Contrast in vulnerability to freezing-induced xylem embolism contributes to divergence in spring phenology between diffuse- and ring-porous temperate trees

1.   Introduction

Early spring frosts are particularly important as a natural selection factor for temperate trees in light of their high susceptibility to frost in the early stages of growth (Lenz et al., 2013; Kollas et al., 2014). With the end of ecodormancy, the potential for hardening in response to cold temperatures decreases dramatically and reaches a minimum as bud break, during which the frost resistance of bud is the weakest (Neuner, 2007; Vitra et al., 2017). Under the backdrop of global climate change, warmer spring temperatures lead to the advance of phenology in trees (Augspurger, 2013; Ge et al., 2015; Piao et al., 2019), but spring frosts may not advance at the same pace, which can potentially result in an increased probability of freezing damage in early flushing trees (Vitasse et al., 2014b; Chamberlain and Wolkovich, 2021). For example, the occurrence of freeze events such as the "Easter Freeze" of 2007 and the "Mother's Day Freeze" of 2015 resulted in the freezing of leaf buds, newly formed leaves, and shoots, causing catastrophic damages to trees (Gu et al., 2008; Augspurger, 2009; Arora and Taulavuori, 2016). The phenological periods of deciduous tree species growing in temperate climates exhibit substantial inter-specific difference (Takahashi et al., 2013; D'Orangeville et al., 2021), which can be related to their differences in cold tolerance, growth strategy, and competitiveness (Myneni et al., 1997; Hasenauer et al., 1999; Chuine and Beaubien, 2001; Chuine, 2010; Vitasse et al., 2014a). For example, broad-leaf tree species in temperate deciduous forests show a trade-off between leafing out early to maximize resource acquisition through longer growing periods and leafing out late to reduce the risk of frost damage (Lockhart, 1983; Saxe et al., 2001). While the spring phenology of different temperate tree species can strongly be linked to their responses to early spring cold waves, there is a lack of comprehensive research on the relationship between tree spring phenology and cold resistance and the underlying physiological mechanisms.

In addition to damaging buds, young leaves and cambium tissues that seriously affect tree growth, the early spring frost can also induce xylem embolism, resulting in hydraulic dysfunction and damage to trees that break dormancy and grow in advance. The freeze-thaw cycle is one of the main environmental threats that induces xylem embolism and can be more important than drought in threatening the hydraulic function of woody plants in the temperate zone of higher latitudes (Granda et al., 2014; Charrier et al., 2015; Niu et al., 2017; Yin et al., 2022). Temperate broad-leaf tree species have different adaptive strategies in dealing with embolism caused by freeze-thaw cycles. Some species have evolved root and/or stem pressures to repair winter embolized xylem conduits (Evert, 2006; Yin et al., 2018; Savage and Chuine, 2021), while others meet the water conduction demands primarily through the growth of new conduits in the spring (Cochard et al., 2001; Améglio et al., 2002; Niu et al., 2017; Dai et al., 2020). Many studies have shown that in the context of climate warming spring phenologies have been significantly advanced in temperate tree species, which can increase the water demand of trees that enter the growing period in advance (Root et al., 2003; Gordo and Sanz, 2005; Savage and Chuine, 2021). If cold waves induce xylem embolism at this phase, water transport from roots to new shoots will be hampered (Hacke and Sperry, 2001; Pittermann, 2010), which can lead to tree damage and even death (Hoffmann et al., 2011). Contrary to the fact that xylem embolism caused by drought in the context of climate change has been well documented as an important mechanism for accelerated tree mortality on a global scale, investigations on the impacts of early spring cold wave-induced xylem embolism on temperate tree species are still scarce.

Ring-porous and diffuse-porous species that often coexist in temperate forests show distinct differences in phenology and related adaptive strategies in terms of dealing with frost stress. Field observations have shown that ring-porous species usually leaf out later in spring than diffuse-porous species of the same communities (Lechowicz, 1984; Wang et al., 1992; Taneda and Sperry, 2008; Yu et al., 2016), which may be related to lower cold resistance in the former since buds of the late-flushing tree species usually have lower frost resistance than early-flushing species when compared at the same stage of development (Lenz et al., 2013; Vitasse et al., 2014a; Hänninen, 2016; Muffler et al., 2016). Moreover, the large vessels in earlywood of ring-porous tree species may be more susceptible to cold wave damage in early spring because vulnerability to freeze-thaw induced xylem embolism has a strong positive correlation with vessel diameter (Sperry and Sullivan, 1992; Davis et al., 1999; Feild and Brodribb, 2001; Pittermann and Sperry, 2006). The late spring phenology of ring-porous species relative to diffuse-porous species is likely an adaptation for the avoidance of freezing damages both to bud tissues and to the stem hydraulic systems. Together with early leaf drop in the autumn (Wang et al., 1992; Panchen et al., 2014; Gričar et al., 2020), the ring-porous species would have shorter periods of photosynthetic carbon assimilation. Ring-porous species may compensate for shorter annual periods of leaf carbon assimilation by higher rates of photosynthetic carbon assimilation (Chabot and Hicks, 1982; Umebayashi and Fukuda, 2018). It has been found that ring-porous tree species have substantially more efficient shoot hydraulic system than sympatric diffuse-porous species that would permit greater transpiration rates and photosynthetic rates due to the hydraulic-photosynthetic coordination (Meinzer, 2002; Yang et al., 2019; Xu et al., 2021). Considering the substantial intrinsic differences in spring phenology and freezing tolerance between ring-porous and diffuse-porous species, climate change factors may have contrasting influences on the two functional groups. Particularly, the more frequent cold waves are likely to be more threatening to the water transport systems of the ring-porous species since their large earlywood vessels are more prone to freeze-thaw induced embolism (Zimmermann, 1983; García-González and Fonti, 2006). Nonetheless, it is not clear whether the divergence in spring phenological and cold resistance between the two species groups are functionally coupled. In addition, a comprehensive comparative study involving spring phenology and xylem hydraulics between diffuse- and ring-porous trees would shed light on the understanding in the potentially divergent climate change responses between the two functional groups.

