Understanding how trees respond to environmental conditions (e.g., climate, soil, etc.) is crucial for an accurate prediction of future changes to forest dynamics caused by global warming, especially in the Northern boreal ecosystems (Vaganov et al., 2006; Zhang et al., 2020). A latitudinal gradient is associated with consistent temperature differences that can act as a natural laboratory, helping us to understand forest responses to global warming (De Frenne et al., 2013). Although there are studies on tree growth response to latitude, most of them focus on the effects of climatic factors on ring width and wood density for a limited number of species (Wettstein et al., 2011; Björklund et al., 2017), while there are few inter-specific studies on the xylem anatomical response to various environmental conditions. Conduit dimensions of xylem have historically been important traits for both angiosperms and gymnosperms given their multiple functions vital to tree growth (Hacke, 2015). Similar to angiosperm vessels, tracheid dimensions can provide us with valuable information about how conifer species adapt to various environments and their possible responses to climate change (Vaganov et al., 2006). However, the latitudinal patterns of tracheid traits have been surprisingly understudied compared to vessel traits (Brodribb et al., 2012).

Conifer xylem has less cellular diversity than angiosperm xylem, and consists of two cell types: tracheids (approx. 90–93% of the xylem surface area) and parenchyma (approx. 7–10% surface area) (Hacke, 2015). Temperate conifer tree rings are mainly composed of two tracheid types: (1) large and thin-walled earlywood tracheids, which are produced early in the growing season and are primarily responsible for water transport, and (2) small but thick-walled latewood tracheid, which are produced after the earlywood cells and serving for mechanical support (Fonti et al., 2013). Therefore, tracheid diameter and wall thickness represent useful proxies to examine tracheid cell profiles within a growth ring and across xylem. This is especially true in studies using old trees as samples, since tracheid size tends to be constant as the tree ages (Vysotskaya and Vaganov, 1989; Zhou and Jiang, 1994; Vaganov et al., 2006). In recent years, quantitative wood anatomy (QWA) emerged as a growing field of dendrochronology that allows obtaining a large number of parameters of xylem cells and structure, highlighting the adjustments of trees to their environment and relationship between cell structure and functions (Arzac et al., 2021). Understanding the temporal pattern of these traits and how the environment shapes them are of high importance for forest management.

Tracheid cell development processes are influenced by environmental conditions, especially climatic conditions (Castagneri et al., 2017). Recent studies on wood formation dynamics provide some insights into the process of xylogenesis, i.e., xylem formation (Rathgeber et al., 2016). For instance, Rossi et al. (2016) found that the timing of xylem phenology events and mean annual temperature of different sites were related linearly for 10 pine species in the Northern Hemisphere. The latter authors suggested that the uniformity of the process of wood formation was mainly determined by the climatic conditions occurring at the time of growth resumption (Rossi et al., 2016). In short, cell enlargement and secondary cell wall deposition and lignification (wall thickening) are the two fundamental sub-processes of xylogenesis. The complex interplay between the duration and rate of xylogenesis determines the changes in tracheid dimensions, e.g., cell and lumen diameter, lumen area and wall thickness, which in-turn creates the anatomical structure driving the wood density profile (Cuny et al., 2014). So far, our understanding of how environmental conditions affect xylogenesis is still incomplete and many relevant results were often species-specific and site-dependent with a strong focus on temperature effects (Cuny et al., 2014; Rossi et al., 2016; Buttò et al., 2019). There are relatively few interspecific studies on the effect of other environmental factors on xylem anatomical traits. Furthermore, previous studies on tracheid dimensions generally focused on radial tracheid diameter and wall thickness within-rings (Vaganov et al., 2006). Including tangential tracheid diameter and wall thickness for various conifer species could provide more valuable information in a comparative study.

