examined pit characteristics, the anatomical and photosynthetic traits of 13 cycads from a common garden, to determine if pit traits and their coordination are related to water relations and carbon economy. We found that the pit traits of cycads were highly variable and that cycads exhibited a similar tradeoff between pit density and pit area as other plant lineages. Unlike other plant lineages (1) pit membranes, pit apertures, and pit shapes of cycads were not coordinated as in angiosperms;

(2) cycads exhibited larger pit membrane areas but lower pit densities relative to ferns and angiosperms,but smaller and similar pit membrane densities to non-cycad gymnosperms; (3) cycad pit membrane areas and densities were partially coordinated with anatomical traits, with hydraulic supply of the rachis positively coordinated with photosynthesis, whereas pit aperture areas and fractions were negatively coordinated with photosynthetic traits; (4) cycad pit traits reflected adaptation to wetter habitats for Cycadaceae and drier habitats for Zamiaceae. The large variation in pit traits, the unique pit membrane size and density,and the partial coordination of pit traits with anatomical and physiological traits of the rachis and pinna among cycads may have facilitated their dominance in a variety of ecosystems from the Mesozoic to modern times.

The origin of the cycads can be dated to the late Paleozoic, and they later dominated and prospered with dinosaurs in the Mesozoic (Mamay, 1969, Norstog & Nicholls, 1998). Cycads are considered living fossils because extant cycads are morphologically similar to their fossil ancestors (Brenner, 2003, Mamay, 1969), although all modern cycads seemed to have recently and rapidly diversified during the Yu-Kun Pang and Lan-Li Qin contributed equally to this work.

Neogene, 12 million years ago (Nagalingum et al., 2011). Cycads are now distributed in tropical and subtropical regions across a range of habitats, from very moist tropical rainforests to drylands and from lowlands to high elevations (Hill et al., 2004, Norstog & Nicholls, 1998, Zhang et al., 2015). Ten genera and 367 species are currently accepted (Calonje et al., 2022), with nearly 70% of these species listed on the IUCN red list (IUCN, 2022). Given the current threats of climate change and human disturbance ( Álvarez-Yépiz et al., 2019), there exists increasing urgency for the conservation of this relict plant lineage.

Two families of cycads are currently accepted-Cycadaceae and Zamiaceae (Chang et al., 2020, Christenhusz et al., 2011, Jiang et al., 2016). Cycadaceae have diversified earlier than Zamiaceae and are distributed in colder and higher precipitation habitats than Zamiaceae (Meng et al., 2021). The breadth of climates occupied by each family and the differences in climate between families suggest that there may be differences in functional anatomy that influence cycad leaf water balance. Like other gymnosperms, cycads possess xylem tracheids for water transport, although vessels have also been reported in some species (Huang et al.,2010, Huang & Zhang, 1999, Jacobsen,2021, Xu et al., 2005). The xylem organization in the rachis of cycads is typically shaped like an "omega," resulting in the distal leaflets being irrigated by abaxial bundles, thus guaranteeing uniform water supply along the entire leaf length (Tomlinson et al., 2018).

Cycad leaflets are highly diverse, with Cycadaceae having a single primary vein (midvein) and Zamiaceae exhibiting multiple primary veins (Stevenson et al., 1996). Furthermore, Zamiaceae leaflets have been shown to be thicker and tougher than Cycadaceae leaflets (Meng et al., 2021). Although hydraulic coordination between stem and leaf has been well studied in angiosperms (Brodribb & Feild, 2000, Hao et al., 2011, Jiang et al., 2017, Pivovaroff et al., 2014), it remains largely unknown in cycads. Some leaf functional traits of cycads generally align with the global leaf economic spectrum, though in contrast to other vascular plants, leaf hydraulic conductance has been shown to be unrelated to the photosynthetic rate in cycads (Zhang et al.,2015). Additionally, in contrast to other major groups of vascular plants, the stomatal density of some cycad leaves does not change with elevated atmospheric CO 2 , with the exception of the stomatal number in Zamia furfuraceae, which increased with higher atmospheric CO 2 , further suggesting that cycad leaves are diverse and may have species-dependent physiological responses to changing climate (Haworth et al., 2011, Steinthorsdottir et al., 2021). These differences between cycads and other extant vascular plants suggest that xylem structure-function relationships in cycads may also differ from those observed in other vascular plants.

Plant species with low water transport capacity may also exhibit reduced gas exchange through their stomata because water loss from the leaves is likely also limited by stems' hydraulic supply and hydraulic architecture (Brodribb & Feild, 2000, Santiago et al., 2004). Thus, the structure and water transport properties of the vascular system affect the balance between water supply and total transpiring leaf area (Cardoso et al., 2020, Jiang et al., 2022, Sorek et al., 2021). Previous studies have suggested that a larger interconduit pit aperture diameter may confer higher rates of mass-based photosynthesis in conifers (Song,Sterck, et al., 2022). In contrast, pit membrane diameter was reported to be negatively correlated with mass-based photosynthetic rate among congeneric angiosperms (Liet al., 2019). These two contrasting results suggest that the effect of interconduit pit trait variation on transpiration and photosynthesis may differ among plant lineages (Li et al., 2019, Song, Sterck, et al., 2022).