Here, under a common garden condition, we investigated the early spring leaf phenology and freezing tolerance in five ring-porous and five diffuse-porous deciduous tree species commonly found in temperate regions of China. The effects of freezing on young leaves and the hydraulic function of the stems were studied by simulating early spring cold wave events in the laboratory. In addition, stem hydraulic conductivity, leaf stomatal conductance in the growing season as well as branch radial growth rate of the 10 species were investigated. Specifically, the following hypotheses were tested: (ⅰ) the ring-porous species leaf out later in spring and their young leaves have lower resistance to damages by frosts; (ⅱ) the shoot hydraulic function of the ring-porous species would be more sensitive to simulated early spring cold waves due to the high vulnerability of their large earlywood vessels to freeze-thaw induced embolisms; (ⅲ) the ring-porous species would have greater hydraulic efficiency and photosynthetic carbon assimilation rate to compensate for shorter active growing period that would lead to comparable growth rate in comparison with the diffuse-porous species.

2.   Materials and methods

2.1   Study site and tree species

The study was carried out in the Kepu Park (41°11′51″‒43°02′13″ N, 122°25′09″‒123°48′24″ E), located in Shenyang, Liaoning Province, Northeast (NE) China. The region has a temperate semi-humid continental monsoon climate with strong seasonality in precipitation and temperature (Xu et al., 2014). During the period of 2000–2020, the mean annual temperature of the region is 8.8 ​± ​0.8 ​℃, with the lowest and highest monthly mean temperature occurring in January and July, i.e. −12.3 ​± ​0.5 ​℃ and 25.6 ​± ​0.2 ​℃, respectively. In the same period, the mean annual precipitation is 554.8 ​± ​22.2 ​mm, most of which occurs from June to August. The first frost day in Shenyang is in late September at the earliest, and the last frost day is in early May at the latest (Mu et al., 2018). The study site is located in a region sensitive to climate warming with the risk of spring frost damage gradually increasing over the past decades (Zhao et al., 2016; Deng et al., 2020). In recent years, the highest temperature in Shenyang could reach about 20 ​℃ in late March, but the lowest temperature in April still hovered around 0 ​℃, showing a strong "spring cold wave" feature (Wang et al., 2015). We selected five ring-porous species and five diffuse-porous species that are commonly found in temperature forests of NE China (Table 1). All trees sampled were mature and growing under a common garden, providing a homogeneous environment for the detection of intrinsic differences in frost resistance among these species of the two functional groups.

Table  1.  List of the 10 studied tree species belonging to the two different functional groups, i.e. diffuse-porous (DP) and ring-porous (RP) tree species. The semi-ring-porous species (SRP) is put into the RP functional group for data analyses considering its similarity in functional traits with the RP group.

Group code	Species	Species code	Symbol	Family	DBH (cm)
DP	Acer negundo L.	An		Aceraceae	22.32 ± 1.00
DP	Acer truncatum Bunge.	At		Aceraceae	14.38 ± 1.33
DP	Euonymus maackii Rupr.	Em		Celastraceae	8.92 ± 0.52
DP	Tilia mandshurica Rupr.et Maxim.	Tm		Malvaceae	17.32 ± 0.82
DP	Salix matsudana Koidz.	Sm		Salicaceae	29.92 ± 2.36
RP	Ailanthus altissima (Mill.) Swingle	Aa		Simaroubaceae	21.96 ± 1.16
RP	Catalpa ovata G.Don	Co		Bignoniaceae	18.93 ± 1.20
RP	Fraxinus mandshurica Rupr.	Fm		Oleaceae	24.45 ± 1.65
SRP	Juglans mandahurica Maxim.	Jm		Juglandaceae	22.05 ± 1.53
RP	Quercus mongolica Fischer ex Ledebour.	Qm		Fagaceae	27.80 ± 2.38

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2.2   Phenological observations in early spring

Leaf phenology of all the species was recorded from mid-March to early May of 2021 and 2022. Four mature and healthy trees were observed for each tree species. Branches at mid-height on the sun-exposed sides of the canopy of the trees were observed. Spring phenologies including bud swelling period (BSP), bud opening period (BOP) and the beginning of leaf expansion period (BLE) were visually monitored 1–2 times a week. All data were expressed in days of the year (DOY) by averaging the data of different individuals of the same species. Leaf phenologies showed no significant difference between the two consecutive years and were thus averaged. The BSP was defined by a swollen bud with visible leaf primordia, the BOP was defined by a visible petiole and leaflets and the BLE was defined by the date of 50% leaf visibility (Vitasse et al., 2009; Amico Roxas et al., 2021). The diameter at breast height (DBH) for each tree was also recorded.

