Intra-Annual Wood Formation of Cryptomeria fortunei and Cunninghamia lanceolata in Humid Subtropical China

Monitoring cambial activity is important for a better understanding of the mechanisms governing xylem growth responses to climate change, providing a scientific basis for tree-ring-based climate reconstructions and projections about tree growth under future climate scenarios. It plays an even more important role in investigating evergreen tree growth in regions with less distinct seasonal cycles. Subtropical evergreen forests have been studied in recent years for their sensitivity to climate change, but it remains unclear how xylem growth is driven by subtropical climates. To further understand the climate-growth response strategies of subtropical conifers, we micro-cored Cryptomeria fortunei and Cunninghamia lanceolata weekly in 2016 and 2017 at the humid subtropical Gushan Mountain in southeastern China. Our weekly growth monitoring showed that the vegetation periods of these two species were both approximately 2–3 months longer than trees in temperate and boreal forests. The growth of C. fortunei in 2016 and 2017 and C. lanceolata in 2017 showed a bimodal pattern of xylogenesis, which was induced by summer drought. The results also indicated that the earlier end of the xylem formation was related to the yearly drought stress. These findings provide more specific information about tree growth and evidence of how climate influences wood production at the cellular level in subtropical regions.


INTRODUCTION
A high portion of the carbon in a forest ecosystem is deposited in the xylem, and its dynamics play a crucial role in the forest carbon cycle (Pan et al., 2011;Cuny et al., 2015). Consequently, a better understanding of xylem growth in various ecosystems, which can be characterized by its onset, growth rate, and anatomical features, has helped improve predictions of forest responses to climate change (De Luis et al., 2011;Deslauriers et al., 2017;Ren et al., 2019).
The process of xylem growth, that is, xylogenesis, describes the phenological phases of various cellular development processes involved in wood formation, as well as the factors driving growth rates (Deslauriers et al., 2017). The key parameters of xylogenesis include cell division, cell enlargement, cell wall thickening, lignification, and programmed cell death (Gričar et al., 2015;Rossi et al., 2016;Deslauriers et al., 2017). Previous studies on cambial phenology (both observations and controlled experiments) in temperate and boreal forests have demonstrated the crucial role of temperature in the onset of cambial growth (Rossi et al., 2008Begum et al., 2013;Bryukhanova et al., 2013;Chen et al., 2019). In the Mediterranean forests, the onset of xylogenesis is mainly determined by temperature (Gričar et al., 2006;Camarero et al., 2010;Vieira et al., 2014). Moreover, cambial activity may have a bimodal pattern, with a low growth rate in the summer due to heat and drought stress (Balducci et al., 2013;Garcia-Forner et al., 2019). In semiarid alpine forests, low spring precipitation combined with low temperatures delays the onset of the growing season (Ren et al., 2015;Amoroso et al., 2017). The mechanism of xylogenesis cessation is still relatively unexplored Marchand et al., 2020). In boreal forests, low temperature or a short photoperiod plays an important role at the end of xylogenesis (Hamilton et al., 2016;Guada et al., 2020). In contrast, the end of xylogenesis in the dry or semidry regions is more related to drought stress (Delzon et al., 2015;Ren et al., 2015;Novak et al., 2016;Zhang et al., 2018;Fonti et al., 2020).
Among the various ecosystems investigated in most previous studies, subtropical forests are underrepresented. However, it has been suggested that subtropical forests are sensitive to climate change , and could suffer more frequent or severe heat and drought stresses in the future (Stocker et al., 2013;Liu Q. et al., 2019). The phenology of xylem growth and how it is affected by climatic drivers remain uncertain in subtropical forests (Dhirendra Singh and Venugopal, 2011;Singh and Venugopal, 2011;Pumijumnong and Buajan, 2012;Huang et al., 2018;Rahman et al., 2019;Zhang et al., 2019). Indeed, the xylem growth of subtropical forests exhibits different dynamic patterns as observed in a limited number of existing studies. For example, Yan et al. (2013) revealed a long growing season, which spanned almost a whole year in the subtropics, while Huang et al. (2018) observed an inactive semidormancy period in January. There are also subtropical species (e.g., Cunninghamia lanceolata), which show bimodal growth due to seasonal drought as visible in stem radial variation .
Additionally, most current studies in the subtropics have only explored the intra-annual variability of tree growth (Huang et al., 2018;Liu et al., 2018), but comparative studies on the interannual differences in intra-annual wood formation are limited . Based on the few climate conditions investigated in the existing studies, only limited generalizations could be made regarding xylem growth in subtropical forests and their responses to environmental changes. Therefore, high-resolution monitoring of cambial phenology is needed, especially with more climatic conditions included. This will improve our understanding of xylem growth dynamics in subtropical forests and subsequently help reduce the uncertainty in predicting growth trends in the context of climate change (Huang et al., 2018).
Cryptomeria fortunei and Cunninghamia lanceolata are fastgrowing conifer species inhabiting subtropical China and are widely planted because of their high-economic value. In addition, these two species provide important ecological benefits, including carbon sequestration, soil erosion conservation, and diminishing atmospheric pollutants (Tian et al., 2011;Zhao et al., 2017). We aimed to compare the onset and end of xylogenesis as well as the growth rate between C. fortunei and C. lanceolata in relation to climate variables during the two sampling years. We hypothesized that (i) summer drought triggers bimodal growth, and (ii) drought stress induces the earlier end of xylem formation. The results of the present study provide more specific information about subtropical tree growth and could help improve the modeling strategy for the carbon cycle in subtropical forest ecosystems.

