The “chi-chi” of Ginkgo biloba L. grows downward with horizontally curving tracheids having compression-wood-like features
Journal of Wood Science volume 69, Article number: 27 (2023)
The old Ginkgo biloba L. trees often develop cylindrical woody structures that grow downward from the underside of the large branches near the trunks. This structure is traditionally called “chi-chi” (a breast) in Japan. The structure of chi-chi has not been investigated in detail because of the rarity of such old trees of G. biloba. This study examined the chi-chi from wood anatomy and chemistry viewpoints. After debarking, there were many woody bulges and latent buds. There were hollows corresponding to these latent buds on the inner side of the bark. In the transverse section obtained from the tip part of the chi-chi sample, we found tracheids curving in a horizontal plane, and the parenchymatous latent bud tissue is the center of the swirl. Microscopic observations and X-ray micro-computed tomography suggest the growing course of the chi-chi due to several swirls contiguous to each other. From these observations, the downward growth of the chi-chi starting from the cambium cell division might be driven by the woody bulges with latent buds. The cell walls of the curved tracheids were not thickened, not rounded, and had no intercellular spaces, but their S3 layers disappeared. Furthermore, the results of thioacidolysis and acetyl bromide analyses exhibited p-hydroxyphenyl subunits deposition to lignin and high lignin content on the tip part of the chi-chi. These results suggest that the curved tracheids in the chi-chi have compression-wood-like properties to some extent.
The old Ginkgo biloba L. trees often develop cylindrical woody structures that grow downwards from the underside of the large branches near the trunks (Fig. 1). Traditionally, this tissue is called “chi-chi” (a breast) and has been a folk belief on childbirth and lactation in Japan. After an extended period, they grow downward to over 1 m long. When grown down to the ground, they take roots and produce vegetative shoots  (Additional file 1: Fig. S1). The same cylindrical woody structures of ginkgo are called “zhongru” (stalactite) in China. Li and Lin examined the chi-chi anatomically, found that some of the tracheids were arranged in whirls in the tangential section, and described it as the abnormal growth of coniferous trees . Barlow and Kurczyńska used the word “chi-chi” as a name of the tissue and concluded that a chi-chi uses the vascular cambium not only for its widening growth but also for its elongation . Del Tredici studied the chi-chi found at the underground level (“a basal chi-chi") and concluded that vegetative regeneration using the basal chi-chi may have played a role in the remarkable survival of the genus Ginkgo since the Cretaceous. He also discussed that a basal chi-chi is identical to an aerial chi-chi . The aerial chi-chi is the same one called "chi-chi” in our study.
Regarding anatomical characteristics of gymnosperms, they usually produce specific tissues, so-called compression wood, to push stems upwards on the underside of the leaning stems. The branches of the gymnosperm also have compression wood on their underside. G. biloba is similar to modern conifer and develops compression wood [5,6,7]. Compression wood tracheids are thick-walled and rounded in cross-section, resulting in intercellular spaces between the corners of adjoining cells. While the cellulose microfibril angle (MFA) of normal wood tracheids is low (~ 10°), the thickened S2 cell wall layer has an increased MFA (30–50°) [7, 8]. The S3 wall layer is frequently absent in compression wood.
As for the compositional differences in compression wood, secondary cell walls reduce polysaccharides and increase lignin. Lignin is an indispensable substance for vascular plants. The principal function of lignin in plants is to assist in water movement; lignin forms a barrier for evaporation and, thus, helps channel water to critical areas of the plant. Lignin is an aromatic heteropolymer built up by the combination of three basic monomer types of p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S). Generally, H-unit is a minor structural unit found in the compression wood and grasses, G-unit is the main structural unit in softwood (gymnosperm) lignin and the second most unit in hardwood (angiosperms) lignin, and S-unit is the most abundant structural unit in angiosperm lignin . G. biloba is a gymnosperm, so it mainly has a G-unit and contains H-unit only in the compression wood. However, as Uzal et al. found the presence of S-unit in G. biloba cell cultures , the biosynthetic mechanisms of plant lignin are flexible and complex.