Among plant lineages, pit membranes fall into one of two categories: the torus-margo type found in most gymnosperms and the homogenous pit membranes characteristic of angiosperms (Choat et al., 2008, Pittermann et al., 2005), ferns (Carlquist & Schneider, 2007), and several cycad species (Jacobsen, 2021). More permeable pit membranes are found in basaltracheophytes, as well as in vesselless angiosperms and conifers, possibly to partially compensate for the higher hydraulic resistances imposed by a tracheid-based vascular system (Brodersen et al., 2014, Hacke et al., 2007, Pittermann et al., 2005). This suggests that there could be a tradeoff among pit membrane, tracheid, and vessel characteristics across different plant lineages. Because pit membranes have the potential to act as the nexus of both cavitation safety and hydraulic transport efficiency, pit anatomical traits may influence both safety and efficiency. Pit traits include the thickness and size of pit membranes, the size and arrangement of pit membrane pores (Lens et al., 2011), and the number of interconduit pits per vessel (Hargrave et al., 1994). For example, lower pit density, larger pit membrane area, larger pit aperture area, and higher aperture fraction (aperture area per pit membrane area) may be associated with higher sapwood hydraulic conductivity and decreased embolism resistance (Brodersen et al., 2014, Jacobsen et al., 2016, Lens et al., 2011, Pittermann et al., 2010). Additionally, pit aperture shape is also correlated with embolism resistance, with more cavitation-resistant species exhibiting narrower and more ellipticalpit apertures (Lens et al., 2011, Scholz et al., 2013, Zhang et al., 2021).

From the hydraulic efficiency perspective, larger diameter conduits are more vulnerable to embolism because of their intertracheid pits. Larger conduits may have more pit membranes that allow for higher hydraulic efficiency. This higher total pit membrane area would increase the chance of possessing a large membrane pore through which air may seed to an adjacent vessel. This idea is known as the rare-pit hypothesis (Hargrave et al., 1994, Lens et al., 2011, Wheeler et al., 2005). Consequently, angiosperms adapted to drier habitats might be expected to exhibit smaller vessel diameters, thicker, denser, and more elliptical pit apertures, and smaller pit membranes than plants inhabiting wetter environments (Hacke et al., 2007, Jansen et al., 2009, Lens et al., 2011, Scholz et al., 2013, Wheeler et al., 2005). However, results demonstrating vessel diameter is the mechanistic link to hydraulic safety are lacking, suggesting a more nuanced role of interconduit pit anatomy in influencing both hydraulic safety and efficiency (Lens et al., 2022). However, the connection between interconduit pit anatomy among cycads and their physiological and habitat affinities remains unclear. Although several cycad species have been shown to possess homogenous pit membranes similar to angiosperms (Jacobsen,2021), these studies have been limited to just a few species without analyzing the possible connections between anatomical and physiological traits (Jacobsen, 2021). Comparative studies have revealed large intraspecific and interspecific variations in pit membrane and pit aperture size, shape,density, and the ratio of total pit aperture area to vessel wall area,likely reflecting differences in environmental adaptation and community filtering processes (Ellmore et al., 2006, Lens et al., 2011, Schmitz et al., 2007, Zhang et al., 2017). For example, polyploid Betula species have more but smaller pits, possibly giving them a greater ability to withstand harsh climate conditions relative to their diploid counterparts (Zhang et al., 2017). Given the broad ecological habitats of cycads,as well as their unique evolutionary history (Meng et al., 2021, Zhang et al., 2015), they make an ideal plant group to study pit trait variation.

In this study, we examined intertracheid pit traits of eight Cycadaceae and five Zamiaceae species in individuals growing in a common garden. Scanning electron microscopy (SEM) was used to quantify pit traits, and light microscopy (LM) was used to quantify rachis and leaf traits. We tested for bivariate correlations among these anatomical and functional traits (e.g., photosynthetic traits, theoretical hydraulic conductivity) of the rachis and leaf (Table 1). By combining LM and SEM anatomical traits and photosynthetic measurements, we addressed the following questions: (1) How do pit traits vary among longer axis μm A pit Intertracheid pit surface area or Intertracheid pit membrane surface area μm 2 A pa Intertracheid pit aperture surface area μm 2 F pf Pit membrane fraction = proportion of pit membrane area per tracheid wall area % F pa Pit aperture fraction = pit aperture surface area/pit membrane surface area % R pit Pit shape = ratio of the longest axis of outer pit membrane to the shortest axis -R pa Pit aperture shape = ratio of the longest axis of outer pit aperture to the shortest axis -D p Pit density = number of intertracheid pits per tracheid wall area No. μm 2 Anatomical traits of rachis from light microscopy A l Tracheid lumen area = total tracheid lumen area of a cross section for rachis mm 2 A w Tracheid wall area = total wall area of all tracheids of a cross section for rachis mm 2 A phl Tracheid phloem area = total phloem area of a cross section for rachis mm 2 A x Tracheid xylem area = total xylem area of a cross section for rachis mm 2 D Tracheid diameter μm D h Hydraulically-weighted tracheid diameter μm F x Fraction of xylem = proportion of xylem area to the cross-section area for rachis % F phl Fraction of phloem = proportion of phloem area to the cross-section area for rachis % N Number of tracheid = total tracheid number of a cross section for rachis -R lx Ratio of lumen area to xylem area of rachis = A l /A x -T d Tracheid density = total tracheid number of a cross section for rachis/area of a cross section for rachis No. mm 2 T w Double tracheid wall thickness μm Anatomical traits leaf from light microscopy D s Stomatal density No. cm 2 D v Vein density mm mm 2 S s Stomatal size μm 2 Physiological functional traits A a Light-saturated photosynthetic rate per area μmol m 2 s 1 A m Light-saturated photosynthetic rate per mass nmol g 1 s 1 C i Intercellular CO 2 concentration μmol mol 1 G s Stomatal conductance mol m 2 s 1 K th Theoretical hydraulic conductivity kg m 1 MPa 1 s 1 cycads and differ from other plant lineages (angiosperms, ferns, and noncycad gymnosperms)?

