwelling species were
 less distinct. Conclusions: Environmental filtering shaped the functional
 traits combination and spatial distribution of bryophytes along the
 vertical gradients. Bryophyte species from  upper canopy of cloud
 forests show narrow  variation in functional traits under intense
 light , whereas species in the lower vertical strata associated with low
 levels of light intensity exhibited contrasting, but more diverse
 ecological strategies.
description: <strong>Study area and species</strong>
 The study area was in a subtropical, evergreen broad–leaved
 montane cloud forest in the Xujiaba region (23°32′N, 101°01′E) of the
 Ailao Mountains National Nature Reserve in Yunnan, China, where mean
 annual precipitation is c. 1859 mm, 86% of which falls from May to
 October, and mean annual relative humidity (RH) and temperature are 82.6%
 and 11.6 °C, respectively (Fig. 1). Mean monthly photosynthetic active
 radiation at 18 m above forest ground layer between September 2014 and
 August 2017 was higher than at 1.5 m, whereas mean relative humidity was
 lower (Fig. 1). There is a rich and abundant bryophyte flora in the area,
 including 176 species belonging to 38 families and 83 genera (Ma et al.,
 2009), so we selected six dominant species from each of three
 microhabitats in the forest (ground, tree trunk, and branch) (N = 18)
 based on species frequency data (Ma et al., 2009; Ma et al., 2011), and
 classified them according to life–form (after Bates, 1998) (Table 1). The
 canopy height is 18-25m (Li, 2010) and we mainlycollected samples from
 dominant trees (<i>Lithocarpus hancei, Castanopsis rufescens, L.
 xylocarpus</i>, etc.) in the study area and collected bryophyte
 samples from tree trunks at 0.5-1.5m, and branches at 10-15m above ground,
 and ground-dwelling bryophytes were collected from forest floor around
 host trees. Five 20 × 20 m quadrats were randomly placed in a 6-ha plot
 near Ailao Mountain Station for Subtropical Forest Ecosystem
 Studies.. <strong>Leaf and cell morphological
 traits </strong> We selected four leaves from
 five shoots of each species that were mounted and photographed (Olympus
 IX51 microscope and Olympus DP 80, Tokyo, Japan) at 20–400× magnification.
 We measured leaf area (LA), leaf length (LL), leaf width at the widest
 part (LW), length/width ratio (L/W), costa length (CL), and
 perimeter<sup>2</sup>/area ratio (P<sup>2</sup>/A)
 using ImageJ software (U.S. National Institutes of Health, Bethesda,
 Maryland, USA), and 4–6 cells from the central area of the lamina were
 measured to determine cell lumen area (CLA), cell lumen length (CLL), cell
 lumen width (CLW), and cell wall thickness (CWT); cells per area (CPA) was
 measured from 15–20 cells by circling a center area (diameter=0.05mm) of
 leaves. <strong>Photosynthetic light response
 traits </strong> We
 collected six replicate samples of shoot material from each species, from
 March to April 2017, from which photosynthetic light response curves were
 measured using a portable photosynthesis system (LI–6400, Li–Cor, Inc.,
 Lincoln, NE, USA). After dirty and dead tissue had been removed, 2.5 to
 4.5 cm long samples of stem material were cut from the apex or whole plant
 by removing the rhizoids; the stem material was then submerged and
 saturated in distilled water for 1 h. Then, excessive superficially
 adherent water was carefully removed using a paper towel (Zotz et al.,
 1997), before samples were placed in a whole plant chamber (LI–6400–17)
 attached to a portable infrared gas analyzer (LI–6400XT) to determine net
 photosynthetic rates; a full spectrum light source (LI–6400–18) that was
 placed on top of the chamber provided cold illumination to avoid dramatic
 changes in temperature. Photosynthetic rate based on bryophyte area and
 mass is lower than in vascular plants; therefore, we set the flow rate at
 300 µmol m<sup>−2</sup> s<sup>−1</sup> under
 ambient environmental conditions (14–20 °C; 50–70% RH). Light response
 curves were determined using 12 photosynthetically active radiation (PAR)
 gradients (300, 250, 200, 150, 125, 100, 80, 60, 40, 20, 10, and 0 µmol
 m<sup>−2</sup> s<sup>−1</sup>); after measurement