2.3   Resistance of leaf membrane integrity to low temperatures

The leaf resistance to frost damage during late spring was evaluated using the electrolyte leakage method (Wilner, 1960). In early May 2021, three to five sun-exposed branches from different individuals per species were sampled. The sampled leaves were labeled and put into black plastic bags containing wet paper towels and transported to the laboratory immediately. The leaves were washed with distilled water twice, and the water on the leaf surfaces was wiped off with a paper towel. Leaves were tiled and kept non-overlapping in zip-lock bags, and the bags were then fixed inside a refrigerator and exposed to a temperature gradient of 21, 8, 0, −6, −11, −17 and −21 ​℃, with different sets of leaves being exposed to each target temperature. After leaves were treated at each target temperature for 2 ​h, about 0.2 ​g of the leaves were then taken out from each bag with a hole punch and put into a test tube with 40 ​mL distilled water. After the solution in each tube reached room temperature, the electrical conductivity (C₁) was measured with an electrical conductance meter (DDS-11A, INESA Instrument, Shanghai, China). Then the tubes were placed in a boiling water bath for 20 ​min to completely destroy the cell membrane system. The maximum electrical conductivity (C₂) of the solution with killed leaf tissues in each tube was then measured after the tubes cooled down. The electrical conductivity of distilled water was set as C₀. Leaf relative electrical conductivity (EC) at each treated temperature was calculated as EC (%) ​= ​[(C₁ ‒ C₀)/(C₂ ‒ C₀)] ​× ​100. The lethal temperature at 50% relative EC (LT₅₀) was calculated by fitting the EC values versus the corresponding temperatures and was used as a proxy for freezing resistance (Zhang et al., 2016).

2.4   Hydraulic conductivity and xylem embolism in perennial branches

The hydraulic conductivity measurements were conducted on three branches each sampled from a different individual for each species in July 2021. Straight, sun-exposed, long perennial branches ca. 1 m in length were sampled before dawn. The samples were quickly placed in black plastic bags with wet paper towels and immediately transported to the laboratory. Upon arrival at the laboratory, the branches were inserted into water and a ca. 10 ​cm segment was cut off from each branch at the bottom and the branches were allowed to rehydrate for about an hour. An unbranched stem segment ca. 25 ​cm in length was then cut off from the middle part of each branch under water to measure hydraulic conductivity (K_h-perennial). After the two cut ends were smoothed with a sharp razor blade, the stem segment was connected to a tubing apparatus with filtered and degassed 20 ​mmol·L⁻¹ KCl solution for hydraulic conductivity measurements. A hydraulic head of 50 ​cm was used to generate a hydrostatic pressure that drove water flowing through the segment. The flow rate was determined by recording the volume of liquid flowing through the segment using a graduated pipette during a certain period of time. The hydraulic conductivity of the stem segment (K_h-perennial) was calculated as follows: K_h-perennial ​= ​J_v/(ΔP/ΔL), where J_v is the flow rate through the segments (kg·s⁻¹) and (ΔP/ΔL) is the pressure gradient across the segment (MPa·m⁻¹). Following the measurements of native hydraulic conductivity, stem segments were flushed over 20 ​min using filtered (0.2 ​μm) and degassed 20 ​mmol·L⁻¹ KCl solution under a pressure of 0.1 ​MPa to remove xylem embolism. The maximum hydraulic conductivity (K_{max-perennial}) was subsequently measured. The percentage loss of hydraulic conductivity (PLC) was calculated as: PLC ​= ​100 (K_{max-perennial} ​− ​K_h-perennial)/K_{max-perennial.}

Stem sapwood area was determined using the dye-staining method on the same stem segments for hydraulic conductivity measurements. After hydraulic conductivity measurements, stem segments were connected to a tubing apparatus allowing basic fuchsin dye (0.1%) to flow through under a hydrostatic pressure of 5 ​kPa overnight. Short dye-stained segments ca. 1 ​cm in length were then cut from both ends of the stem and after shaving the cut ends with a sharp razor blade the transverse sections were scanned with a scanner (HP Scanjet G3110, Hewlett-Packard Development Co., Beijing, China). The wood cross-sectional area (WA) and the stained sapwood area (SA) were calculated using Image J software (US National Institutes of Health, Bethesda, MD, USA). Leaves distal to the stem segment were also scanned to estimate the leaf area (LA) using the Image J software. Leaves were then dried at 65 ​℃ for 48 ​h for dry mass determination, and LMA was calculated as the ratio of leaf dry mass to fresh leaf area. Leaf-to-sapwood area ratio (LA/SA) was calculated based on leaf area and sapwood area measurements. Wood-specific hydraulic conductivity (K_w, kg·m⁻¹·s⁻¹·MPa⁻¹), sapwood-specific hydraulic conductivity (K_s, kg·m⁻¹·s⁻¹·MPa⁻¹) and leaf-specific hydraulic conductivity (K_l, kg·m⁻¹·s⁻¹·MPa⁻¹) were calculated as K_h-perennial divided by WA, SA, and LA, respectively.