As described above, the cell wall structures and lignin chemical structures provide essential information about the tissue. However, the detailed cell wall structures of the chi-chi have yet to be reported as far as we know, although abnormal tracheid shapes were reported . Also, there is no study on the lignin of the chi-chi. Therefore, this study examined the chi-chi using microscopic observations using scanning electron microscopy (SEM), field-emission SEM (FE-SEM), optical microscopy with staining, and X-ray micro-computed tomography (X-ray μCT). Further, the content and the chemical structures of lignin were evaluated using the acetyl bromide and thioacidolysis methods, respectively. Our purpose is to reveal how the chi-chi grow downward and how are the cell wall structure and lignin chemical structures in the tissue.
Materials and methods
Generally, most elder ginkgo trees growing on the premises of Shinto shrines and Buddhist temples are designated as national monuments by the government or natural monuments by local governments. Therefore, it is difficult to obtain a chi-chi as an experimental material in Japan. Fortunately, we got an opportunity to sample the chi-chi, which was not a designated natural monument (Fig. 1).
On June 21st, 2012, over 20 cm long chi-chi was cut from the trunk of a G. biloba tree growing in the premises of Akiba shrine (35°22′18″ N, 136°45′42″ E) in Kasamatsu town, Gifu prefecture, Japan (Fig. 1a). The height and the diameter at breast height of the ginkgo tree were evaluated as about 25 m and 119 cm, respectively, by the Educational Committee of Kasamatsu town in 2016. The tree age was not correctly evaluated, but the committee estimated it as over 200 years old. In addition, lateral shoots near the chi-chi were also sampled. The samples were carried to Nagoya University and frozen with liquid nitrogen. Next, the chi-chi of the tip part was cut to 9.5 cm long (Fig. 1b) and cut in half longitudinally. Then, several samples were cut from the chi-chi and named samples A, B, C, D, and E (Fig. 2). The sample E surface was polished with a polishing paper (Sankyo Chemical, Super Precision Polishing Film #1000). The polished surface of the longitudinal half block (sample E) is shown in Fig. 2.
Sample A was observed by SEM and then was cut into four pieces, A-1, A-2, A-3, and A-4 (Fig. 3a and b). The samples A-1 and A-3 were sliced to tangential sections of 0.1 mm thickness with sliding microtome (REM-710, Yamato kohki, Asaka, Japan) and used for lignin analysis after extraction by ethanol/benzene (1:2, v/v) over 10 h in a Soxhlet extractor. Sample A-2 was used for FE-SEM observation. The upper part of the chi-chi (sample B) was used for SEM observation after debarking. After SEM observation, sample B was similarly used for lignin analysis after the same procedures used for sample A-1. The lowest tip part of the chi-chi (sample C) was cut to a 1 cm × 1 cm diamond-shaped × 3 cm height block and used for X-ray μCT, optical microscopy, and SEM observations.
One-sixth of the tip part of the chi-chi (sample D) was debarked and cut into 17 pieces of 5 mm thickness with a jigsaw (Fig. 4a). Then, No.1, No.5, No.9, No.13, and No.17 boards suggested in Fig. 4a were cut to small blocks of Da, Db, Dc, Dd, De, Df, Dg, Dh, Di, Dj, Dk, and Dl as schematically illustrated in Fig. 4b. Each block was similarly used for lignin analysis after the same procedures used for the sample A-1. Next, lateral shoots were cut to 30 mm in length and debarked. Then, the shoots were cut to separate the lower and upper parts. Each sample was similarly used for lignin analysis after the same procedures used for sample A-1.
Scanning electron microscopy (SEM) and field-emission (FE) SEM
The samples were hydrated in the water at room temperature and planed off their surfaces with a sliding microtome. After drying in a vacuum oven at 40 ℃, samples were observed using SEM (S-3400N-T3, Hitachi, Japan) without any surface metal coating. FE-SEM (S-4500, Hitachi, Japan) was used to confirm the existence of the S3 layer in the middle part of the chi-chi (sample A-2). The sample was prepared in the same manner as that described above. Before the observations, the surface was coated with Pd–Pt (E-1030 Ion Sputter, Hitachi, Japan). Then, the coated sample was observed with FE-SEM.
