Geographic and longitudinal variations of anatomical characteristics and mechanical properties in three bamboo species naturally grown in Lombok Island, Indonesia
Sampling sites and sample preparationCulms of three- to four-year-old of Bambusa vulgaris Schrad. ex J.C., B. maculata Widjaja, and Gigantochloa atter (Hassk) Kurz ex Munro were collected from naturally bamboo forests at four sites in Lombok Island, Indonesia23. The culm age was estimated based on some morphological features (the presence of culm sheath, color, and sound created by tapping with fingers) checked by an experienced bamboo farmer. Figure 1 shows the map of sampling sites and climatic conditions of the sites. Ten individual culms in each species at each site were collected from different clumps and cut 20 cm above the ground (Fig. 2). A total of 120 culms (three species × four sites × 10 individual culms from 10 individual clumps) were collected in the present study (Fig. 2). To determine the longitudinal variations of the anatomical characteristics and mechanical properties, the internode section was collected at 2-m intervals from 2 to 8 m above the ground; a total of 480 internode sections. (120 culms × four heights) were obtained from three species (Fig. 2). The collection of bamboo culms was permitted by Indonesian Institute of Science (Reference no. B-206/SKIKH/KS.02.04/X/2020) and complied with relevant guidelines and regulations of Indonesian CITES Management Authority, Ministry of Environment and Forestry, Indonesia. In addition, the voucher specimen was deposited at the Herbarium Lesser Sunda, University of Mataram, Indonesia under the voucher number of DSR01, 02, and 03 (specimens were identified by Mr. Niechi Valentino). Table 1 shows the culm diameter at 1.3 m above the ground, total culm height, and mean value of culm thickness at four positions23.Figure 1Locations and climate conditions of sampling sites in the present study23. Note: Site I, Tempos (8°41′59″ S, 116°8′40″ E); Site II, Kabul (8°47′21″ S, 116°10′21″ E); Site III, Keruak (8°45′45″ S, 116°28′54″ E); Site IV, Genggelang (8°23′16″ S, 116°15′35″ E). *, mean annual precipitation. The value in the bracket is the mean annual temperature. Climate data were provided from Nusa Tenggara River Basin Management I, Indonesia. Mean monthly temperature and precipitation were calculated by averaging monthly values from 2016 to 2018. Bars indicate the mean values of precipitation. Circles indicate the mean values of temperature. The graph was originally created by R27 (version 4.0.3, https://www.R-project.org/).Full size imageFigure 2Photographs of the clumps in three bamboo species (a–c) and schematic diagrams of experimental procedures (d). Note: a, B. vulgaris; b, B. maculata; c, G. atter. The specimens of fiber area measurement and mechanical properties have the whole culm thickness (including the cortex and inner part of the culm) in the radial direction.Full size imageTable 1 Mean values and standard deviations of growth characteristics in three bamboo species at each site23.Full size tableAnatomical characteristicsThe internode sections were split into two parts: the strips (10 mm in the longitudinal direction) and the small blocks (10 [T] mm by 10 [L] mm by culm thickness in the radial direction) (Fig. 2). The strips and small blocks were the samples for measuring fiber length and fiber area, respectively. In the present study, the fiber area was defined as the sheaths area around the vascular bundles24.To determine the fiber length, small sticks (not including the cortex and the most inner part of the culm) were obtained from the strips with a razor blade (Fig. 2). Randomly selected sticks from each height position (without separation of collected positions of the samples within the radial direction of the culm in a height) were macerated with Schultze’s solution (100 mL of 35% nitric acid containing 6 g potassium chloride) at 70 °C for two hours. The length of 50 fibers was measured in each sample with a digital caliper (CD-15CX, Mitutoyo, Kawasaki, Japan) on a microprojector (V-12B, Nikon, Tokyo, Japan).To measure the fiber area, one block was taken at each height position on each individual culm (Fig. 2). The transverse sections of the blocks were polished with sandpaper sheet (#180, 3 M Japan, Tokyo, Japan), and then their images were captured using a microscope digital camera (DS-2210, Sato Shouji Inc., Kawasaki, Japan) attached to a stereo microscope (SZX12, Olympus, Tokyo, Japan). The fiber area was determined by ImageJ25 (version 1.53e). Binarized images were prepared by ImageJ to distinguish as clearly as possible between the vascular bundle and the background (Fig. 3). The darker area of binarized images in Fig. 3 was identified as fiber sheaths. The fiber area was calculated as follows:$$FAleft( % right) , = A_{fs} /A_{c} times {1}00$$