2.5   Freeze-thaw embolism induction in current-year shoots

For each of these 10 species, vulnerability to freezing-induced stem xylem embolism was evaluated on current-year shoots during the mid-growing season of 2021. The current year stems instead of the perennial ones were used because we aimed at investigating the damage of spring frosts to the newly formed stem xylem. Moreover, the high degrees of native embolism in the perennial branches, particularly in the ring-porous species, would prevent the precise measurement of PLC induced by the freeze-thaw treatment. We did not do the experiment in the spring because the current year branches were small and not completely lignified at that time and cannot technically meet the requirements of hydraulic measurements. Considering that the formation of embolism in vessels is primarily a process that is not affected much by the physiological state of branches (Hölttä et al., 2002; Secchi and Zwieniecki, 2011), the results of the freeze-thaw simulation conducted in the summer would not differ if it were done in the spring. A perennial branch about 1.5 ​m in length with at least two current-year shoots was cut off from each individual using a clipper and put into black plastic bags containing wet paper towels and transported to the laboratory immediately. Two current-year shoots were selected from each sampled branch and paired, for which one was used as a control sample and the other was used for freeze-thaw treatment. For the control current-year shoot, a stem segment about 15 ​cm in length was cut off under water, and the two ends of the stem segments were trimmed with a razor blade. A hydrostatic pressure driving water flow through the stem segment was generated by a hydraulic head of 20 ​cm. After the initial measurement of K_h-control, the stem segments were flushed at 0.1 ​MPa for 20 ​min to remove embolized vessels. Then the maximum hydraulic conductivity (K_max-control) was measured. The percentage of loss of hydraulic conductivity in the control group (PLC_control, %) was calculated as follows: PLC_control ​= ​100 ​× ​(K_max-control ​− ​K_h-control)/K_max-control.

The branch with the remaining current-year shoots was placed into a plastic bag, sealed, and then placed in a refrigerator (BC/BD-100HD, Qingdao Haier Special Refrigerator Co. Ltd, Qingdao, China) for 4 ​h under a preset temperature of ca. −20 ​℃. The actual air temperature in the refrigerator was monitored using a T-type thermocouple (Omega Inc., Norwalk, CT, USA) that was connected to a CR1000 data logger (Campbell Scientific, USA) and the data were logged at an interval of 1 ​min. The actual air temperature fluctuated between −19 and −25 ​℃ during the treatment. For the freeze-thaw treatment, substantially lower temperature than what plants may experience during early spring cold waves was used to guarantee the occurrence of freezing in the stem xylems, considering the fact that stems may freeze at a temperature substantially lower than 0 ​℃ due to the phenomenon of supercooling. This is justified for the purpose of investigating the impact of early spring frost on stem hydraulic functions since it has been demonstrated that PLC is determined by the number of freeze-thaw cycles rather than the absolute temperature during the freezing (Sperry et al., 1994; Mayr and Sperry, 2010). After the freezing treatment, the branches were placed on laboratory benches while kept in plastic bags for about 4 ​h to allow thawing and recovery to room temperature, and the initial hydraulic conductivity (K_{h freeze-thaw}) and the maximum hydraulic conductivity (K_{max freeze-thaw}) of the current-year shoot were then measured. The percentage loss of hydraulic conductivity in the freeze-thaw-treated current-year shoot (PLC_freeze-thaw, %) was calculated as follows: PLC_freeze-thaw ​= ​100 ​× ​(K_{max freeze-thaw} ​− ​K_{h freeze-thaw})/K_{max freeze-thaw}. The degree of xylem embolism induced by the freeze-thaw treatment was estimated as the difference (ΔPLC, %) between PLC_freeze-thaw and PLC of the paired control (non-treated) current year shoots (PLC_control, %).

2.6   Wood anatomy

A portion (ca. 2 ​cm in length) of each stem segment used for hydraulic conductivity measurement was used for xylem anatomical measurements. Transverse sections of approximately 20 ​μm thick were sampled using a sliding microtome (Model, 2010–17, Shanghai Medical Instrument Corp, Shanghai, China) and stained with 0.1% methylene blue solution to increase visual contrast. Images were taken under a magnification of 40× using a digital camera mounted on a light microscope (Leica ICC50, Wetzlar, Germany). All the sections were taken from non-current-year rings (the 3rd and/or 4th year rings), and three to six images were selected for each individual. Image J software was used to analyze the number of vessels and the area of each vessel in randomly chosen sectors from each cross-section. The diameter of each vessel was estimated based on its area, assuming a circular shape, and then the average vessel diameter (D, μm) of each tree species was calculated. Vessel density (VD, no.·mm⁻²) was determined as the number of vessels per unit xylem area. The hydraulically weighed vessel diameter (D_h) of a stem was calculated based on diameter measurements of all the measured vessels using the following equation: D_h ​= ​2(Σr⁵/Σr⁴), where r is the radius of a conduit.