(2) Are pit traits correlated with other anatomical and physiological traits across cycad species, such as theoretical hydraulic conductivity of the rachis and photosynthetic capacity of the pinna? (3) Are cycad pit traits linked to variation in habitat? We hypothesized that pit traits would exhibit high variation based on the observation that cycads are highly diverse in their overall morphology, habitats (Whitelock, 2003), and leaf physiology and structure (Coiro et al., 2020, Coiro et al., 2021, Glos et al., 2022, Zhang et al., 2015, Zhang et al.,2017). We also hypothesized that pit traits and the coordination of anatomical and physiological traits of the rachis and leaf would be different for cycads compared with other plant lineages, and that the strength of this coordination would depend on the relationships between leaf hydraulics and photosynthesis (Zhang et al.,2015).

The studied cycad species (Table 2) included eight Cycadaceae species (Cycas segmentifida, Cycas guizhouensis, Cycas hainanensis, Cycas revoluta, Cycas ferruginea, Cycas parvula, Cycas debaoensis, and Cycas elongata) and five Zamiaceae species (Z. furfuracea, Macrozamia moorei, Ceratozamia latifolia, Encephalartoslaurentianus, and Encephalartos tegulaneus), representing 5 of the 10 genera of cycads. All plants were sampled at the Nanning Botanical Garden, southern China (for more information, please see Meng et al.,2021). The native habitats of the species range from wet to dry zones (Table S1). Three to five randomly selected individuals per species were selected for sampling. On each plant, a sun-exposed rachis with pinna was cut and put in a sealed black plastic bag with wet tissues in the evening or early dawn, then transported back to the laboratory within half an hour.

The collected rachises were cut into 1-3 cm long pieces and placed in 100 mL of 5% FAA fixative (90:5:5 ratio of 70% ethanol, acetic acid, formaldehyde) at room temperature (25 C) to prevent expansion or shrinkage. A 1-cm segment of the rachis was cut and longitudinal sections of the segments were made with a sliding microtome (RM225, Leica Inc.) at a thickness of 2-3 mm.Then the sections were fixed to aluminum sample holders with carbon double-sided tape (NISSHIN EM Co., Ltd.), air-dried for 12 h at room temperature, and coated with gold using a sputter coater (Cressington 108Auto) for 40 s at 0.08 mA to get a 20-nm thick gold layer, under an argon atmosphere. A conventional SEM (FEI Quattro S) with a voltage of 2 kV was used to visualize intertracheid pit parameters according to standard protocols (Jansen et al., 2009, Lens et al., 2011, Zhang et al., 2021).

ImageJ (Rueden et al., 2017) was used to determine the following pit characteristics: intertracheid pit aperture surface area (A pa ), T A B L E 2 Pit traits in 13 cycad species. Abbreviations are as defined in Table 1.

Habitat Species D pms (μm) D pml (μm) A pit (μm 2 ) D pas (μm) D pas (μm) A pa (μm 2 ) F pa (%) R pa R pit F pf (%) D p (no. μm 2 ) Cycadeceae Wet Cycas debaoensis 7.62 ± 0.33 9.20 ± 0.93 55.67 ± 6.50 1.58 ± 0.28 5.89 ± 0.62 5.64 ± 0.97 10.39 ± 0.44 4.66 ± 1.07 1.23 ± 0.11 55.93 ± 6.90 0.011 ± 0.0027 Dry Cycas elongata 7.91 ± 0.45 7.04 ± 0.38 44.95 ± 4.50 2.10 ± 0.12 4.18 ± 0.01 4.09 ± 0.26 9.14 ± 0.92 2.26 ± 0.06 0.91 ± 0.03 55.80 ± 3.20 0.016 ± 0.0012 Dry Cycas ferruginea 6.92 ± 0.44 7.79 ± 0.07 42.33 ± 2.90 1.37 ± 0.22 5.05 ± 1.15 5.84 ± 1.99 13.96 ± 4.82 3.75 ± 0.45 1.13 ± 0.07 58.09 ± 5.20 0.012 ± 0.0009 Wet Cycas guizhouensis 7.70 ± 0.56 8.22 ± 0.71 51.46 ± 7.46 1.12 ± 0.17 4.85 ± 0.52 4.38 ± 0.94 7.98 ± 1.66 4.65 ± 0.56 1.06 ± 0.03 56.65 ± 4.24 0.013 ± 0.0025 Wet Cycas hainanensis 9.39 ± 0.29 9.39 ± 0.80 70.27 ± 7.98 2.16 ± 0.08 5.92 ± 0.20 9.96 ± 0.12 14.68 ± 1.47 2.89 ± 0.16 1.02 ± 0.08 54.74 ± 5.24 0.008 ± 0.0016 Wet Cycas parvula 7.45 ± 0.42 8.65 ± 0.52 53.08 ± 6.53 2.40 ± 0.12 6.71 ± 0.23 12.92 ± 1.08 20.39 ± 2.24 2.83 ± 0.15 1.18 ± 0.01 51.60 ± 4.93 0.010 ± 0.0013 Wet Cycas revoluta 7.70 ± 0.63 8.64 ± 0.61 53.55 ± 8.32 1.72 ± 0.16 4.49 ± 0.14 6.12 ± 0.50 11.74 ± 1.26 2.76 ± 0.33 1.14 ± 0.05 46.61 ± 8.54 0.009 ± 0.0015 Wet Cycas segmentifida 7.77 ± 0.35 8.27 ± 0.32 52.90 ± 3.36 1.46 ± 0.04 4.93 ± 0.81 5.74 ± 0.89 13.48 ± 0.74 3.56 ± 0.71 1.07 ± 0.03 51.96 ± 5.95 0.010 ± 0.0007 Zamiaceae Wet Ceratozamia latifolia 7.04 ± 0.29 8.50 ± 0.06 47.41 ± 1.72 1.60 ± 0.13 5.30 ± 0.05 6.20 ± 0.71 14.35 ± 1.53 3.49 ± 0.34 1.10 ± 0.06 54.84 ± 1.55 0.012 ± 0.0004 Dry Encephalartos laurentianus 6.52 ± 0.57 8.50 ± 0.86 44.49 ± 7.90 1.20 ± 0.14 6.59 ± 0.77 6.38 ± 1.44 14.12 ± 0.87 5.65 ± 0.38 1.16 ± 0.10 64.68 ± 1.32 0.015 ± 0.0027 Dry Encephalartos tegulaneus 7.95 ± 0.42 8.61 ± 0.22 54.23 ± 3.11 1.50 ± 0.11 6.11 ± 0.40 7.20 ± 0.60 13.43 ± 0.62 4.20 ± 0.44 1.15 ± 0.04 62.47 ± 2.75 0.012 ± 0.0012