X-ray micro-computed tomography (X-ray μCT)
The computed tomographic images of the lowest tip part of the chi-chi (sample C) were obtained by X-ray μCT analysis (Sky Scan 1272, Bruker, Belgium). The measurement conditions are as follows: beam source, 50 kV 200 μA; exposure, 481 ms; rotation step, 0.1 deg; image pixel size, 3.0 μm; measurement time 15 h 45 min; reconstruction time of 8802 slices, 35 h 37 min; result image size, 4904 × 4904 pixels. Slice images were obtained from the reconstructed data as 964 × 917 pixels in gif format. The gif animation file was converted to a movie file and embedded in the Additional file 2.
Lignin content evaluation by the acetyl bromide method
The extractive-free sections sliced 0.1 mm thickness was used for lignin determination by the acetyl bromide method [11, 12]. The lignin content of the samples was determined by measuring the absorbances at 280 nm using a lignin absorption coefficient of 21.0 L g−1 cm−1 which was obtained by using the milled wood lignin (MWL) of ginkgo. The MWL was prepared from the stem of a ginkgo 19-year-old tree grown in the Nagoya University experimental farm (Nagoya, Japan). The absorbance was measured by a UV spectrometer (V-530, JASCO, Japan).
Thioacidolysis and gas chromatography–mass spectrometry (GC–MS)
Thioacidolysis selectively cleaves the β-aryl ether bonds of lignin. Therefore, the analysis of the lignin-derived monomeric products can evaluate the type and amount of the lignin structural units only involved in β-aryl ether bonds . Furthermore, the lignin-derived dimers are obtained after desulfurization of the thioacidolysis products over Laney nickel [14, 15].
The extractive-free sections sliced 0.1 mm thickness was used for thioacidolysis. Thioacidolysis and desulfurization were carried out according to the method reported by Lapierre et al. [14, 15]. Approximately, 5 mg of the different sliced dry sections were placed in a 5 mL dioxane/ethanethiol mixture (8.75:1, v/v) and 0.2 M BF3 etherate in a tube fitted with a Teflon-lined screwcap. An internal standard (docosane, 0.05 mg) was added to the sample tube. Then, the sample was heated at 100 °C for 4 h, shaking mildly for a minute at intervals of 30 min. The reaction mixture was cooled on ice, and 4 mL NaHCO3 aqueous solution (0.4 M) was added to stop the reaction. The pH of the mixture was adjusted to 2–3 with HCl aqueous solution (1:3, v/v), and the entire mixture was extracted using CH2Cl2 (5 mL × 3 times). The combined organic extracts were dried over Na2SO4 and evaporated under reduced pressure at 40 °C. The final residue was diluted in 1.0 mL CH2Cl2 and stored at 4 °C in a vial. Three replications were performed for every sample.
For desulfurization, 0.5 mL of the thioacidolysis CH2Cl2 solution was treated with 1.5 mL Raney nickel aqueous slurry and 2.5 mL dioxane in a glass tube fitted with a Tefron-lined screwcap. Desulfurization was carried out at 50 °C for 4 h with shaking at intervals of 30 min. The tube was cooled on ice, and the supernatant solution of the reaction mixture was moved into another tube. Then the pH of the solution was adjusted to 3–4 with HCl aqueous solution (1:3, v/v), and the entire mixture was extracted using CH2Cl2 (5 mL × 3 times). The combined organic extracts were dried over Na2SO4 and evaporated under reduced pressure at 40 °C. The final residue was diluted in 1.0 mL CH2Cl2 and stored at 4 °C in a vial. Three replications were performed for every sample.
The final CH2Cl2 solution of lignin monomeric or dimeric products was silylated with N,o-bis(trimethylsilyl)trifluoroacetoamide and pyridine. The silylated derivatives were analyzed by GC–MS (QP2010, Shimadzu, Japan). Measurement conditions for monomeric products: capillary column, Rtx-1 of inner diameter, 0.32 mm × 30 m, film thickness 0.25 μm; injection temperature, 250 °C; temperature program, from 180 to 230 °C at 2 °C/min, to 250 °C at 15 °C/min, and hold 10 min; carrier gas, He (1.36 mL/min). For dimeric products: temperature program, from 180 to 240 °C at 2 °C/min, to 280 °C at 1 °C/min, and hold 10 min. A response factor 1.5 was used for monomeric products, and 1.0 was used for dimeric products. Three measurements were conducted for every sample.