Study Site
This study was conducted in a subtropical evergreen forest on the north-facing slopes of the Gushan Mountains at an elevation of 450 m a.s.l. (26.05 • N, 119.38 • E) (Figure 1). The forest canopy is dense, and the overstory is dominated by conifer species (C. lanceolata, Pinus massoniana, and C. fortunei) mixed with a small number of Castanopsis carlesii and Schima superba. The soil is classified as Humic Acrisols with high moisture content. The climate is subtropical, with an annual mean temperature of 19.9 • C and total annual precipitation of 1,366 mm (according to the records of the near Fuzhou meteorological station from 1954 to 2017). The Western Pacific Subtropical High and the Spring Persistent Rains strongly affect the region, and it usually has wet springs and dry summers. Spring precipitation (April to June) accounts for 40.6% of the annual precipitation, while the typhoon season (from July to September) accounts for 31.8% of annual precipitation, but these percentages have high inter-annual variability. July is usually the driest month with an average precipitation of 137 mm and the highest average temperature of 28.72 • C. Extreme hightemperature events most often occurred in July and August (Supplementary Figure 1A).
To assess weather conditions during 2016 and 2017, daily meteorological data, including daily mean temperature ( • C), total precipitation (mm), and sunshine duration (h), were obtained from the Fuzhou weather station (Figure 1). There was a comparable mean air temperature of 23.8 and 23.9 • C during March to November in 2016 and 2017. However, the mean air temperature from March to June in 2016 was 0.7 • C higher than in 2017 (21.4 vs. 20.7 • C), and the mean air temperature from July to November was 0.7 • C higher in 2017 (Figure 2A and Table 1). From March to November, the sum of precipitation was 1,969.9 mm in 2016 and 1,282.3 mm in 2017. However, the total precipitation from March to June in 2016 was 256.8 mm lower than it was in 2017 (659.9 vs. 916.7 mm). Few precipitation events were observed from July to November in 2017, and the mean precipitation during September to November was 41.5 mm, indicating a serious drought ( Figure 2B and Table 1). The lower   Long-term means for the full period of operation of the monitoring  are reported in parentheses.
precipitation from July to November 2017 was consistent with the higher vapor-pressure deficit (VPD) (Figures 2C,D and Table 1).