Dry Macrozamia moorei 6.31 ± 0.53 8.00 ± 0.43 40.50 ± 5.38 1.49 ± 0.10 5.37 ± 0.17 6.48 ± 0.64 17.21 ± 1.69 3.68 ± 0.16 1.29 ± 0.06 47.28 ± 2.65 0.012 ± 0.0017 Dry Zamia furfuracea 5.42 ± 0.41 6.67 ± 0.79 29.13 ± 3.56 1.67 ± 0.08 6.30 ± 0.52 8.79 ± 0.90 18.63 ± 3.58 3.83 ± 0.43 1.35 ± 0.19 33.39 ± 3.01 0.014 ± 0.0027 intertracheid pit surface area or intertracheid pit membrane surface area (A pit ), pit aperture fraction (F pa ), pit membrane fraction (F pf ), pit aperture longest diameter (D pal ), pit aperture shortest diameter (D pas ), pit aperture shape (R pa ), pit membrane longest diameter (D pml ), pit membrane shortest diameter (D pms ), pit shape or pit membrane shape (R pit ), and pit density (D p ) (Table 1). Mean values for intertracheid pit aperture surface area (A pa ), area or intertracheid pit membrane surface area (A pit ), pit aperture longest diameter (D pal ), pit aperture shortest diameter (D pas ), pit aperture shape (R pa ), pit membrane longest diameter (D pml ), pit membrane shortest diameter (D pms ), pit shape or pit membrane shape (R pit ) were based on at least 50 measurements from SEM images of various intertracheid walls per individuals.

All measurements were made on a fully expanded, healthy, sunexposed rachis with pinna for each individual. Cross-sections of the rachis were made using a sliding microtome (RM225, Leica Inc.)at a thickness of 40-60 μm. Sections were bleached for 10 min, then rinsed in water, and then stained with Safranin O (0.5% wt/vol in water) for 5 min and with Alcian Blue (1% wt/vol in 3% acetic acid)

for 20 s to 1 min, and then mounted on glass slides.

Pinna samples, avoiding the margin and midrib, were cut into sections of about 120 mm 2 and incubated at 70 C in a 1:1 solution of H 2 O 2 (30%) and CH 3 COOH (100%) until all pigments had been removed. The sections were removed from this solution and placed in a petri dish with clear water for 3 min, then the epidermises were separated with forceps from the mesophyll and veins, allowing these three layers (upper epidermis, lower epidermis, and mesophyll with veins) to be stained and mounted separately.To increase contrast,all samples were stained with Safranin O (0.5% wt/vol in water) for 5 min and Alcian Blue (1% wt/vol in 3% acetic acid) for 10 s to 20 min,then washed in water and mounted on microscope slides. In the following analysis, we used only the abaxial (bottom surface) to estimate stomatal density.

Images were taken at 5, 10, and 20, with fields of view of 3.99 mm 2 , 0.89 mm 2 and 0.22 mm 2 , respectively, using a compound microscope outfitted with a digital camera (DM3000, Leica Inc.).For the rachis cross-sections, 50-80 images were taken for each rachis where D is the diameter of a circle with the equivalent area of tracheids whose area was calculated from perpendicular long and short diameters, and N is the number of tracheids measured.

According to the Hagen-Poiseuille law,theoretical hydraulic conductivity was calculated as:

where T d is tracheid density (no. mm 2 ), ρ w is water density (998.2 kg m 3 at 20 C), ƞ is water viscosity (1.002 10 9 MPa s 1 at 20 C).