Although previous research has suggested that hydraulic traits like tracheid dimensions are correlated with both precipitation and temperature, temperature is being increasingly recognized as the primary driver of conifer growth reactivation in a cold climate. However, most studies have only focused on temperate and boreal ecosystems in which snowmelt provides abundant water especially at the beginning of spring and summer, and where water availability is not a limiting factor for conifer xylem formation (Cuny and Rathgeber, 2016; Rathgeber et al., 2016; Rossi et al., 2016). Given that xylem cell expansion is a turgor-driven process depending on cellular water uptake and solute accumulation, water availability can affect xylogenesis (Kozlowski and Pallardy, 2002). Researchers have shown that both cell division and expansion are sensitive to water potential (Fonti et al., 2010), and that water deficit is the primary constraint for xylogenesis of Pinus pinaster in a Mediterranean climate (Vieira and Campelo, 2020). A study on Juniperus przewalskii provides additional evidence that soil moisture is more important than temperature at initiating xylem growth under cold and dry conditions (Ren et al., 2018). These results suggest that wood formation and xylem structure of various conifers could be controlled by genetic and multiple environmental factors such as temperature, soil moisture content and other soil properties, thus an interspecific study in a phylogenetic context and along a wide latitudinal gradient could shed light on this important research topic.

Although conifer xylem mainly consists of tracheids, the distribution of extant conifer species is globally as wide as angiosperms (Yang et al., 2017). Meanwhile, boreal forests at high latitudinal regions consist mainly of a few conifer species whereas in low latitudinal regions most conifer species generally tend in inhabit mountainous regions. The latter indicates that conifer trees are more adapted to stresses (i.e., coldness and drought) than angiosperm trees, allowing them to thrive in habitats unsuitable for angiosperm trees (Brodribb et al., 2012). Moreover, the adaptation of conifers is due to differences in life history strategies between them and angiosperms, such as traits that confer greater resistance to freezing and drought events and a high nutrient use efficiency (Brodribb et al., 2012). Therefore, adaptation to environmental conditions would be reflected in their xylem structure, especially in tracheid dimensions in both earlywood and latewood due to construction cost and water transportation constraints in addition to highly specialized bordered pits (Bouche et al., 2014). As tracheids of earlywood and latewood function differently, we hypothesize that the xylem structure of conifer species is mainly a consequence of adaptation to cold and/or drought stress, and that differences in tracheid dimensions between earlywood and latewood could be viewed as an ecological strategy to changing environmental conditions along a latitudinal gradient, especially temperature and precipitation. Specifically, we address the following questions: (1) do tracheid dimensions always differ between earlywood and latewood? (2) What is the role of phylogeny in associations among tracheid dimensions? (3) Are there clear trends of xylem structure along a latitudinal gradient? (4) How much of the xylem structure variance can be explained by environment and phylogeny, respectively? To the best of our knowledge, there is currently no cross-species anatomical study of conifers along such a wide gradient, making our study an important contribution to plant science.

Our results show that conifers adapt to their environmental conditions through changes in their xylem structure. Tracheid diameter and tracheid wall thickness for latewood decreased with a rise in latitude. Tracheid wall thickness, but not diameter for earlywood, also showed a decreasing latitudinal trend. Climatic condition and soil properties contributed to this latitudinal pattern, which explains about half of the variance for tracheid diameter and wall thickness. Meanwhile, phylogeny was found to play an important role in variance of conifer xylem structure, with Pinaceae species showing strong divergence in contrast to the conservatism of species from Cupressaceae, Podocarpaceae and Taxaceae. Our comparative study of 78 conifer species provides evidence of xylem adaptation to environmental conditions when factoring for phylogeny.

There is a widely held idea that earlywood is composed of wide, thin-walled tracheids in contrast to the narrow thick-walled tracheids of latewood. Through a phylogenetic paired t-test, our results showed that tracheid diameter in earlywood was larger than in latewood (Figures 2B,C and Supplementary Table 4), which partly supports the latter idea. An explanation could be that the duration of latewood tracheid development was shorter than for earlywood, while a large proportion of carbohydrates produced in the previous and current growing season had been consumed by earlywood cell wall thickening (Kagawa et al., 2006; Rathgeber et al., 2016). Since the principal function of tracheids in earlywood is water transport, in contrast to latewood, which functions more in mechanical support and water storage (Domec and Gartner, 2002), the xylem structure presented by the tracheid diameter in earlywood and latewood were consistent with their functions. A large tracheid size in earlywood results in a large lumen diameter, enabling higher water conductivity while also increasing the risk of hydraulic failure associated with freezing-induced embolism. By contrast, a small tracheid diameter (<30 μm) is assumed to protect against freezing-induced embolism for conifers at high latitudes or altitudes (Pittermann and Sperry, 2003).