Sample Collection and Preparation
We selected Cryptomeria fortunei and Cunninghamia lanceolata in a subtropical evergreen forest in southeastern China for observation and collected microcore samples for two consecutive years to investigate the intra-annual variability of the xylem growth of these two species. Microcores were collected weekly from C. fortunei and C. lanceolata trees from March to November 2016 and from February 2017 to January 2018. Three healthy and mature trees of each species were randomly selected and micro-cored at breast height (1.3 m) using a Trephor tool (Rossi et al., 2006). The monitored C. fortunei and C. lanceolata trees had an average diameter at breast height of 32.8 ± 1.4 cm and 23.7 ± 1.9 cm, a height of 10.9 ± 1 m and 13.1 ± 0.9 m, and an age of 36 ± 1 years and 24 ± 1 years, respectively. Micro-cores were sampled at least 5 cm apart to avoid wound reactions from nearby sampling points. The cores were 2 mm in diameter and 20-30 mm in length, containing two to five annual rings and a cambium zone with adjacent phloem (Rossi et al., 2006). The microcores were stored in formalin-ethanol-acetic acid solution (mixing ratio: 5:90:5) immediately after sampling in the field . In the laboratory, the microcores were dehydrated in an ethanol gradient (70, 90, 95, and 100%), cleaned in a clearing solvent of D-limonene, and embedded in paraffin. Transverse sections (9-12 µm thick) were cut with a rotary microtome (YD315, Jinhua YIDI Medical Appliance Co., Ltd., China) and stained with safranin (3 in 95% ethanol) and Astra blue (0.5 in 95% ethanol).

Microscopic Observations
To assess the dynamics of annual xylem ring formation, we counted the number of tracheids during the stages of enlargement, wall thickening, and maturation on each collected microsection along with three representative radial files (Rossi et al., 2006;Gärtner et al., 2015;Deslauriers et al., 2017). We identified (i) cells in the phase of enlargement when the cell was at least two times larger than the cambial cells, (ii) cells in the phase of wall thickening when the birefringence of the secondary walls appeared under polarized light (Abe et al., 1997), and (iii) cells in the phase of matured when the tracheid walls were lignified and cell protoplasts disappeared (Rossi et al., 2006;Deslauriers et al., 2017) (Supplementary Figure 2). The onset of xylem formation in spring was defined when at least one cell entered the enlargement phase, while the end of the season in autumn was determined when there were no more wall thickening tracheids (Rossi et al., 2006).

Data Analysis
Cambial phenology was computed for days of the year (DOY), corresponding to dates of (i) the onset and (ii) the end of cell enlargement, (iii) the onset, and (iv) the end of wall thickening. Micro-cores were taken from different positions within the tree circumference during the growing season (Rossi et al., 2003). Circumferential variability in cambial activities, including ring width, cell number, cell diameter, and wall thickness, exist at different positions of the stem (Creber and Chaloner, 1984). Thus, standardization of the cell number was required : In Equation 1, where nc i is the standardized number of xylem cells, ncm i is the measured number; of xylem cells, rw m is the mean ring width of previous rings for all samples, and rw s is the ring width of previous rings for each sample. We conducted ANOVA and Tukey's tests for the timing of the onset of each xylogenesis phase between years and used the results to assess the dynamics of intra-annual xylem ring formation (Queen et al., 2002).
We then fitted the total number of xylem cells (enlargement + wall thickening + mature cells) of each tree and year over the whole growing season with the Generalized Additive Model (GAM). Subsequently, we averaged at the tree and year level to indicate the intra-annual variation in the progress of wood production (Cuny et al., 2013).
In Equation 2, where y is the total number of xylem cells, x 1 is the day of the year (DOY), and f represents the smoothing function. The Poisson link function was used, given that the response variable is a count variable. To fit GAMs, the mgcv package was used within the R statistical software environment (R Core Team, 2013). The non-negative value of the derivative of GAM was used to obtain the rate of cell division (Huang et al., 2018;Yu et al., 2019).
We used Pearson coefficients to evaluate the relationship between growth rate, cambium cells, enlargement cells, and climatic variables. The number of cambium and enlargement cells was chosen during the onset and the end of enlargement, while the growth rate was calculated as the mean value between successive sampling dates. Three climatic variables were included: daily mean temperature, precipitation, and VPD. All factors were calculated as averaged values, except for precipitation for which we used summed values between successive sampling dates (7 days). For C. fortunei, the sample sizes of cambium cells and enlargement cells were 87 and 76 in 2016 and 2017, respectively. For C. lanceolata, the sample sizes of cambium cells and enlargement cells were 58 and 67 in 2016 and 2017, respectively. We also used Pearson coefficients to evaluate the relationship between the duration of wood formation and climatic variables (annual mean temperatures, precipitation, and VPD).