 of 3-4 PAR gradients, samples were submerged in distilled water for 1 min
 to maintain hydration. Light response curves were fitted to a modified
 rectangular hyperbola (Ye, 2007; Ye and Yu, 2008) using equation
 (1): <i>A</i><sub><i>n</i></sub><b>=α</b><i>1</i><sub>-</sub>β<i>I</i>1<sub>-</sub>γ<i>I</i><i>I<b>-</b></i><i>R</i><sub><i>area</i></sub> <b>  </b> (1) where <i>A<sub>n</sub></i> is the net photosynthetic rate derived from irradiance (µmol m<sup>−2</sup> s<sup>−1</sup>); <i>I</i> is light intensity (µmol m<sup>−2</sup> s<sup>−1</sup>); <i>R<sub>area</sub></i> is dark respiration rate (µmol m<sup>−2</sup> s<sup>−1</sup>); and α, β and γ are coefficients. Model fitting of the measured net photosynthetic rate to irradiance was completed using Sigma Plot 11.0 (Systat Software, Inc., San Jose, CA, USA); the software was used to directly fit the coefficients (α, β, γ, and R<sub>d</sub>). Subsequently, these coefficients were used to calculate the main photosynthetic parameters, including the light–saturated net photosynthetic rate (A<sub>area</sub>, µmol m<sup>−2</sup> s<sup>−1</sup>), light saturation point (I<sub>s</sub>, µmol m<sup>−2</sup> s<sup>−1</sup>) and light compensation point (I<sub>c</sub>, µmol m<sup>−2</sup> s<sup>−1</sup>). Shoot mass by area (SMA) was derived as the shoot dry mass divided by the projected area, while net photosynthetic rate by mass (A<sub>mass</sub>) and dark respiration by mass (R<sub>mass</sub>) were calculated as A<sub>mass</sub> = A<sub>area</sub>/SMA and R<sub>mass</sub> = R<sub>area</sub>/SMA, respectively. Projected shoot area was measured using an Epson Perfection V700 Photo flatbed scanner (Seiko Epson Corp., Nagano, Japan) and determined using WinRHIZO Pro 2009b (Regent Instruments, Quebec, Canada). Samples were oven–dried for 48 h at 70 °C after trait measurement to determine dry mass. <strong>Extraction and determination of chlorophyll pigments</strong> Five replicates of approximately 0.3 g of dry leaf tissue per bryophyte species were collected and stored at 4 °C, and then ground under dim light in 95 % ethanol with quartz sand and CaCO<sub>3</sub> powder (0.5g approximately respectively) using a mortar and pestle that had been pretreated at 4 °C. Extracted chlorophyll pigments were measured at 470, 649, and 665 nm absorbance using a spectrophotometer (UV–B 2501, Shimadzu, Japan), and concentration of Chl a, Chl b, carotenoid (Car), and total chlorophyll (Chl) were calculated according to the extinction coefficients of Arnon (Nasrulhaq–Boyce and Mohamed, 1987). <strong>Chlorophyll fluorescence </strong> Chlorophyll a fluorescence signals were measured using a PAM fluorometer (FMS 2, Hansatech, Instruments Ltd., Norfolk, UK) from five replicate leaf samples per species that were saturated in distilled water and dark–adapted for 23 min prior to measurement. Two or three overlapped leaves were selected growing in shoots to fill the measuring area (Φ=0.5cm) (Chen, 2016). Leaf material was exposed to a weak modulated beam to assess initial minimal fluorescence efficiency in the dark–adapted state (F<sub>0</sub>), and then a saturation pulse of c. 5500 µmol m<sup>−2 </sup>s<sup>−1</sup> for 0.7 s was applied to assess maximal photochemical efficiency when PSII reaction centers were closed (F<sub>m</sub>). Minimal and maximal Chl fluorescence efficiency and the steady–state Chl fluorescence efficiency in the light–adapted state ((F<sub>0</sub>′, F<sub>m</sub>′ and F<sub>s</sub>) were measured using actinic illumination (c. 110 µmol m<sup>−2</sup> s<sup>−1</sup>) and saturating illumination, respectively. Maximal photochemical efficiency of PSII in the dark-adapted state (F<sub>v</sub>/F<sub>m</sub>), where F<sub>v</sub> is variable fluorescence yield, was calculated as F<sub>v</sub> = F<sub>m</sub>−F<sub>0</sub>; actual photochemical efficiency of PSII in the light–adapted state (φPSII) was calculated as φPSII = (F<sub>m</sub>′−Fs)/F<sub>m</sub>′; photochemical quenching (qP) was calculated as qP = (F<sub>m</sub>′−F<sub>s</sub>)/(F<sub>m</sub>′−F<sub>0</sub>′); and non–photochemical quenching (NPQ) was derived from NPQ = (F<sub>m</sub>−F<sub>m</sub>′)/F<sub>m</sub>′.