During July-August 2020, we measured mature and healthy pinnae from three different individual plants of each species using a portable gas exchange system (LI-6800, LI-COR). Leaves were measured on sunny days between 08:30 and 11:30 h solar time, at a photosynthetic photon flux density of 1500 μmol m 2 s 1 , ambient temperature (30 C -36 C), and CO 2 concentration of 400 ppm, allowing VPD to range from 1.2 to 1.7 kPa. For species with pinna area smaller than the cuvette (6 cm 2 ), we normalized the measurements by the leaf area in the chamber, which was measured afterwards using a LI-3000A leaf area meter (Li-Cor).Pinnae were then oven dried at 70 C to constant mass and weighed. Photosynthetic traits, including light-saturated photosynthetic rate per leaf area (A a ), light-saturated photosynthetic rate per mass (A m ), stomatal conductance (G s ), and intercellular CO 2 concentration (C i ) were obtained.

All statistical analyses were performed using R (version 4.1.3, R Development Core Team). We used linear regression and standard major axis regression (R package "smatr") to determine the relationships between traits (Warton et al., 2012), and the statistical tests were considered significant at p < 0.05.Principal component analysis (PCA) was carried out with the "vegan" package. Mean values were z-scaled and centered prior to calculating principal components, and we removed strongly correlated traits with similar functional implications to prevent having more traits than the number of taxa. Differences among cycads and other plant lineages were analyzed using one-way ANOVA, followed by Tukey post hoc tests. Paired t-tests were used to determine differences in PC scores between Cycadaceae and Zamiaceae,and between wet and dry habitat species. Published data for intertracheid pit membrane surface area (A pit ) and pit density (D p )

were obtained from the literature listed in Table S2.

To account for the statistical non-independence of relations between the species sampled, we incorporated phylogenetic covariance into our regression analyses. We used the phylogenetic tree from (Nagalingum et al.,2011) by keeping the 13 species in this study.

We calculated phylogenetically corrected generalized least squares regressions (Symonds & Blomberg,2014) for pairwise trait combinations using the R packages "ape" (Paradis & Schliep, 2018) and "caper" (Orme et al., 2012), "geiger" for analysis of evolutionary diversification with test statistics reported in the Supplementary Data S1.

3.1 | Variation of pit traits among cycad species SEM images were used to examine intertracheid pits in Cycadaceae (Figure 1A-C) and Zamiaceae (Figure 1D-F). Pit membranes were typically homogeneous within species.Pit apertures were generally oval to elliptical in shape and sealed with a pit membrane (Figure 1). We observed more than two columns of pits on the tracheid wall surfaces, and scalariform perforation plate-like structures were observed for some species (Figure 1).

There was wide variation in many intertracheid pit traits: S3). D pas varied about 2.2-fold (from 1.12 ± 0.12 μm 2 in C. guizhouensis to 2.4 ± 0.17 μm 2 in C. parvula), whereas A pa varied

3.2-fold (from 4.09 ± 0.26 μm 2 in C. elongata to 12.92 ± 1.08 μm 2 in C. parvula). F pa also varied about 2.6-fold (from 7.98% ± 1.66% in C. guizhouensisto 20.39% ± 2.24% in C. parvula), and R pa varied about 2.1-fold (from 2.26 ± 0.06 in C. elongata to 4.66 ± 1.07 in C. debaoensis)among Cycadaceae species. In addition, D pas varied about 1.39-fold (from 1.2 ± 0.14 μm in E. tegulaneus to 1.67 ± 0.08 μm in Z. furfuracea), A pa varied about 1.42-fold (from 6.2 ± 0.71 μm 2 in C. latifolia to 8.79 ± 0.9 μm 2 in Z. furfuracea),F pa varied about 1.39-fold (from 13.43% ± 0.62% in E. laurentianus to 18.63% ± 3.58% in Z. furfuracea), and R pa varied about 1.6-fold (form 3.49 ± 0.34 in C. latifolia to 5.65 ± 0.38 in E. tegulaneus) among Zamiaceae species, indicating that intertracheid pit traits were more diverse in Cycadaceae.

Pit density D p was negatively related to D pms , D pml , A pit (Figure 2A-C). No correlations were found between A pit and A pa (Figure 2D), R pa and R pit (Figure 2E),or between D p and pit aperture traits D pas , D pal , and A pa (Figure S1).Interestingly, F pa was positively correlated with R pit (Figure 2F). Almost all of these correlations remained the same after accounting for shared evolutionary history, except for D p and A pa which became significantly negatively correlated (Table S6).

In order to compare pit packing density and pit area among the plant lineages (ferns, cycads from this study, non-cycad gymnosperms, and angiosperms), A pit and D p data were collected from the published references (Table S2). Results showed that A pit was significantly negatively correlated with D p among the four plant groups (Figure 3),with ferns, angiosperms, and cycads being closer to the theoretical packing limit (1/A pit ) than non-cycad gymnosperms (Figure 3).Pit area A pit of cycads was significantly larger than angiosperms and ferns, but significantly smaller than non-cycad gymnosperms ( p < 0.05, Table 3). In contrast, the pit density D p of cycads was smaller than for angiosperms and ferns, but similar to non-cycad gymnosperms (p < 0.05; Table 3).

Pit density D p was found to be positively correlated with tracheid lumen area A l (Figure 4A) and phloem area A phl (Figure 4B) of the rachis, as well as with vein density D v (Figure 4E) in leaves, suggesting that larger rachis lumen area was associated with more numerous pits (Figure 4E). No relationship was found between D p and anatomical trait of xylem area A x of the rachis (Figure 4C), stomatal density D s (Figure 4F),nor with physiological traits such as theoretical hydraulic conductivity K th (Figure 4D), leaf area-based photosynthetic rate A a , or stomatal conductance G s (Figure 4G,H).