Contrary to tracheid diameter, the difference in tracheid wall thickness between earlywood and latewood was insignificant when taking phylogeny into account (Figures 2C,D and Supplementary Table 4). A possible explanation for our result was that the difference in tracheid wall thickness between earlywood and latewood was species-specific and may not always manifest the same pattern for numerous conifers. Although conifer species in boreal forests may have thicker cell walls in latewood compared to earlywood, conifer species in warm and wet regions, e.g., species of Podocarpaceae and Araucariaceae in the Southern Hemisphere, may not behave in the same way (Carlquist, 2017). Previous studies also found that tracheid wall thickness in intra-ring cells was surprisingly constant for some Pinaceae species in temperate regions (Cuny et al., 2014). However, the majority of conifer species are distributed throughout subtropical climates in our study (Figure 1), which may explain the insignificant differences in tracheid wall thickness between earlywood and latewood. From a functional point of view, mechanical strength enhancement of latewood tracheids was achieved not by cell wall thickness but due to the increased wall-to-range ratio from reduced tracheid diameter, especially in the radial direction (Pittermann et al., 2006).

Although many anatomical traits of woody angiosperms have shown a latitudinal trend (Wheeler et al., 2007; Zheng et al., 2019), studies showing similar results on gymnosperm wood have been less abundant until recently (Rossi et al., 2016; Björklund et al., 2017). In our cross-species analyses, we revealed a decrease in tracheid wall thickness for both earlywood and latewood together with tracheid diameter in latewood along a latitudinal gradient in China (Figure 3). In such a large latitudinal gradient under a monsoonal climate, the carbon investment for conifer xylem formation would decrease in the northern regions owing to a shortened photoperiod and a lower temperature during the growing season, resulting in a reduction in tracheid diameter and tracheid wall thickness in latewood. The latter finding is in good agreement with the fact that tree growth declines toward high latitudes. Besides, tracheid radial diameters of latewood were generally lower than 30 μm (Figure 5B), which is consistent with the threshold diameter against freezing-induced embolism (Sperry et al., 1994; Pittermann and Sperry, 2003). Therefore, the decreased stem hydraulic capacity in a cold region might be a consequence of the evolution of reduced vulnerability to freezing and drought-induced embolism, representing an ecological strategy for conifers distributed across colder regions (Creese et al., 2011). For instance, the wide distribution of Pinus species in the Northern Hemisphere was thought to be due to adaptation to cold temperature during the Eocene (Millar, 1993).

Climatic conditions (i.e., MAT, MAP, and TSEA) were correlated with different tracheid traits to varying degrees (Supplementary Table 3), which was largely in line with findings that climate could strongly control wood anatomy and formation (Pandey, 2021). Previous studies on boreal conifers showed that the period of wood formation lengthened linearly with MAT in a range of 14°C and contributed to increased tracheid dimensions for 10 conifer species in the Northern Hemisphere (Rossi et al., 2013). However, our results showed that temperature, precipitation and temperature seasonality all contributed to lumen diameter and tracheid wall dimensions for multiple conifer species across various climate conditions (i.e., temperate, subtropical, and tropical climate) after taking phylogeny into account. We are aware of the limitations of the climate data extracted from WorldClim, as we could not analyze the effect of climatic conditions on wood formation for each location without introducing large bias. In addition, soil properties (i.e., PH, SILT, and CLAY) could explain part of the variance in tracheid traits, especially for tracheid diameter in earlywood that did not demonstrate a latitudinal trend (Table 2). Although conifers are largely confined to high latitudes and elevations or nutrient-poor soils, some conifer species nearly always grow better on more fertile soils than on infertile soils (Bond, 1989; Beets et al., 2001). A possible explanation for the role of soil on tracheid dimensions was that a high soil fertility could increase the growth rate of conifers, resulting in a larger tracheid diameter and thinner tracheid wall thickness (Bergh et al., 1999). As there are only a few studies on the effect of soil condition on tracheid dimensions compared to growth rate or wood density, how the interaction of soil and climate influences wood anatomy requires further investigation.