Cambial Phenology
In general, Cryptomeria fortunei had a higher growth rate and longer growth duration than Cunninghamia lanceolata ( Table 2). For C. fortunei, the onset of xylogenesis occurred on 8 April ± 1.7 days (mean ± SEs; DOY, 99.3 ± 1.7) and 4 April ± 6.2 days (DOY, 94.3 ± 6.2) in 2016 and 2017, respectively. The cambial cells reached the peak of 4-6 cells soon after the onset and then gradually decreased until they were stable within the range of 2-4 cells. Cell wall thickening started on April 15 ± 5 days (DOY, 106 ± 5) in 2016 and April 13 ± 4.  Table 2).
The cell wall thickening showed a bimodal pattern, with two peaks of cell activity in spring (April-June) and late summer to autumn (September-November) in both species across the study period except C. lanceolata in 2016 (Table 2 and Figures 3B,C). The duration of xylogenesis was significantly (p < 0.01) different between species, but not between years (Supplementary Table 1).
In 2016, C. fortunei and C. lanceolata showed a relatively higher growth rate and increased cell growth in summer. In 2017, both species showed a bimodal pattern of the growth rate, with a relatively high growth rate in spring and a relatively low growth rate in summer (Figure 4).

Wood Formation and Climate
Both Cryptomeria fortunei and Cunninghamia lanceolata have obvious inter-annual differences in the relationship between intra-annual growth and climate. Due to the large sample sizes, the correlations are significant, although their values are not that high. For C. fortunei, temperature and VPD showed significant negative impacts on the cambium and the enlargement of cells in 2016, and precipitation showed significant positive impacts on the cambium cells. In 2017, precipitation and VPD had no significant effect on the phase of xylogenesis, while temperature had significant negative effects on the enlargement of cells (Figure 5). For C. lanceolata, temperature and VPD showed significant negative impacts on cambium cells in 2016, and there was no significant correlation between the enlargement of cells and climatic factors. In 2017, temperature and VPD had significant negative impacts on the cambium and the enlargement of cells. Precipitation had no significant impact on the cambium and cell enlargement in C. lanceolata from 2016 to 2017 (Figure 6). There was no significant correlation between climate factors, growth rate, and duration of xylogenesis in the two species in 2016-2017 (Supplementary Figures 3, 4).