Pit shape was positively correlated with tracheid thickness (T w ; Figure S2) and the fraction of xylem (F x ; Figure S2). In contrast, pit shape was negatively correlated with the ratio of lumen area to xylem area (R lx ; Figure S2) in the rachis, indicating that more elliptically shaped pits were associated with thicker tracheid walls.

Pit membrane area (A pit ; Figure 5A) was not correlated with K th among cycads, though A pit was negatively correlated with K th only among the Zamiaceae species (Figure 5A). No other correlations were found between pit membrane traits and anatomical or physiological traits of the rachis among the 13 cycad species (Table S4). While A pit was negatively correlated with D v and D s (Figure 5B,C), it was not correlated with other functional traits related to gas exchange, for example, G s , A a , and C i (Table S4).It is worth noting that the correlation between A pit and D v was driven largely by differences in both traits between the two families (Figure 5B). Thus, while rachis pit membrane traits may be coordinated with some leaf anatomical traits, these pit traits may not be linked to leaf function. All of these correlations remained the same after accounting for shared evolutionary history, and only the significant negative relationships of R pit and R lx became marginally significant ( p = 0.0525; Table S6).

The functional trait K th was positively correlated with the lightsaturated photosynthetic rate per mass A m (Figure 6A). Because tracheid diameter D h is the major driver of K th (Table S4),this pattern suggests that water supply from the rachis was positively coordinated with pinna photosynthesis. Like pit membrane traits, pit aperture traits were found to be independent of anatomical and physiological traits of the rachis (Table S4). Only pit aperture area (A pa ; Figure 6B) and fraction (F pa ; Figure 6C) were positively correlated with K th in Zamiaceae, suggesting that pit aperture size influences theoretical hydraulic conductivity in Zamiaceae, but not in Cycadaceae.

Although pit aperture was not correlated with leaf anatomical traits (Table S4), significant negative correlations were found between F pa and A m (Figure 6D),D pal (Figure 6E) and stomatal conductance G s , for which species were reported with primitive vessels (Huang et al., 2010;Huang & Zhang, 1999;Jacobsen, 2021;Xu et al., 2005). No torusmargo structures were found in pits of the studied cycad species.

as well as A pa (Figure 6F) and G s , indicating that gas exchange rates may decrease with increasing pit aperture size. All the correlations mentioned above remained the same after accounting for shared evolutionary history (Table S6).

Although pit traits were overall very similar among both Cycadaceae and Zamiaceae, except for the difference in pit membrane short diameter (Table S3),PCA using species' z-scaled and centered trait values of pit revealed that the first two components explained 36.99% and 32.97% of the total variation, respectively. The first PC was most strongly associated with A pa , F pa , R pit , F pf , and A pit . (Figure 7). The second PC was driven mainly by A pit , A pa , R pa , R pit , and D p (Figure 7). Eigenvalues and loadings are listed in Table S7. Among all species,Z. furfuracea had the most elliptical pit shape, whereas the pit aperture area of C. parvula, and the pit diameter of C. hainanensis were significantly largerthan those of the other species.Furthermore,the seven wet habitat species and the six dry habitat species were separated mainly along the second PC (Figure7). Wet species had significantly larger A pit and significantly fewer pits (D p ) than dry habitat species (t-test, p < 0.05; Table S8). S2).

Cycads are highly diverse in morphology, leaf structure, and physiological traits (Whitelock, 2003;Zhang et al., 2015), with nearly 70% of living cycads listed as endangered (IUCN, 2022). Threats to cycad survival and mortality may depend on their physiological capacity to withstand future climate change, such as prolonged droughts. Anatomical and physiological traits associated with both xylem hydraulic efficiency and xylem hydraulic safety may be important traits that determine the thresholds of plant mortality to drought-induced hydraulic failure (Brodersen et al., 2014;Kaack et al., 2021;Lens et al., 2011;Pittermann et al., 2010). Here, we surveyed 13 species of cycads to determine (1) how traits vary among cycad families and compare them to other plant lineages, (2) the relationships between xylem anatomical traits and leaf physiological traits, and (3) whether these xylem anatomical traits are linked to differences in habitat affinities.

We found that intertracheid pit traits of the rachis varied markedly among cycads (Table 2). Further, cycads differed from other lineages in pit membrane area and density, and we report, for the first time, a clear tradeoff between these traits across lineages. Although cycads deviate in relationships among pit traits and between pit traits and other anatomical and physiological traits, there are some pit characteristics relationships across cycad species that are similar to relationships reported among angiosperms (Jacobsen et al., 2016;Li et al., 2019).

We observed wide variation in intertracheid pit characteristics of the 13 cycad species we studied (Table 2), which was consistent with our hypothesis that pit traits would be species-dependent. There is generally a tradeoff between pit membrane area and density across vascular species such that pit aperture area and shape are coordinated with pit membrane traits (Hacke et al., 2004;Jacobsen et al., 2016;Li et al., 2019;Orians et al., 2004). However, we found that cycads only partially supported this trend, with pit membrane area being correlated with pit density (Figure 2), while pit aperture remained independent of pit membrane traits concerning size and shape. Importantly, the large variation in pit traits we report here is from species growing in a common garden, indicating that this variation in traits is due to genetic differences among species that may result in different habitat preferences (Monson,1996).