The chi-chi sample extended downward from a branch near the trunk and was 25 cm long. It was cylindrical and covered with bark to the tip as described by Fujii  (Figs. 1b, c, 2). After debarking, there were many woody bulges and latent buds (Fig. 2D). In Fig. 2D, the bulges have plural buds; therefore, the buds seemed to be in the upper part but not always at the tip of the bulges. The latent buds are parenchymatous tissue and diminished after debarking and drying (Additional file 1: Fig. S2, S3). On the inner surface of the bark, there were hollows corresponding to the position where the latent buds were present (Additional file 1: Fig. S2d). Sample A, used for SEM, FE-SEM, and lignin analysis, also had downward extending woody bulges before the lower part was cut (Additional file 1: Fig. S4).
Horizontally curving tracheids
A clear boundary between the light brown and dark brown areas was observed in the left 1/3 part of sample A (Fig. 3a). Figure 5a shows a radial-view SEM image of the boundary, and a white arrow indicates the direction of gravity. The same sample block of A-2 was observed from a different angle and shown in Fig. 5b. The observation angles and relationships are schematically illustrated in Fig. 5c. The SEM image in Fig. 5d is an enlarged image of the area framed by the white rectangle in Fig. 5b and shows that the tracheids have bordered pits.
These images show that the tracheids in the growing part of chi-chi (block A) are horizontally (perpendicular to the gravity direction) curving. Furthermore, as shown in Additional file 1: Fig. S3, the center of the tracheid swirling might be the parenchymatous latent bud tissue.
In the case of sample B, taken from the upper and bark-side part of the chi-chi sample (Fig. 2), the tracheids aligned parallel to the gravity direction (Fig. 6). Therefore, ca. 10 cm far from the tip of the chi-chi, gravity-parallel tracheids are generated by the cambium, similar to those in normal trunks.
Cell wall structures of the curving tracheids
An FE-SEM image of sample A-2 taken from the lumen side is shown in Fig. 7. The image shows no S3 layer in the cell wall layer structure and that the S2 layer microfibril orientation is 45°. However, as shown in Fig. 5, other features commonly observed in a gymnosperm compression wood, such as cell wall thickening, the rounded tracheid shape, and the development of intercellular spaces, were not observed. The comparable images of the cell walls of vertical (in sample B) or horizontal (in sample A-2) tracheids are summarized in Additional file 1: Fig. S5.
Wood formation with swirls
Sample C was taken from the central tip of the chi-chi (Fig. 2). SEM images of the horizontal plane at 30 mm apart from the tip (sample C) are shown in Fig. 8. In Fig. 8a, tracheids curved and spirally aligned as a swirl. In the same sample block C, a wide-angle view displays that several swirls are contiguous to each other (Fig. 8b). The center of the swirl in Fig. 8a has an aggregate of parenchyma cells (Enlarged and displayed in Additional file 1: Fig. S6). However, it was unclear how the curving started or whether it had a pith-like tissue.
The enlarged SEM image of the area suggested by white rectangles in Fig. 8c shows that curving tracheids have bordered pits (Fig. 8d). In Fig. 8d, cytoplasmic debris are seen in uniseriate ray. Here, these ray cells intersected the horizontally curving tracheids; therefore, the axis of the ray cells was parallel to the gravity direction.
To understand the internal 3D wood formation in the chi-chi, sample C was observed by X-ray µCT. An animation created by moving the horizontally sliced plane is shown in Additional file 2: Fig. S7. Several swirls, in which tracheids were assembled in a spiral shape, were observed to be continuous over millimeters long in the vertical direction and forming wood as multiple swirls contiguous to each other. As for the swirl center, Additional file 1: Fig. S3 shows that the parenchymatous latent bud tissue is the center of the tracheid swirling.
Lignin chemical structures
The leading tip part of the chi-chi sample C was cut to a 20 µm thick section in the longitudinal direction. The phloroglucinol HCl-stained optical microscope image is shown in Fig. 9. On the left side of the image, the tracheids with bordered pits are formed horizontally. The tracheids in different directions can be seen on the right side of the image. The lower part of the image is the bark side, and the reddish-purple color becomes darker toward the upper part, reflecting the progress of lignification.