Climatic Control on the Onset and Termination of Cambial Activity
The two species (C. fortunei and C. lanceolata) included in this study started growing between March and April, while temperate  . Thus, the duration of the growing season of our studied species was approximately 2-3 months longer than that in the temperate and boreal zones, but shorter than that in the Mediterranean zones. Compared to other research in subtropical regions, the later onset and the earlier termination of xylem formation in this study lead to a shorter growing season (Huang et al., 2018), which may be caused by the geographic location and local climate differences. The onset of cambium activity of the two species in 2017 occurred earlier than in 2016, but this difference was not significant. The temperature has long been recognized as the major trigger for growth initiation in coniferous and broadleaf trees. This is true across temperate and boreal forests, with the physiological threshold daily minimum temperature being more than 4-5 • C (Rossi et al., 2008;Li et al., 2016). Higher temperatures in early spring can induce earlier reactivation and differentiation of secondary xylem (Begum et al., 2009(Begum et al., , 2017. For example, the higher temperatures in the spring of 2016 compared with 2015 induced an earlier onset cambium of Quercus variabilis on Qinling Mountain (China) . Moreover, monitoring results of Taiwan pines at different altitudes in the Lushan Mountains show that trees at low altitude sites will start activity earlier than those grown at high altitudes due to higher air temperatures (Liu X. et al., 2019). In this study, mean air temperatures during the 7 days before the onset of cambium activity of C. fortunei were 18.9 • C in 2016 and 16.7 • C in 2017. Temperatures for C. lanceolata in the week before the onset of cambium activity were 21.3 • C in 2016 and 19.5 • C in 2017 (Table 3). However, the increase in temperature in 2016 did not induce the earlier onset of cambium activity. Similar results have been found in other studies in the subtropics (Liu X. et al., 2019;Zhang et al., 2019). Consequently, our results indicate that temperature may not be the only factor influencing wood formation in C. fortunei and C. lanceolata.
Trees need water to generate turgor pressure during cell expansion, so precipitation could also drive the onset of cambium activity, and the effect may depend on the geographic location and local climate (Trouet et al., 2012). Previous research has found that, in arid and cold regions, precipitation is a trigger for the onset of xylogenesis in Juniperus przewalskii (Ren et al., 2015(Ren et al., , 2018Zhang et al., 2018). In the present study, total precipitation during the 7 days before the onset of cambium activity in C. fortunei was 14.8 mm in 2016 and 40.1 mm in 2017. In the case of C. lanceolata, the total precipitation measured was 53.6 mm in 2016 and 36 mm in 2017 (Table 3), as cambium activity was not delayed in 2017 despite the lower precipitation before cambium cell division of C. lanceolata. This is consistent with previous studies showing that precipitation has a weak influence on xylem growth resumption for Quercus variabilis in the subtropical region . Indeed, the influence of precipitation on the  onset of xylogenesis in hot and moist regions remains uncertain. For instance, in tropical regions, the seasonal variability of precipitation regulates cambial activity. Active cambium in several hardwood species has been associated with a rainy season (high rainfall), and dormancy is associated with the dry season (low rainfall) (Rahman et al., 2019). However, Trouet et al. (2012) revealed that the beginning and the end of cambial growth of Brachystegia spiciformis did not coincide with the beginning and the end of the rainy season. It is worth noting that, although both tree species in our study started to onset earlier in 2017, considering the ideal and similar spring hydrothermal conditions in 2016-2017, it is difficult to determine which climatic factors play an important role in initiating cambium activity. In 2017, the study area experienced severe consecutive droughts in late summer and autumn, and the average monthly precipitation from September to November was only 41.5 mm. Both C. fortunei and C. lanceolata ended wood production earlier in 2017. Worbes (1995) stated that an annual dry season of 2-3-month long and monthly precipitation of less than 60 mm would lead to cambial dormancy in tropical regions, which is consistent with our results. Underwater stress conditions, the extracellular water potential pressure suddenly drops even lower than the osmotic pressure of the enlarged cells. This causes the loss of turgor pressure, and the cell activity in the cambium area is weakened, thereby inhibiting the division of new cells (Abe et al., 2003). During periods of drought, cell wall synthesis and protein synthesis are substantially affected (Choat et al., 2012;Bader et al., 2013). Additionally, irrigation and natural experiments on mature trees found that the unirrigated trees/xericsite finished wood formation earlier than the irrigated trees/dry-mesic site Eilmann et al., 2011), confirming the critical role of water availability in stem growth cessation. Generally, drought-stressed termination of cambium activity has been observed in arid regions such as the Qilian Mountains in northwestern China (Zhang et al., 2021) and the Mediterranean region with summer drought stress (Bader et al., 2013;Garcia-Forner et al., 2019). In 2017, the earlier cessation of the cambium activities of C. fortunei and C. lanceolata indicates that, although in relatively humid subtropical regions, seasonal droughts may also limit tree growth.

Effects of Climate Factors on the Bimodal Pattern of Xylogenesis
Cryptomeria fortunei and Cunninghamia lanceolata showed different intra-annual growth dynamics between 2016 and 2017. We detected a bimodal pattern of xylogenesis and the growth rate for C. fortunei and C. lanceolata in the relatively drier year (2017). It is worth noting that this bimodal phenomenon in the research was more obvious when looking at the number of wall-thickening cells. The process of wall thickening depends mainly on the carbohydrates produced by photosynthesis (Cuny and Rathgeber, 2016). Such bimodal patterns of xylogenesis are in contrast to the studies in temperate and boreal forests where a bell-shaped annual pattern in cambium cell numbers was commonly found (Rossi et al., 2008); Ren et al., 2015;Chen et al., 2019). However, our finding is similar to the studies in the Mediterranean, where a bimodal pattern of cambium activity was reported as a result of distinct seasonal water availability (Camarero et al., 2010;Pacheco et al., 2016).
A warm and humid climate enhances photosynthetic ability, carbon fixation capacity (Kellomäki, 2001), and the activity of enzymes in relation to the photosynthesis of leaves. Consequently, photosynthetic capacity is improved, and the promotion of radial growth of the stem occurs (Strand et al., 2002). This can explain why C. fortunei and C. lanceolata could maintain a relatively high growth rate and cell increment during the summer of 2016, and a higher growth rate during spring in 2017 (Figure 4 and Supplementary Figure 5). In 2017, the high VPD accompanied by high temperatures in the study area from July to August is likely to aggravate the transpiration of the trees, which may lead to a decrease of stomatal conductance and limited photosynthesis (Mathur et al., 2014;Zhou et al., 2015;Ouyang et al., 2018;Grossiord et al., 2020;Song et al., 2020). These conditions may cause a decrease in carbohydrate availability and a decreased growth rate (Michelot et al., 2012;Zhang et al., 2021). Relevant studies based on tree rings and stable carbon isotope have also confirmed the negative effect of summer drought on tree growth on an interannual scale (Li et al., 2019;Bai et al., 2020;Dong et al., 2020).
We found that the percentage of net cell increment to the total xylem cells during 2016-2017 was consistent with the seasonal distribution of precipitation, and the final number of xylem cells was similar even though the precipitation showed such a remarkable difference. This result suggests that xylem growth in the subtropical zone can adjust its physiological activities to deal with environmental changes. Such plastic growth characteristics also offset the influence of climate to a certain extent.