Xylem structure is a canvas for the evolutionary strategies of opposing functions through the production of two kinds of tracheid cells each year (Carlquist, 2017). Previous studies on conifer species from Pinaceae (Martínez-Vilalta et al., 2004), Cupressaceae (Pittermann et al., 2012), and Podocarpaceae (Turner and Cernusak, 2011) suggested that there are at least three evolutionary directions for conifer xylem adapting to different stresses in a geological time scale: cold adaptation, drought adaptation, and shade adaptation (Figure 6). For instance, both Cupressaceae spp. and Pinaceae spp. mainly grow in temperate climates. Cupressaceae are often restricted to more stressful cold and dry environments and show higher resistance to drought damage than coexisting Pinaceae. In a previous study on two coexisting conifers, Pinus halepensis and Juniperus thurifera, growing in a continental Mediterranean climate, it was shown that the two species significantly differ in the wood-anatomical features, such as distribution of tracheid lumen area and cell wall thickness (Pacheco et al., 2015). The authors explained their different vulnerability to drought-induced die-off by the fact that Cupressaceae have evolved toward a drought resistant xylem, while Pinaceae evolved more toward cold resistant xylem. However, since Cupressaceae and Pinaceae are temperate conifer families, including many species with varying capacity of drought and cold resistance, comparative studies between the two families are rare. A similar situation also applies to the tropical conifer families Podocarpaceae and Araucariaceae (Zimmer et al., 2015).

During the evolution of conifers, selection acted to optimize xylem structure to fulfill both safety and efficiency of water transport (Hacke, 2015). Conifers must adapt to diverse and often difficult environments by adjusting their xylem structure at different levels from micro-to-macro, e.g., the pit structure at the pit level (Bouche et al., 2014) through to tracheid dimensions at the cell level (Pittermann et al., 2006), and perhaps the amount of ray parenchyma at the tissue level (Olano et al., 2013). There are some hypotheses suggesting that coordinated traits combined – not one trait alone – determines the safety/efficiency requirements and ultimately the species distribution (Hacke, 2015; Carlquist, 2017). Recent physiological and anatomical studies on bordered pits provide new insights into the mechanisms and evolution of conifer’s drought resistance (Choat et al., 2008; Hacke, 2015). However, the eco-physiological functions and evolution behind earlywood and latewood tracheids has been overlooked. We postulated that tracheid dimensional differences between earlywood and latewood have evolved to allow conifers to adapt to various environmental conditions, especially freezing conditions and drought.

Resistance to embolism is a crucial trait in trees to cope with cold and drought stresses, which further determines the geographic distribution of conifer species (Choat et al., 2012). Embolism resistance might be influenced by the structure and function of bordered pits and tracheids in xylem as well (Losso et al., 2018). A small alteration in xylem anatomy can lead to different performances in terms of water transport efficiency, embolism resistance and hydraulic capacitance (Hacke, 2015). Our phylogenetic analyses provide some evidence for functional optimization of xylem structures for species from different clades (Figures 4, 6). However, due to a currently incomplete understanding of embolism in tracheids, it is unclear whether anatomical traits such as tracheid dimensions, pit structure (Roskilly et al., 2019) and amount of axial parenchyma (Olano et al., 2013), evolved in parallel during conifer evolution, making it an interesting topic for further study.

In this comparative study, variance of conifer xylem structure provides further evidence of conifer adaptation to various environmental conditions. In a latitudinal range from 18° N to 54° N, tracheid dimensions of 78 conifer species showed a consistent xylem pattern, where approximately half of the variance was found to be attributable to climatic and soil factors in tandem with phylogeny. The strong xylem structural divergence along phylogeny in Pinaceae species in contrast to Cupressaceae, Taxaceae and Cupressaceae further suggest that tracheid dimensions are an evolutionary outcome for conifers adapting to cold and drought stresses. Further work on the interspecific variability of conifer xylem would be required to better understanding hydraulic traits and functional adaptation to different environment for conifers. Given the fast development of image processing techniques and automatic climate and soil monitoring methods, we believe that a combination of information from radial growth, wood density and xylem anatomy will open up a new research area for tree-ring science and allows for the detection of large structural, biogeographical and environmental patterns as well as their interpretation, providing valuable references for future forest management under climate change.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

JZ conceived the study. YL and JZ collected the data and did the statistical analyses. JZ, HM, FV, and SJ wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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