2.7   Stomatal conductance

In July 2021, stomatal conductance was measured with a steady-state leaf porometer (SC-1 Leaf Porometer, Meter Inc., Pullman, USA) between 9:00 and 11:00 on sunny days. The sensor head was calibrated as per the company's protocol before taking the measurements. Three to five individuals were selected for each tree species, and three mature and sun-exposed leaves at positions of 1.5–3.0 ​m above the ground were measured for each individual.

2.8   Branch radial growth rate

For each tree species, three to five sun-exposed branches with each from a different individual were sampled for the measurements of stem radial growth rates. The branches sampled were six years old in all the studied species except Qm, for which 4-year-old branches were sampled since the 6-year-old branches would be too big to be allowed for sampling. Short segments ca. 1 ​cm in length were debarked and perfused with 0.1% methylene blue dye solution. After shaving the cut ends with a sharp razor blade, images of the transverse sections were taken using a scanner. Image J software was used to calculate the transverse sectional area of each growth ring and the pith area. The stem cross-sectional area increment (SAI, cm²·year⁻¹) and cumulative stem area (CSA, cm²) of each branch were separately calculated as follows:

where SAI_i and CSA_i is the value of SAI and CSA at the ith year. S₀ is the area of the pith, S_i is the cross-sectional area of previous i years containing the area of the pith, and S_i is the cross-sectional area of previous (i‒1) years containing the area of the pith. The same branches were then used for the determination of wood density (WD, g·cm⁻³) using the water displacement method. The mean branch growth rate (G_dw, kg·m⁻¹·year⁻¹) was calculated as the product of SAI and WD (Sterck et al., 2012).

2.9   Statistics

One-way analysis of variance (ANOVA) was used to test for differences in measured traits within each functional group, and the independent sample t-test was used to compare the differences between ring-porous and diffuse-porous species groups. The bivariate relationship between functional traits was evaluated by linear regression. Sigmoid curves were fitted to the relationship between EC and temperature for each species. The Pearson correlation coefficients between pairwise functional traits were used in the heatmap analysis, and principal component analysis (PCA) was used to analyze the multivariable relationship among traits.

3.   Results

The spring leaf phenology distinctly differed between the two functional groups (Fig. 1, Table S1), with the species having diffuse-porous wood sprouting and leafing earlier than the species having ring-porous wood. Although there was substantial interspecific variation across both functional groups, diffuse-porous species on average had bud phenology one to two weeks earlier than ring-porous species, with similar patterns in the timing of bud swelling, bud opening, and leaf expansion. The two diffuse-porous species Em and Sm had bud phenology earlier than any other species, whereas An and At of the same functional group had bud phenology overlapping with the ring-porous species having earlier bud phenologies. Notably, the semi-ring-porous Jm had the earliest bud phenology compared with the true ring-porous species and the bud phenology of the two ring-porous species Aa and Co was substantially later than that of any other species (Fig. 1).

Figure  1.  Average days of the year for the occurrence of different phases of bud development (1, bud swelling period, BSP; 2, bud opening period, BOP; 3, the beginning of leaf expansion period, BLE) in five diffuse-porous (crimson) and five ring-porous (blue) tree species. Symbols for each species are as defined in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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The hydraulic characteristics of the two functional groups differed significantly with the ring-porous species being less resistant to freeze-thaw induced embolism but having higher stem hydraulic conductivity in the growing season. While there was no significant difference in LT₅₀ between the two functional groups (P ​ > ​0.05, one-way ANOVA; Fig. 2b, Fig. S1), xylem vulnerability to freeze-thaw embolism expressed as ΔPLC was significantly higher in ring-porous species than that in diffuse-porous species (P ​ < ​0.05, one-way ANOVA; Fig. 2a). Consistently, perennial branches that had undergone recurring freeze-thaw cycles on average had a higher PLC in ring-porous species than in diffuse-porous species, i.e. 45.9% and 23.4%, respectively (P ​ < ​0.05, one-way ANOVA; Fig. 3c). There was no significant difference in PLC_control in the current year branch between the two functional groups (P ​ > ​0.05, one-way ANOVA; Fig. S2a). Following one simulated freeze-thaw event, however, the PLC_freeze-thaw in the current year branch increased from 21.3% to 38.3% in diffuse-porous species while it increased from 26.0% to 86.7% in ring-porous species (P ​ < ​0.05, one-way ANOVA; Fig. S2b). The two functional groups overall clearly diverged in stem xylem hydraulic efficiency in the growing season, with the K_s being, on average, significantly higher in ring-porous species than that in diffuse-porous species (P ​ < ​0.01, one-way ANOVA; Fig. 3a). When hydraulic conductivity was expressed on per unit leaf area basis, the values in the diffuse-porous species were comparable to those in the ring-porous species except for Aa (P ​ > ​0.05, one-way ANOVA; Fig. 3b).

Figure  2.  (a) Percentage loss of conductivity induced by a freeze-thaw cycle treatment (ΔPLC) in the current-year shoots of the 10 tree species belonging to the two functional groups, (b) subzero temperature leading to 50% of membrane leakage (LT₅₀). Error bars show 1 SE (n ​= ​3–5). Different capital letters above the horizontal lines indicate significant differences between diffuse-porous and ring-porous functional types (P ​ <  ​0.05, independent sample t-test). Different lower-case letters above the bars indicate significant differences between species within the same functional type (P ​ <  ​0.05, one-way ANOVA). Species name abbreviations are as defined in Table 1.