(1)
where FA = fiber area (%), Afs = the transverse-sectional area of fiber sheath in bamboo culm (mm2), and Ac = the transverse-sectional area of bamboo culm (mm2).Figure 3The photomicrographs of transverse section in B. vulgaris (a and d), B. maculata (b and e), and G. atter (c and f). Note: a, b and c, original image; d, e and f, binarized image processed by ImageJ25 (version 1.53e, https://imagej.nih.gov/ij/). The darker area in photomicrographs (d, e and f) is fiber sheath area.Full size imageMechanical propertiesThe following mechanical properties of culm were measured: bending properties (MOE and MOR), CS, and tensile properties (TM and TS). A total of 480 specimens (one specimen × four heights in an individual × ten individuals × three species × four sites) without node were obtained in each property (Fig. 2).The strips (10 [T] mm × 200 [L] mm × varied culm thickness in the radial direction) were prepared as the specimens for the static bending test (Fig. 2). The static bending test was conducted using a universal testing machine (MSC 5/500–2, Tokyo Testing Machine, Tokyo, Japan). A load was applied to the center of the specimen on the outer cortex surface with 180 mm span and 3 mm min−1 load speed. Due to larger thickness (exceeded 12.9 mm = 180 mm of span / 14) in the radial direction, the span / depth ratio in some specimens was less than 14, indicating that MOR in some specimens might be underestimated due to the occurrence of the shearing strength26. Of 480 specimens, the large culm thickness exceeded 12.9 mm was total 19 specimens from B. vulgaris species collected at 2 m height position from different sites (Site I = four specimens, Site II = six specimens, Site III = four specimens, and Site IV = five specimens). However, all these 19 specimens were broken at the tension side of the specimens during static bending test, which was the normal breaking forms of bending specimens with span / depth ratio less than 14.The load and deflection were recorded with a personal computer, and then MOE and MOR were calculated by the following formulae:$$MOE , left( {GPa} right) , = Delta Pl^{3} / , 4Delta Ybh^{3} , times 10^{ – 3}$$
(2)
$$MOR , left( {MPa} right) , = , 3Pl/ , 2bh^{2}$$
(3)
where ΔP = difference between upper and lower proportional limit within the range of elasticity (N), l = length of the span (mm), ∆Y = deflection due to ∆P (mm), b = width of the specimen (mm), h = height of the specimen (mm), and P = maximum load (N).The compressive test specimen (10 [T] mm × 20 [L] mm × culm thickness in the radial direction) was also prepared (Fig. 2). The test was conducted using a universal testing machine (RTF-2350, A&D, Tokyo, Japan) with a load speed of 0.3 mm min−1. The compressive strength parallel to grain (CS) was calculated by the following formula:$${text{CS }}left( {{text{MPa}}} right) , = P/A_{0}$$
(4)
where P = maximum load (N), and A0 = the cross-sectional area of the specimen (mm2).The tensile tests were conducted using bone-shaped specimens (Fig. 2). The specimen length was 230 (L) mm with a 20 (T) mm width of the specimen grip. The cross-sectional area of the specimen was 2 mm in the tangential direction by culm thickness in the radial direction. A strain gage type extensometer (SG25-10A, A&D, Tokyo, Japan) was used to detect the elongation in the test specimen. The specimen grip sections were attached to small boards (75 mm in length × 40 mm in width × 5 mm in thickness) and then were clamped between the metal grip of a universal testing machine (RTC-2410, A&D, Tokyo, Japan). The tensile load was applied at 1 mm min−1. The tensile strength (TS) and Young’s modulus (TM) were calculated by the following formulae:$${text{TS }}left( {{text{MPa}}} right) , = P/A_{0}$$
(5)
$${text{TM }}left( {{text{GPa}}} right) , = Delta Pl/A_{0} Delta l times {1}0^{{ – 3}}$$
(6)
