Above and below ground functional traits in Xishuangbanna tropical rainforest

Locations containing streams or large rocks were excluded when selecting seedling plot sites. Each station consisted of a seed trap accompanied by three 1 m × 1 m seedling plots positioned 2 m from the sides of the trap. Within each seedling plot, all woody plants with DBH < 1 cm were classified as seedlings, tagged, and identified to species.

Seedling height was measured from the ground surface to the terminal apical bud and used to estimate height growth. The initial census of seedlings was conducted in November 2007, followed by repeated surveys twice annually at the end of the dry season (May) and the end of the rainy season (November) over the subsequent ten years. Seedling traitsIn November 2021, we quantified 23 functional traits (including eight defense traits) for 51 species using standard methodologies for plant trait measurements (Poorter & Markesteijn 2008; Perez-Harguindeguy et al. 2013).

We selected three seedlings < 50 cm in height per species for trait measurements and measured their leaf and root functional traits. Lateral roots were traced and carefully excavated from target seedlings to their finest first-order root tip using a sterilized hand trowel (wiped with 75% ethanol). Roots with rhizosphere soil intact were transported back to the lab in sterilized plastic bags (BKMANLAB Inc., China) and stored in a refrigerator (4 ℃) until processing (typically within 2 hours).

Root samples were processed by delineating roots into intact entire systems of the first 3 root orders (McCormack et al. 2015). Root systems were thoroughly washed, weighed (fresh mass), and then scanned using a double-sided optical scanner (EPSON Perfection V800, Epson America Inc.) in black and white at high resolution (600 dpi). Each root system was placed in an acrylic scanning tray, submerged in water, positioned flat, and scanned.

Scanned images were analyzed morphologically using WinRHIZO (2016 version, Regent Instruments, Quebec, Canada). WinRHIZO returns data on average diameter (mm), length (mm), volume (cm3) and surface area (mm2). From these three measurements and dry mass, five important root functional traits were determined (root average diameter: RD, root tissue density: RTD, root dry matter content: RDMC, specific root area: SRA, and specific root length: SRL). For seedling leaves, we measured leaf thickness with a precision micrometer screw gauge (Sanliang Inc., China).

We scanned all leaves using a digital scanner (EPSON Perfection V19, Epson America Inc.) and determined individual leaf area (LA; cm2) using Image J (Schneider et al. 2012) via the LeafArea R package (Katabuchi 2017). Leaf toughness was measured as the force required to penetrate the leaf using a needle-based force gauge penetrometer (Hp-10, Puyan Inc. China).

Next, we dried leaves from each seedling for 48 h at 65 ºC and then weighed each component to calculate the leaf dry matter content (LDMC 100 × dry mass per unit fresh mass; %). We calculated the specific leaf area (SLA; leaf area/unit dry leaf mass; m2/kg). After measuring morphological traits, dry leaf and root tissues were homogenized by Freeze Grinding Machine (MM400, Retsch, Germany). For the homogenized tissue samples, we measured total carbon (C) and nitrogen (N) concentration (mg/g) of both root and leaf tissue with a Dumas-type combustion C-N elemental analyzer (Vario MAX CN, Elementar Analysensysteme GmbH, Hanau, Germany).

We also measured defense-related traits for both homogenized root and leaf tissues, those being the concentration of tannins (mg/g), flavonoids (mg/g), non-structural carbon (NSC; mg/g), cellulose (mg/g), lignin (mg/g). All chemical components were measured using corresponding kits produced by Shanghai Enzyme-linked Biotechnology Co., Ltd. (www.mlbio.cn).