Pit size and density also differed between cycads and other major clades. Cycads had larger A pit than ferns and angiosperms,but smaller than non-cycad gymnosperms (Figure 3). Cycad pit density D p was also much lower among cycads than among ferns and angiosperms, but higher than among non-cycad gymnosperms. The smaller, more densely packed pits of ferns and angiosperms bring them closer to the maximum packing limit,while cycads and other gymnosperms tend to be farther away from the maximum packing limit (Figure 3; Table 3).

Larger but fewer homogenous pits have been reported to confer higher hydraulic efficiency (Brodersen et al., 2014;Hacke et al., 2007;Pittermann et al., 2005), which may provide a potential anatomical explanation for the dominance of cycads across a variety of terrestrial habitats in the Mesozoic. On the other hand, cycads have short tracheids, similar to non-cycad gymnosperms,which should result in low xylem-specific conductivity. However, non-cycad gymnosperms have more permeable torus-margo pits (Pittermann et al., 2005), which would likely give non-cycad gymnosperms a hydraulic advantage over cycads,all else being equal. Although ferns generally have more permeable pit membranes (Brodersen et al., 2014), they are usually smaller compared with cycad pits and may therefore result in relatively lower hydraulic conductance. Thus, different xylem anatomical features of pits and xylem conduits may either reinforce or counteract each other to influence hydraulic conductivity and vulnerability to embolism. Without more data on hydraulic conductivity, it is difficult to resolve the effects of each individual trait alone.

Pit characteristics are frequently related to other xylem anatomical and physiological traits (Lens et al., 2011;Li et al., 2019;Song, Sterck, et al., 2022). For example, fewer and larger pits or pit apertures provide greater hydraulic efficiency (Lens et al.,2011;Mrad et al., 2018;Zhang et al., 2022), whereas smaller surface area and higher density of intervessel pits increase embolism resistance and lower hydraulic efficiency (Choat et al.,2005;Ellmore et al.,2006;Hacke et al., 2006;Wheeler et al., 2005). Larger diameter and longer conduits increase hydraulic efficiency, whereas more numerous pits on the conduit wall increase hydraulic safety (Lens et al., 2011). While angiosperm shrubs tend to have lower pit density and greater hydraulic efficiency (Jacobsen et al.,2016), in some cases, higher pit density can confer greater hydraulic efficiency and higher rates of gas exchange; for example, in nonhemiepiphytic compared with hemiepiphytic Ficus species (Liet al., 2019). These patterns suggest that pit traits are, in some cases,linked to species-habitat associations. For the cycad species studied here, pit density (D p ) and area (A pit ) were not correlated with tracheid diameter or theoretical hydraulic efficiency (K th ), or with other physiological traits (Table S4). The one exception was that pit membrane area was negatively correlated with K th in Zamiaceae (Figure 5A).Pit membrane area was only coordinated with a few anatomical traits of the rachis and pinna (Figure 5B,C and Table S4). One possible reason why pit membrane area was not coordinated with other anatomical traits may be because cycads universally have relatively low pit density and large pit membranes. However, pit density (D p ) increased with pinna vein density (Figure 4E),suggesting that D p might be associated with adaptation to warmer and drier habitats among the Zamiaceae (Table S8; Qin et al., 2022). Consistent with this possible effect of habitat, Zamiaceae also had relatively larger xylem fractions (Brenner, 2003) and more elliptical pits (R pit ; Table 2).

Additionally, among all Zamiaceae,R pit was positively correlated with tracheid double-wall thickness, which is thought to confer higher hydraulic safety (Figure S2; Lens et al., 2011, Scholz et al., 2013, Zhang et al., 2021).

A negative correlation between pit membrane area A pit and K th in Zamiaceae (Figure 5A) is likely associated with compensatory changes in other pit traits, for example, larger pit apertures were associated with higher K th in Zamiaceae (Figure 6B,C). Pit aperture area in cycads was relatively larger than in angiosperm species (Li et al., 2019), which may contribute to higher K th in Zamiaceae. In other plant lineages, the pit aperture diameter represents a smaller and constant fraction of the pit membrane diameter, with a pit aperture of around 10% of the pit membrane area (Lens et al., 2011;Scholz et al., 2013). However, the higher fraction (F pa ) among cycads was negatively correlated with mass-based photosynthesis (A m ; Figure 6D), and, among all pit traits, only pit aperture area and fraction were correlated with the physiological traits (but not to K th ) in cycads, consistent with previous results that sapwood hydraulic conductivity is not correlated with pit traits among conifers (Song, Poorter, et al., 2022).

Some pit traits were coordinated with physiological traits among cycads, particularly among the Zamiaceae. The negative relationship between F pa and both A a and A m (Table S4) is consistent with Ficus species (Li et al.,2019) but opposite a previous report showing that a larger pit aperture was positively associated with carbon assimilation and increased hydraulic vulnerability among conifers (Song, Sterck, et al., 2022). Although A pa and F pa were not correlated with K th across all 13 cycad species, these traits were correlated among only the Zamiaceae species,with increasing K th being associated with higher mass-based photosynthesis (A m ; Figure 6A). These results are consistent with the coordination between stem hydraulic capacity and leaf photosynthetic rates (Brodribb & Feild, 2000;Santiago et al., 2004) and suggest that this coordination was achieved at least in part by adjusting the pit aperture area. However, in contrast to both theory and prior results, area-based leaf hydraulic conductance was reported to be unrelated to A a among cycads (Zhang et al., 2015). Though some pit traits exhibit similar relationships among cycads as among other lineages,other relationships deviate, suggesting that further work on cycads could help elucidate important mechanistic links between traits that might not be possible by examining only non-cycad lineages.