First, a quantitative comparison was made by the acetyl bromide method (Additional file 1: Table S1). The Dh, Dj, Dk, and Dl samples resulted in the lignin content of 30.8–33.7%, with an average value of 32.6%, which tended to be higher than the average value of 31.5% obtained for the other part of samples, where no H lignin units were detected as described below. The amount of lignin in the lower part of the Lateral shoots was 35.6%, while that in the upper part was 30.8%.
Then, thioacidolysis and GC–MS analysis were performed on samples A-1, A-3, B, and lateral shoots. The yields of thioacidolysis monomeric products obtained are shown in Fig. 10 and Additional file 1: Table S1. The chi-chi samples each yielded ca. 400–550 μmol/g of G-unit, and the lateral shoot samples yielded about 400 μmol/g of G-unit. In terms of H-unit yield, no H-unit was detected in sample B, in which the growth axes of the tracheids and ray cells were similar to that of the normal trunk. In contrast, approximately 30 μmol/g was detected in samples A-1 and A-3, where the curving tracheids were observed. In the lateral shoot samples, H-unit yield was higher in the lower part, i.e., usual ginkgo compression wood, with 51 μmol/g. The H-unit composition ratio [= H-unit/(G-unit + H-unit)] was 12%. This value is close to the H-unit composition ratio of 14% reported for spruce compression wood . The ratio of H-unit in the chi-chi sample was 5.9% and lower than that in the lateral shoot.
Thioacidolysis results from small blocks are summarized in Fig. 10 and Additional file 1: Table S1. G-unit was detected in all blocks, ranging from 300 to 480 μmol/g. No H-unit was detected in the upper small blocks (Da, Db, Dc, Dd, De, Df, Dg, and Di). H-units were detected in 12–19 μmol/g in small blocks (Dh, Dj, Dk, and Dl) sampled from the lower part. In the blocks where H-units were detected, the composition ratio was about 4%.
Finally, the thioacidolysis dimeric product analysis result is listed in Additional file 1: Table S2. There were no significant differences in the composition of dimer linkage types (5–5ʹ, 4-O-5ʹ, β-1ʹ, β-5ʹ, and β–βʹ) within the chi-chi and lateral shoot samples.
Barlow and Kurczyńska  proposed a growth mechanism in which chi-chi elongates downward by the cambium. They described “tracheids show variable orientations” and suggested “a swirl of tracheids may indicate initiation of a woody nodule.” Here, a “nodule” was reported as a tissue having disturbed and horizontal arrangements of tracheids, and it was estimated as a result of the specific growing behavior of the chi-chi tissue. They called the small object on the chi-chi surface as woody spines and did not mention the relationships with growing mechanisms.
These descriptions of the chi-chi tissues agree with our results in this study to some extent. In this study, the internal microstructure of the chi-chi was observed by microscopic observations and X-ray µCT. Here, we take a step forward about the chi-chi elongation mechanisms. There are three significant points. First, the surface small object was not woody (lignified) spines but parenchymatous ones; we called the object as latent buds. Second, the center of the tracheid swirling might be the parenchymatous latent bud tissue. Third, the swirls are continuous over a millimeters scale, and several swirls construct a woody bulge. From these observations, we estimated that the cambium around the woody bulges with latent buds drives the downward growth of the chi-chi.
Generally, compared to normal wood, compression wood tracheids are rounded in the transverse section, and intercellular spaces between the corners of adjacent cells occur in it. These tracheids are thick-walled and have higher lignin content with significant p-hydroxyphenyl subunits. The S3 layer is absent, and the S2 layer microfibril orientation is close to 45°. For the curving tracheids in the chi-chi, there was no S3 layer, and the S2 layer microfibril orientation was 45°. This is not the helical cavity but should be the spiral drying check of S2 layer due to drying of the sample, as reported previously [6, 7], or the possible physical stresses within the sectioning processes. To the best of our knowledge, this is the first reported case of the absence of S3 in the cell wall structure of a curved tracheid. The absence of the S3 layer is an essential early anatomical change in the transition of normal wood to compression wood . Although cell wall thickening and shape rounding of the curving tracheids were not observed, some compression-wood-like features are thought to be expressed.
Another parameter that differentiates the chi-chi tissue from normal ginkgo wood is the lignin chemical structure. The lignin composition and lignin content of the chi-chi was heterogeneous and differed between the upper and lower parts, with the lower tip tending to have more H-unit and lignin content, indicating that it has compression-wood-like features. This result is consistent with the SEM image in Fig. 6, which shows that the tracheids of sample B in the upper part of chi-chi are aligned parallel to the gravity direction and similar to normal wood.