Xylogenesis Difference Between
Cryptomeria fortunei and Cunninghamia lanceolata Marked differences were observed in the responses of xylogenesis to climatic factors between 2016 and 2017, especially in C. lanceolata. The negative effects of temperature (p < 0.01) and VPD (p < 0.01) on the enlargement stages of C. lanceolata were significant in 2017 but not in 2016. Moreover, the positive effect of precipitation on the enlargement stage of C. lanceolata was also enhanced. Both high VPD and low soil moisture are considered characteristics of drought (Liu et al., 2020). When the plant is in a high VPD situation, it increases the absorption of soil water by the root system to cope with the loss of water (Liu et al., 2020). Thus, we can infer that, in the relatively arid 2017, the xylogenesis process of C. lanceolata was affected by high temperature and drought. Lower drought tolerance caused by a smaller root system and less fine roots (Sun et al., 1990) may explain why C. lanceolata was more sensitive to moisture restrictions in 2017 than C. fortunei.
Wood production was determined by the duration of the growing season and the growth rate. The growth rate and duration of the growing season of C. fortunei are much higher than those of C. lanceolata, which explains the much higher number of xylem cells. Previous research found that the growth rate of C. lanceolata will decrease significantly after the mature stage, while C. fortunei can maintain a high growth rate even at the mature stage (Wu et al., 2001). A higher growth rate can increase the competitive ability, survival, and long-term success of a species, especially in severe environmental conditions (Silvertown, 2004;Grotkopp and Rejmánek, 2007). These observations confirmed that C. fortunei had a higher competitive advantage than C. lanceolata in mixed forests (Wu et al., 2001).

CONCLUSION
This study presented 2 years of weekly monitoring of xylem formation in two subtropical conifers (C. fortunei and C. lanceolata). The growing season in the humid subtropics was observed to last 2-3 months longer than in temperate and boreal forests. Observations also highlighted that drought was the dominant factor ruling the earlier end of xylem formation. Drought during summer induced bimodal xylogenesis in drier years. C. fortunei appeared to be less sensitive to drought and thus performed better than C. lanceolata. These findings provide evidence of how climate influences wood production at the cellular level in subtropical regions.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
KF, ZZ, and FZ designed the project. ZZ, FZ, and ZD performed field-based microcores. ZZ performed lab-based experiments, data analysis, and drafted the manuscript. PF, PR, XL, and GM contributed to the final version. All authors reviewed the manuscript.

FUNDING
This study was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB26020000), National Science Foundation of China (41822101, 41971022, 41888101, and 41772180), the State Administration of Foreign Experts Affairs of China (GS20190157002), fellowship for the National Youth Talent Support Program of China (Ten Thousand People Plan), the Natural Science Foundation Project of Fujian Province (2017J0101), and fellowship for Youth Talent Support Program of Fujian Province and the innovation team project (IRTL1705).

ACKNOWLEDGMENTS
We thank anonymous reviewers for their careful work and thoughtful suggestions that have helped improve this manuscript substantially.