2.1. Study site
The study was performed at the Qingdao forest ecosystem national positioning observation and research station in Shandong province, China (36°10’N, 120°37'E). This region is a typical representative area of warm temperate forests in China, with a climate type of warm temperate continental monsoon climate, an altitude of 389–700 m, an average annual precipitation of 900 mm, and an average annual temperature of 13℃. After conducting a preliminary vegetation survey in the station, it was found that plant species in the forest generally exhibit insect feeding, leading to a common phenomenon of leaf damage in the forest ecosystem (Fig. S1). The sample plot for this study was set according to the unified standard of long-term positioning observation of forest sample plots of the Chinese Ecosystem Research Network. Based on preliminary investigations, this study focuses on five common species in the sample plots of the research station: Q. acutissima, Q. serrata, Q. aliena, Q. dentata, and R. pseudoacacia. These species have sufficient sample sizes in the study plots, where insect defoliation phenomenon is common.
2.2. Experimental design
This study was conducted in September 2022 at the station. Based on a thorough investigation of the species composition, distribution, and insect defoliation in the wild, five representative and widely distributed common species in the warm temperate zone were selected, Q. acutissima, Q. serrata, Q. aliena, Q. dentata, and R. pseudoacacia. For each species, five individuals of similar age and good health status and five individuals with insect defoliation were selected and labeled. The leave samples were taken back to the laboratory for measurement of carbohydrate content, secondary metabolic content, nitrogen and phosphorus content, and other characteristics.
2.3. Non-structural carbohydrates
Non-structural carbohydrates (NSCs) mainly include soluble sugar (SS) and starch (ST). SS was extracted twice from 20 mg of dried tissue in 5 mL of 80% aqueous ethanol. SS from the supernatants was quantified by the anthrone-sulfuric acid method with a spectrophotometer (UV-9000S, Metash, Shanghai, China) at 620 nm. ST was measured after the solid residue of each sample had been extracted twice with HClO₄. Then, the absorbance at 620 nm was measured with the same spectrophotometer after an anthrone-sulfuric acid method. The content of SS and ST (measured as glucose equivalents) were calculated for dry mass (mg g− 1).
2.4. Lignin
The determination of lignin content was carried out using the acetyl bromide method. Weigh 5 mg of dried plant sample for acetylation treatment, measure the UV absorption value of the acetylation solution at 280 nm, and calculate the lignin content.
2.5. Tannin
Tannins have strong UV absorption at 275 nm, and the tannin content is detected by utilizing the properties of activated carbon that can specifically adsorb tannins. Dry the sample to constant weight, crush it, pass through a 40-mesh sieve, weigh about 50 mg, add 1 mL of extraction solution, seal with a sealing film to prevent liquid splashing, and extract in a 70 ℃ water bath for 30 min. Shake several times during this process. Centrifuge at 12000 rpm, 25 ℃ for 10 min, take the supernatant, and make up to 1 mL with the extraction solution for measurement.
2.6. Total phenols
Under alkaline conditions, phenolic substances reduce tungsten molybdate to produce blue compounds with characteristic absorption peaks at 760 nm. The total phenolic content of the sample can be obtained by measuring the absorbance value at 760 nm. Dry the sample to a constant weight, crush it, pass it through a 40-mesh sieve, weigh about 100 mg, add 2.5 mL of extraction solution, and use ultrasonic extraction method for extraction. The ultrasonic power is 300 W, the sample is crushed for 5 s, the interval is 8 s, and it is extracted at 60 ℃ for 30 min. Centrifuge at 12000 rpm, 25 ℃ for 10 min, take the supernatant, and make up to 2.5 mL with the extraction solution for measurement.
2.7. Flavonoids
In an alkaline nitrite solution, flavonoids form a red complex with aluminum ions that have a characteristic absorption peak at 470 nm. By measuring the absorbance value of the sample extract at 470 nm, the flavonoid content of the sample can be calculated. Dry the sample to constant weight, crush it, pass it through a 40-mesh sieve, weigh about 0.1 g, add 1 mL of extraction solution, and use ultrasonic extraction method for extraction. The ultrasonic power is 300 W, the sample is crushed for 5 s, the interval is 8 s, and it is extracted at 60 ℃ for 30 min. Centrifuge at 12000 rpm, 25 ℃ for 10 min, take the supernatant, and make up to 1 mL with the extraction solution for measurement.
2.8. Nitrogen and phosphorus
Grind and sieve the harvested dry leaf samples, and determine the total nitrogen content (%) using the Kjeldahl method. Weigh about 0.2 g of the sample and place it in a digestion tube. Add 0.2 g of copper sulfate, 3 g of potassium sulfate, and 8 mL of concentrated sulfuric acid. Digest at 200 ℃ for 40 min, then heat up to 400 ℃ and digest for 40 min. Rinse the liquid in the digestion tube multiple times and pour it into a 50 mL volumetric flask to volume. Take 10 mL of the liquid into a clean digestion tube and place it in a K9860 nitrogen analyzer (Hanon, K9860, China) to measure the nitrogen content. Take another 5 mL of the test solution and transfer it into a 50 mL volumetric flask. Measure the total phosphorus content (%) using molybdenum antimony colorimetry and UV spectrophotometer (Shimadzu, UV-2550, Japan),
2.9. Statistical methods
All variables were tested for normality of distribution and homogeneity before analysis. To explore the variation of leaf trait indicators between healthy and defoliated trees, we used analysis of independent sample t-test in SPSS 23.0 (IBM Corp., Armonk, NY, USA). Principal component analysis (PCA) was used to describe the relationships between leaf traits, which was performed using R (Oksanen et al. 2019).