Data collection
China is the country with the highest number of conifer species in the world among which more than 90 species are endemics (Li, Shen, Ying, and Fang 2009). Meanwhile, the land mass of China covers a large latitudinal gradient from 18° N to 54 ° N. Besides, although the precipitation is generally higher in lower latitude regions, seasonal drought is common in Southwest China, where a hotspot of conifer species is situated (Yang 2015; Sundaram et al. 2019). Such diverse species and environmental conditions provide an opportunity for a cross-species study on the correlation between xylem structure and climate. We compiled a tracheid trait dataset for Chinese conifer species (Cheng Yang, and Liu 1993, Yang Lu, Liu, and Wu 2001; Jiang Cheng, and Yin 2010). All taxonomic names were verified using the Plant List (http://www.theplantlist.org/) to correct for synonyms, and varieties and subspecies were removed since the study is at the interspecific level. Finally, we obtained wood anatomical data for 79 conifer species across China (Fig.1). Most tree samples were taken from the Eastern Monsoonal climate zone in China, where the temperate, subtropical, and marginal tropical natural forests are distributed (Wu 1995). The maximum plant height for each species was extracted from Fu (2012) to check their potential relationships with conduit size.
Five cell dimensional traits from both earlywood and latewood for the 79 conifer species were selected, including tangential and radial tracheid diameter, tangential and radial tracheid wall thickness, and tracheid length (Fig. 2, Appendix 1, and Appendix 2). The anatomical data was mainly collected from publications by The Chinese Academy of Forestry, which hosts the largest wood collection in China, and the wood anatomical traits were measured following their methods (Cheng et al. 1993; Zhou and Jiang 1994; Yang, Cheng, Yang, and Lu 2009). In brief, most conifer tree species were sampled from mature trees over 60 years old with a DBH of over 20 cm in natural forests, as previous studies suggest that tracheid size is consistent in the sapwood of old trees (Zhou and Jiang 1994). The sampled discs were collected at a height of 1.3m, avoiding compression wood. These discs were cut, from pith to bark, into six equal parts according to the equidistance method and anatomical samples were taken from the middle of the outermost part (closest to the bark). These outermost samples were sectioned with a microtome and the entire span of a randomly selected growth ring analysed. Wood anatomical traits were measured using a light microscope, including tracheid diameter, tracheid wall thickness, and tracheid length for earlywood and latewood respectively. The early-and-latewood subzones were chosen as close as possible to the ring boundaries, and tracheids at the very end of latewood with irregular shapes were avoided. For tracheid diameter and tracheid wall thickness, 100 tracheids were measured randomly in the earlywood and latewood subsection of each sample. For tracheid length, 50 tracheids in earlywood and latewood from a randomly selected growth ring were measured using the maceration method after wood samples were treated with glacial acetic acid and hydrogen peroxide for one to three days (Yang et al. 2001).
As the sampling locations were not recorded for most of the species in the database, geographical coordinates for the distribution of each species in China were retrieved from Atlas of the Gymnosperms of China (Ying, Chen, and Chang 2004) and combined with climatic data extracted from WorldClim (https://www.worldclim.org/) and the aridity index from the global aridity index database (https://cgiarcsi.community/data/global-aridity-and-pet-database/) using the R package ‘raster’ (Hijmans, 2015). The environmental variables included: (1) Location indices: mid-point latitude (LAT, °), longitude (LON, °), and altitude (ALT, m). (2) Annual climatic indices: mean annual temperature (MAT, ℃), mean annual precipitation (MAP, mm), and mean annual aridity index (MAI, mm) which is defined as the ratio of mean annual precipitation and mean annual potential evapotranspiration. Climatic variables retrieved from different locations were averaged for each conifer species for further analysis.
Previous studies demonstrated that xylogenesis and the resulting xylem structure were species-specific (Rathgeber et al. 2016; Chen et al. 2019). Therefore, the effect of phylogeny was taken into account using various phylogenetic models in this cross-species study. We firstly constructed a phylogenetic tree for the species using the largest dated vascular plant phylogeny presently available, the GBOTB mega-tree of Smith & Brown (2018), which includes 79,881 taxa and all families of extant vascular plants based on combined molecular data from GenBank and data from the Open Tree of Life project (Smith and Brown 2018). We then pruned the supertree using the R package ‘V.PhyloMaker’ (Jin and Qian 2019) to generate a phylogeny for 79 conifer species.