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Figure  3.  Functional traits related to water transport and use of the five diffuse-porous and five ring-porous species. (a) sapwood-specific hydraulic conductivity (K_s); (b) leaf-specific hydraulic conductivity (K_l); (c) the loss degree of hydraulic conductivity of perennial branch (PLC); (d) average diameter of vessel (D); (e) leaf area ratio sap-wood area (LA/SA); (f) stomatal conductance (g_s). Bars show the species mean values and error bars show 1 SE (n ​= ​3). Different lower-case letters above the bars indicate significant differences between species within the same functional type (P ​ <  ​0.05, one-way ANOVA). Different capital letters above the horizontal lines indicate significant differences between diffuse-porous and ring-porous functional types (P ​ <  ​0.05, independent sample t-test). Species name abbreviations are as defined in Table 1.

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Consistent with the tendency of ring-porous species having higher hydraulic efficiency than diffuse-porous species, the two functional groups differed in xylem anatomy, leaf-to-sapwood area ratio and gas exchange rate. Ring-porous species had significantly higher D and lower VD than diffuse-porous species (P ​ < ​0.05, one-way ANOVA; Fig. 3d, Table S2). The mean LA/SA was significantly lower in the diffuse-porous species than that in the ring-porous species, i.e. 186.4 ​± ​22.4 and 258.7 ​± ​28.9 ​cm²·mm⁻², respectively (P ​ < ​0.05, one-way ANOVA; Fig. 3e). The gas exchange rate was also significantly different with ring-porous species overall having significantly higher g_s than diffuse-porous species (P ​ < ​0.05, one-way ANOVA; Fig. 3f). In line with the significant difference in gas exchange rate between the two functional groups, the radial growth rate of branches was significantly higher in ring-porous species compared with diffuse-porous species (P ​ < ​0.05, one-way ANOVA; Fig. 4). During the first 4–5 years of branch growth, the mean SAI and CSA for each year were significantly higher in ring-porous species than that in diffuse-porous species (P ​ < ​0.05, one-way ANOVA; Fig. 4a and b, Table S3). Accordingly, ring-porous species also had higher G_dw than diffuse-porous species (P ​ < ​0.05, one-way ANOVA; Fig. 4c).

Figure  4.  (a) Stem area increment (SAI), (b) cumulative stem area (CSA), and (c) the mean branch growth rate (G_dw) for branches of 4–5 years old in the five diffuse-porous (crimson) and five ring-porous species (blue). The symbols are as defined in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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The spring leaf phenology showed consistent patterns of correlation with vessel diameter and xylem vulnerability to freeze-thaw induced embolism across the ten studied species. Species with higher vulnerability to freeze-thaw induced embolism showed later leaf phenologies, as indicated by the consistent positive correlations between ΔPLC and leaf phenological traits (i.e. BSP, BOP and BLE) across the ten studied species (Fig. 5a‒c). Similarly, the D consistently showed significant or marginally significant positive correlations with BSP, BOP and BLE (Fig. 5d‒f). There were significant positive relationships between BSP, BOP and K_s (P ​ < ​0.05; Fig. 7), but no significant relationship between the BLE and hydraulic conductivity was found (P ​ > ​0.05; Fig. 7). Across the ten studied species, the vessel diameter showed positive influence on water transport efficiency, gas exchange rate and branch growth rate but negative effect on resistance to freeze-thaw induced embolism (Fig. 6). Specifically, D was positively correlated with K_s, ΔPLC and g_s across the ten studied species (P ​ < ​0.05; Fig. 6a‒c, Fig. 7). Consistent with the positive correlations between plant water flux characteristics and vessel diameter, the mean branch growth rate (i.e. G_dw) also showed significant positive correlation with vessel diameter (P ​ < ​0.05; Figs. 6d and 7).

Figure  5.  Regressions for (a) bud swelling period (BSP), (b) bud opening period (BOP), (c) the beginning of leaf expansion period (BLE) against percent loss of hydraulic conductivity induced by one freeze-thaw cycle (ΔPLC) across the 10 studied species from two functional types. Regressions for (d) BSP, (e) BOP, (f) BLE against mean vessel diameter (D) across the 10 studied species belonging to the two functional groups. The symbols are as defined in Table 1.

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Figure  6.  Regression for (a) sapwood-specific hydraulic conductivity (K_s), (b) percent loss of hydraulic conductivity in current-year shoots (ΔPLC), (c) stomatal conductance (g_s), (d) the mean branch growth rate (G_dw) against mean vessel diameter (D) across the 10 studied species belonging to the two functional groups. The symbols are as defined in Table 1.