As reported by Sinnott, it has been shown that when a conifer branch is artificially looped in the horizontal plane, compression woods are formed inside the loop . In the horizontal plane of the chi-chi tip, the swirl formation by curving tracheids may have had some stress effect on cell wall formation and contributed to the triggering of the compression wood formation.
For the tracheary element swirls in the secondary xylem, Rothwell et al. reported the presence of the circular tissue in the tangential section of the secondary xylem of 375 million-year-old fossil wood of Archaeopteris, as well as in the present woody Spermatophyta [19, 20]. They attributed this to the formation of auxin whirlpools due to the obstruction of axial polar transport of auxin by buds and branches. The swirls of chi-chi tracheids in the horizontal plane may be caused by latent buds, with a mechanism similar to the auxin whirlpools in the vertical plane proposed by Rothwell et al. .
The enlarged SEM image of the area suggested by white rectangles in Fig. 8c shows that curving tracheids have bordered pits (Fig. 8d). Here, these ray cells intersected the curving tracheids; therefore, the axis of the ray cells was parallel to the gravity direction. Ray cells exist in the horizontal plane in the normal trunk but in the vertical plane at the chi-chi tip. This spatial arrangement may suggest some possible mechanisms in which an orthogonal relationship between tracheids and rays is preferentially maintained in tissue development, regardless of the direction of gravity in the space where the tracheids are located.
The elongation of the chi-chi can be classified into two stages. Near the tip growing part of the chi-chi, multiple aggregates of tracheids curving in the horizontal plane and intersecting ray cells, i.e. swirls, have a parenchymatous tissue of latent bud in the center and form wood. This results in the growth of woody bulges, and the chi-chi grows downward.
Tracheids in the swirl have no S3 layer in their cell wall structure and have H-units in the lignin chemical structure. However, the cell walls of the curved tracheids were not thickened, not rounded, and had no intercellular spaces; therefore, they have only a part of gymnosperm compression-wood-like features. With time, the growing axes of tracheids and ray cells in chi-chi become similar to those in normal wood of the stem, and the lignin is composed only of G-units.
Availability of data and materials
The data sets analyzed during the current study are available from the corresponding author on reasonable request.
Gas chromatography-mass spectrometry
Scanning electron microscopy
- X-ray μCT:
X-ray micro-computed tomography
Milled wood lignin
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The authors thank K. Shinoda, Chairman of Neighborhood Association of Kasamatsu-cho Kamishin-machi, Gifu Prefecture, Japan, for the sampling permission of the chi-chi; S. Sakamoto, Superintendent of Education, Fukaura-cho, Aomori Prefecture, Japan, for the permission for the on-site survey of the elder G. biloba tree in Kitakanegasawa; S. Kimura and R. Fujita, Nikon Instech co. Ltd, Japan, for conducting X-ray μCT analysis. The authors sincerely thank N. Terashima, the professor emeritus, Nagoya University, Japan, for his experimental and research advice.
This work was supported by JSPS KAKENHI Grant numbers 25252032, 18H03959, and 23H02275.
The authors declare that they have no competing interests.
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: Fig. S1. The elder G. biloba tree in Kitakanegasawa (a natural monument). Fig. S2. Latent buds and hollows of the chi-chi. Fig. S3. Photomicrographs of the latent bud in the tip of sample E. Fig. S4. Sample A before cut underpart. Fig. S5. FE-SEM micrographs of the cell wall of the chi-chi sample. Fig. S6. SEM micrographs of the swirls. Table S1. Lignin content and yields of thioacidolysis monomeric products. Table S2. Yields of thioacidolysis dimeric products.
: Fig. S7. Cross-sectional consecutive screen movie of the sample C by X-ray μCT.
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Higuchi, S., Aoki, D., Matsushita, Y. et al. The “chi-chi” of Ginkgo biloba L. grows downward with horizontally curving tracheids having compression-wood-like features. J Wood Sci 69, 27 (2023). https://doi.org/10.1186/s10086-023-02102-4
- Abnormal tracheid shapes
- Gravity direction
- Bordered pits
- Ray cells