The native habitats of the cycad species studied here vary greatly in precipitation, mean annual temperature,and habitat (Table S1). These habitat differences may impact vulnerability to hydraulic failure and traits associated with drought-induced embolism. While there have been numerous reports about different water availability influencing vulnerability to drought among closely related species (Brodribb et al., 2014;Larter et al., 2017;Skelton et al., 2021), there are few measurements of embolism vulnerability among cycads. Because pit anatomical traits can influence embolism vulnerability (Brodersen et al., 2014;Jacobsen et al., 2016;Lens et al., 2011;Pittermann et al., 2010), relationships between pit traits and climate would suggest that cycad species may differ in their vulnerability to embolism.

Indeed, our previous data showed that the embolism vulnerability of three cycads was in the range reported for noncycad gymnosperms; however, M. moorei had a negative hydraulic safety margin, whereas C. revoluta and C.elongata had a positive hydraulic safety margin (Qin et al., 2022). In our dataset, Cycadaceae tended to be from cooler and wetter habitats than Zamiaceae (Meng et al., 2021), and the short diameters of pit membranes were longer in Cycadaceae than in Zamiaceae (Table S3).This was further evidenced by the fact that cycad species from wet habitats tend to have larger but fewer pit membranes (Table S8). This pattern is consistent with other data

showing that species from drier environments often have more elliptical pits (Lens et al., 2011, Scholz et al., 2013, Zhang et al., 2021).

Plants from drier habitats typically have smaller vessels and tracheids, thicker and more densely packed intervessel pits, more elliptical pits and pit apertures,and smaller pit membranes than plants from moist environments (Hacke et al., 2007;Jacobsen et al., 2016;Lens et al., 2011;Scholz et al.,2013). Six of the eight Cycadaceae species (all except C. ferruginea and C.elongata)are found in humid habitats, whereas five of the six Zamiaceae species (all except C. latifolia) are distributed in arid habitats. These wet-habitat and dry-habitat species differed in their pit membrane traits (D pms , D p , and A pit ), pit aperture size (A pa , R pa , and D pas ), and pit shape (R pit ). Species from wet habitats tended to have larger pit membranes and pit apertures and more circular pit apertures (C. hainanensis and C.parvula; Table 2), whereas Zamiaceae species from drier habitats tended to have more elliptical pits and pit apertures, and higher pit densities (Z. furfuracea; Tables 2, S8, and Figure 7). However, these habitat differences in pit traits were driven largely by differences among the two families. Comparing confamilial species that differed in wet-habitat versus dry-habitat affinities revealed smaller differences due to habitat, for example, C. elongata and C. guizhouensis differ in habitat but had similar traits in multivariate space (Figure 7). More data on a broader set of cycad species would help to elucidate the effects of climate on these traits and to what extent pit traits influence xylem hydraulic safety and efficiency.

Cycads first arose ca 250 Myr ago but extant species diversified only very recently (Nagalingum et al.,2011) during a period of declining atmospheric CO 2 and increasing aridity (Beerling & Royer, 2011;Steinthorsdottir et al., 2021;Tanner et al., 2020;Trayler et al., 2020).

Despite this rapid diversification during the Neogene, extinct and extant cycads are morphologically very similar, and some anatomical traits in cycads do not respond plastically to differences in atmospheric CO 2 , suggesting that the trait relationships reported here may reflect the traits of extinct cycads from long-extinct ecosystems (Haworth et al., 2011;Zhang et al., 2015). The fossils of extant Cycadales, however, are limited to the Cenozoic (Coiro & Pott, 2017) when angiosperms started to prosper (Condamine et al., 2015;Simonin & Roddy, 2018). Cycadales have morphologies similar to the Bennettitales and Nilssoniales, two dominant plant groups of the mid-Mesozoic (Coiro & Pott, 2017;Harris, 1961). Extant cycads still grow successfully in a wide range of habitats, from very wet tropical rainforests to dry habitats (Calonje et al., 2022;Norstog & Nicholls, 1998;Whitelock, 2003). However, the lack of plasticity in cycad anatomy suggests that climate-driven changes in traits may occur only during speciation and that modern cycads have limited capacity to acclimate to current and future climate change and must rely instead on adaptive responses.

Our results highlight that studying cycads can reveal important information about the coordination and evolution of anatomical and physiological traits among vascular plants.Cycads exhibit a wide variation in intertracheid pit characteristics and a similar tradeoff between pit density and pit area, as has been reported for other plant lineages, despite deviating in relationships among pit traits and between pit traits and other anatomical and physiological traits. The larger pit membrane area but lower density of pits compared with ferns and angiosperms may provide higher hydraulic efficiency for cycads. On the other hand, the homogeneous but smaller pit membrane area and similar low pit density of non-cycad gymnosperms suggest that the xylem hydraulic capacity of cycads may be low,potentially leading to lower competitive fitness among cycads in present-day terrestrial ecosystems.That pit trait variation among cycad lineages was associated with differences in habitat affinities suggests that selection on hydraulic traits may have driven pit trait evolution among cycads.

Whether the pit traits, including pit membrane thickness and pit resistivity, play crucial roles in the hydraulic system and confer success in