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Figure  7.  Heatmap showing correlations between all possible pairwise functional traits. Asterisks indicate significant correlations at P ​≤ ​0.05 level. BSP, bud swelling period; BOP, bud opening period; BLE, the beginning of leaf expansion period; D, mean vessel diameter; VD, vessel density; D_h, hydraulically weighed vessel diameter; WD, wood density; K_s, sapwood-specific hydraulic conductivity; K_w, wood-specific hydraulic conductivity; PLC, the loss degree of hydraulic conductivity of perennial branch; ΔPLC, the degree of embolism of the current-year shoots; LT₅₀, the low temperature semi-lethal temperature; K_l, leaf-specific hydraulic conductivity; LA/SA, Leaf area-to-sapwood area ratio; g_s, stomatal conductance; LMA, leaf mass per area; G_dw, the mean branch growth rate.

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Results of the principal component analysis based on the 17 traits showed that components 1 and 2 explained 45.8% and 15.1% of the total variation, respectively (Fig. 8). Functional traits related to resource acquisition (e.g. D, g_s, LMA, K_s) were located at the positive side, while traits related to frost resistance (1/BSP, 1/BOP, 1/BLE, VD) were clustered at the negative side of axis 1. Traits associated with water transport safety (e.g. PLC, ΔPLC) were clustered at the positive side, while traits associated with water transport efficiency (e.g. K_s, K_w) were located at the negative side of axis 2. The two functional groups were clearly separated in the space defined by axis 1 with the ring-porous species showing higher resource acquisition capacity in the growing season, and the diffuse-porous species having stronger frost resistance and earlier spring leaf phenology.

Figure  8.  Results of the principal component analysis (PCA) using 17 measured functional traits of the five diffuse-porous (crimson) and five ring-porous (blue) species. The symbols are as defined in Table 1. Abbreviations for the functional traits are as defined in Fig. 7. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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4.   Discussion

Our results showed that there were significant differences in spring leaf phenology and hydraulic characteristics between the ring-porous and diffuse-porous tree species growing under temperate climate conditions. The earlier leaf spring phenology of the diffuse-porous species was related to their strong resistance to xylem embolism, while the later leaf phenology of the ring-porous species is likely an adaptive strategy to avoid frost damage to their hydraulic systems that are more vulnerable to freeze-thaw induced embolism. Consistent with their contrasts in leaf phenology and xylem hydraulics, temperate tree species of the two functional groups diverged in leaf photosynthetic gas exchange and growth characteristics. Diffuse-porous species exhibited a relatively conservative resource acquisition and utilization strategy with lower hydraulic efficiency and leaf gas exchange rates, but they gain carbon paybacks over longer periods of active photosynthesis. Contrastingly, the ring-porous species have shorter growth periods due to late leaf out; however, their significantly higher hydraulic efficiency and photosynthetic gas exchange rates allowed them to compensate for shorter periods of leaf duration that lead to even higher growth rates than diffuse-porous species.

4.1   Correlation between spring phenology and resistance to freeze-thaw induced embolism

There is a significant difference in hydraulic characteristics between the late flushing ring-porous species and the early flushing diffuse-porous species, which indicates a trade-off between hydraulic efficiency and safety in face of the risk of freeze-thaw induced xylem embolism. According to the Hagen-Poiseuille's law, the large earlywood vessels of ring-porous species can ensure high hydraulic conductivity but make these species more susceptible to frosts because the xylem sensitivity to freeze-thaw induced embolism shows a strong positive correlation with vessel diameter (Zimmermann, 1983; Ewers and Fisher, 1989; Cochard and Tyree, 1990; Sperry et al., 1994; Taneda and Sperry, 2008; Jiménez-Castillo and Lusk, 2013; Beeckman, 2016). Ring-porous and diffuse-porous tree species in temperate areas have been found to show contrastingly different degrees of embolism in overwintering stems with PLC being much higher in the former group of species (Christensen-Dalsgaard and Tyree, 2014; Dai et al., 2020).

Our results showed that even a single freeze-thaw cycle can induce serious hydraulic dysfunction in current-year shoots of ring-porous species, which is consistent with the study reporting that after a hard frost the PLC in current year branches of Quercus rubra L. increased from 20% to 90% (Cochard and Tyree, 1990). It has been found that temperate ring-porous species "abandon" winter embolized vessels for water transport and rely mainly on the large vessels in earlywood of the current year ring to fulfill the hydraulic function (Hacke and Sperry, 2001). The much lower levels of embolism in stems of diffuse-porous species caused by the freeze-thaw treatment in the present study is consistent with findings that species of this functional type usually keep low levels of PLC over the winter even after experiencing many freeze-thaw cycles (Niu et al., 2017).