where P = maximum load (N), A0 = the cross-sectional area of the specimen (mm2), ∆P = difference between upper and lower proportional limit within the range of elasticity (N), l = gauge length (mm), and ∆l = elongation of the original gauge length (mm).The moisture content and air-dry density of each specimen were measured after each mechanical testing by the oven-dry method. The moisture content and air-dry density of the specimen at testing were listed in Table S1.Statistical analysisThe statistical analyses were conducted using R software (version 4.0.3)27. To evaluate the longitudinal variations of the measured properties in each species, the y-intercept, linear, and nonlinear mixed-effects models with each measured property value as a responsible variable, the height position as a fixed effect, and site and individual culm as random effects were developed by the “lmer” function in “lme4” packages28 and the “nlme” function in the “nlme” package29. The following four full models were developed and compared:Model I (y-intercept model):$$Y_{ijk} = alpha_{{1}} + Site_{{{1}k}} + Culm_{{{1}jk}} + e_{ijk}$$
(7)
Model II (linear model):$$Y_{ijk} = , (beta_{0} + Site_{0k} + Culm_{0jk} )X_{ijk} + beta_{{1}} + Site_{{{1}k}} + Culm_{{{1}jk}} + e_{ijk}$$
(8)
Model III (logarithmic model):$$Y_{ijk} = , (gamma_{0} + Site_{0k} + Culm_{0jk} ){text{ ln }}left( {X_{ijk} } right) + gamma_{{1}} + Site_{{{1}k}} + Culm_{{{1}jk}} + e_{ijk}$$
(9)
Model IV (quadratic model):$$begin{gathered} Y_{ijk} = , (zeta_{0} + Site_{0k} + Culm_{0jk} )X_{ijk}^{{2}} + , (zeta_{{1}} + Site_{{{1}k}} + Culm_{{{1}jk}} )X_{ijk} hfill \ + zeta_{{2}} + Site_{{{2}k}} + Culm_{{{2}jk}} + e_{ijk} hfill \ end{gathered}$$
(10)
where Yijk is measured property at the ith height position from the jth individual culm within the kth site, Xijk is the ith height position from the jth individual culm within the kth site, α1, β0, β1, γ0, γ1, ζ0, ζ1, and ζ2 are the fixed effects, Site0k, Site1k, and Site2k are the random effect at the site level, Culm0jk, Culm1jk, and Culm2jk are the random effects at the individual culm level, and eijk is residual. Total 36 derived models (three y-intercept models, 15 linear models, nine logarithmic models, and nine quadratic models) were developed. The model selection was conducted using the Akaike information criterion30. The model with the minimum AIC value was regarded as the most parsimonious model among developed models. In addition, the differences in AIC (ΔAIC) ≤ 2 indicate no significant differences between models, and a simpler model with fewer parameters is preferred31. To evaluate the longitudinal variation, estimated values of each property was calculated at 0.1 m interval from 2.0 to 8.0 m above the ground using fixed-effect parameters of the selected models. Mean value and standard deviation were obtained from the estimated values from 2.0 to 8.0 m in each property. In addition, the coefficient of variation was also calculated from the mean value and standard deviation. The longitudinal variation patterns were classified into four types (Types A to D) based on the model selection (Fig. 4). Although model II to IV was selected, longitudinal variation with the coefficient of variation less than 3.0% was regarded as stable (Type A in Fig. 4).Figure 4Classification of longitudinal variation of bamboo culm property. Note: Lines or curves indicate formulae with fixed-effect parameters in the selected mixed-effect model for explaining longitudinal variation (Tables 3, 4, 5). Coefficient of variation calculated from mean values and standard deviation from 2 to 8 m above the ground estimated by fixed-effect parameters values less than 3.0% is regard as stable variation (Type A), even in selected model is Model II to IV.Full size imageGeographic variations in each bamboo property were estimated by evaluating the variance component of sites and culms as random effects by using the intercept-only linear mixed-effects model. The full model is described as follows:$$Y_{ijk} = mu + Site_{k} + Culm_{jk} + e_{ijk}$$
(11)
where Yijk is the bamboo property at the ith height position of the jth individual culm within kth site, μ is the model intercept or grand mean, Sitek is the random effect of the kth site, Culmjk is random effect of jth individual culm within kth site, and eijk is the residual. The contribution of each level of variation was calculated as a percentage of the total random variation in the best model32,33. More