The contrasts between ring-porous and diffuse-porous temperate tree species in current-year stem resistance to freeze-thaw induced xylem embolism suggest that the two functional groups face different selective pressure from early spring frosts, which may at least be partially responsible for their divergence in spring phenology. It has been shown that leaf phenology and vascular cambial phenology are usually highly coordinated, i.e. tree species leaf out earlier also have earlier timing of vascular cambium activity in spring and vice versa (Perrin et al., 2017). Due to relatively high resistance to freezing-induced xylem embolism or effective winter embolism repair through the generation of positive xylem pressures before bud break, overwintering stems of the diffuse-porous species keep high hydraulic conductivity in early spring (Sperry et al., 1994; Hacke and Sauter, 1996; Améglio et al., 2002; Hao et al., 2013; Dai et al., 2020). Diffuse-porous tree species thus do not have to wait until the new growth of early wood to meet the transpirational water demand of leaves (Umebayashi et al., 2008; Takahashi and Takahashi, 2016). In addition, relatively strong freeze-thaw embolism resistance likely makes the cold waves having little impact on the newly formed xylems in stems of diffuse-porous tree species. Differently, the ring-porous species can only meet the water demand of active leaf transpiration after the large vessels in the current year earlywood become functional (Takahashi et al., 2013; Guada et al., 2019). Moreover, the significantly late phenology of ring-porous species contributes to the avoidance of irreversible damage to the hydraulic function of large and vulnerability vessels in earlywood caused by occasional frosts in early spring (García-González and Fonti, 2006; Lopez et al., 2008; Marchin et al., 2015). Notably, compared with the other ring-porous species, Jm's phenology launched earlier that is likely due to its low embolism over the winter since it has a unique winter embolism repair mechanism through the generation of positive pressures (Panchen et al., 2014; Niu et al., 2017).

4.2   Adaptive significance of divergent hydraulic architecture and phenology between the two functional groups

Contrasts in xylem hydraulics and spring phenology between diffuse- and ring-porous temperate tree species may have contributed to the diverging growth strategies between these two functional groups. The comparison between ring- and diffuse-porous temperate tree species can be viewed as the differences in species taking respectively the fast and slow strategies along the economics spectrum (Reich and Cornelissen, 2014). Ring-porous species can achieve much higher xylem water transport efficiency and significantly higher leaf gas exchange rates, which allow them to effectively exploit sufficient resources during the warm and wet growing season under the monsoon climate conditions and thus gain faster annual growth rate despite of late spring phenology (Yang et al., 2019; Castagneri et al., 2020; Yin et al., 2022). Indeed, the number of ring-porous and semi-ring-porous species in the Northern Hemisphere has increased since the Cretaceous, which has been attributed to the increase in climate seasonality (Baas and Wheeler, 2011). Although the ring-porous species have high potential maximum productivity, high vulnerability to freezing induced hydraulic dysfunction limited their ability to dominate harsher environments. Hence, ring-porous species usually dominate environments of lower elevations and lower latitudes, which is in contrasts to the diffuse-porous tree species (e.g. Acer and Betula species) that can often become dominant species at very high elevations and forest communities at the timber line of high mountains (Yang et al., 2019, 2020).

The warmer climate and more frequent extreme cold waves under the background of climate change may have different influences on tree species of the two functional groups. Overall, climate warming causes trees to sprout and grow earlier and trees dehardened in advance can suffer greater impairments in a climate regime having more climate extremes such as late spring cold waves (Zhang and Cao, 2009; Ma and Zhou, 2012; Neuner, 2014). There is great uncertainty about the relative impacts of climate change on these two functional groups of temperate tree species. It is possible that the diffuse-porous species sprouting earlier can be more seriously affected by more frequent cold waves if their spring phenologies are further advanced. Conversely, more frequent cold waves may have a stronger negative influence on the ring-porous species with greater vulnerability to freezing induced xylem embolism if cold waves proceed sufficiently deeper into the spring. More comprehensive ecophysiological comparative studies involving spring phenology and frost stress physiology of different temperate tree species would shed light on predictions of the potentially diverging climate change responses of different functional groups and changes in structure and function of temperate forests.

5.   Conclusions

Our results showed that diffuse- and ring-porous tree species differ significantly in spring leaf phenology, hydraulics, and growth strategy under the temperate climate conditions, where freeze-thaw induced xylem embolism poses a significant selective pressure on tree performance. Spring phenology and resistance to freezing-induced xylem embolism are functionally coordinated across the studied species and the divergence between diffuse- and ring-porous species in spring phenology can at least be partially explained by their contrasts in vulnerability to freezing-induced xylem embolism. The late phenology in ring-porous species for the sake of protecting their more vulnerable hydraulic system from early spring frost does not lead to reduced growth but is rather related to significantly higher growth rate, which is made possible by their substantially higher xylem water transport efficiency and leaf gas exchange rates allowing their leaves to gain even higher carbon paybacks over shorter periods. Climate change factors may likely influence tree species of the two functional groups differently due to their contrasting adaptations with respect to early spring environmental conditions and more comprehensive investigations involving phenology and resistance to climate extremes should be carried out for more thorough understandings on their potential divergences in climate change responses.

Data availability

Data supporting the findings of this study are available in the supplementary material of this article.

Authors' contributions

A.Y.W. and G.Y.H. conceived the ideas and designed the study. H.X.C. and N.W. conducted field and laboratory measurements. H.X.C., A.Y.W. and G.Y.H. analyzed and interpreted the data. H.X.C. and A.Y.W wrote the first draft. G.Y.H., X.W.G., and J.J.G. contributed extensively to the revision of the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Declaration of competing interest

The authors declare no conflict of interests.

Acknowledgements

We thank the staff at the Shenyang Academy of Landscape-gardening for supporting this study. Qiu-Rui Ning, Xiao-Han Yin, Yong-Jiao Zhou and Yue Zheng are acknowledged for assistances with fieldworks and guidance with the laboratory measurements.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fecs.2022.100070.