Philippine Journal of Science 150 (1): 277-289, February 2021 ISSN 0031 - 7683 Date Received: 27 Jul 2020

Morphological and Anatomical Characteristics of Different Variants of Narra ( indicus Willd.) Seedlings

Hannah Mari Carmela M. Flores1,2, Marilyn O. Quimado2, Crusty E. Tinio2, Lerma SJ. Maldia2, and Marilyn S. Combalicer2*

1Community Environment and Natural Resources Office Department of Environment and Natural Resources Lipa City, 4217 2Department of Forest Biological Sciences,College of Forestry and Natural Resources University of the Philippines Los Baños, College, Laguna 4031 Philippines

Pterocarpus indicus, commonly known as narra, is one of the native leguminous trees in the Philippines that has occupied wide geographic distribution resulting in variability of its morphological and structural characteristics. In this study, the morphological and leaf anatomical features of four P. indicus variants (smooth narra – SN, short prickly narra – PNS, medium prickly narra – PNM, and long prickly narra – PNL) in Mount Makiling Forest Reserve (MMFR) were characterized. Based on seven morphological parameters, the mean root length, mean leaf number, and mean leaf area significantly separated the two major P. indicus forms. SN has a higher mean leaf number but shorter mean root length and smaller mean leaf area compared to the three variants of prickly narra (PNS, PNM, and PNL). Within the prickly narra (PN) variants, PNS and PNM showed distinct similarities based on six morphological parameters. At the same time, PNL seedlings have higher root-shoot ratio and mean biomass, indicating the greater capacity of this variant to store photosynthate material in its roots. The leaf anatomical parameters confirmed the similar features of two PN variants (PNS and PNM). Furthermore, the SN and PNL were significantly distinguished by the palisade and spongy mesophyll, xylem, and phloem area, where SN was considerably higher in all parameters except in the phloem area. This indicates that SN has a higher photosynthetic rate and has high storage capacity and ease of transport of water and organic matter. Thus, the morphological attributes and anatomical parameters contribute to further differentiating the two major forms of P. indicus.

Keywords: leaf anatomy, mesophyll, prickly narra, root-shoot ratio, smooth narra

INTRODUCTION endangered by the Philippine National Red List for (DENR-DAO 2017-11) because of the dramatic Narra () is a large , loss of its population over its natural range of distribution. tropical tree species of the family that is It has been mainly influenced by human activities, widely distributed in Southeast and East and the such as the massive exploitation of woods for timber Pacific (Thomson 2006). It is categorized as a critically production, and the extensive destruction of the lowland rain forests from conversion to agriculture, pasture, *Corresponding Author: [email protected] industrial, commercial, residential lands, and kaingin

277 Philippine Journal of Science Flores et al.: Morphology and Anatomy of P. indicus Vol. 150 No. 1, February 2021 activities (PCHMB 2009). The subsequent massive MATERIALS AND METHODS loss of its population leads to a drastic decrease in the variability within the species. For instance, Delos Reyes Study Area and Collection et al. (2016) observed a moderate mean genetic diversity The experiment was conducted from June 2016 up to (0.3183), genetic differentiation (0.0575), and Wright’s May 2017 in the greenhouse and the Microtechnique Fixation Index (0.1528) – suggesting the nearness of the Laboratory of the Department of Forest Biological populations to each other. In their study, they analyzed that Sciences (DFBS), College of Forestry and Natural genetic distance and cluster analysis of P. indicus in the Resources (CFNR), University of the Philippines Los Philippines did not conform to geographical distribution Baños (UPLB), Laguna. The were collected from but revealed the relationships and the possible origin/s of four different mother trees of P. indicus (with identified the individuals of the populations. variation in their forms and ) found within the Distinctive intraspecific variations exist in the perimeters of the MMFR, specifically at the grounds of morphological characteristics of P. indicus. Rojo (1972) the CFNR Quadrangle (14°9.34’ N and 121°14.15’ E), at reported a considerable variation in the morphological the back of the DFBS building (14°9.24’ N, 121°14.16’ E), characteristics of P. indicus in terms of , , at the back of the Social Forestry and Forest Governance size, shape, and hairiness. In the Philippines, there (SFFG) building (14°9.22’ N, 121°14.13’ E), and near are two recognized forms of P. indicus, the SN (P. indicus the Makiling Residence Hall (14°9.18’ N, 121°14.12’ E). forma indicus) and PN (P. indicus forma echinatus). The former has smooth surface fruit and broader and glossy Characterization of Variants and Preparation of the leaves while the latter has prickly fruit and narrower, dull Soil Medium green leaves (Thomson 2006). The four variants of P. indicus were characterized The morphological and anatomical characteristics of according to size, number, and presence of prickles. The plants may serve as a tool in understanding the genetic fruits of P. indicus were air-dried for two weeks before relationship, physiological processes, and ecological seed extraction. The extracted seeds from each variant adaptations in plants (Fahn 1964). The variation is an were labeled and subjected to the floatation test method to adaptation mechanism that could be either a short-term select viable seeds. The viable seeds were dried and stored or long-term response to its changing environment. The at room temperature before planting. The soil medium short-term response is the changes undergone in the used in the seedboxes was a mixture of garden soil, sand, morphological, physiological, and biochemical levels and coir dust in the ratio 1:1:1 (Priyadarshani et al. 2010; while the long-term response of plants is the changes in Yue et al. 2020). To remove soil-borne microorganisms its genetic composition (Bradshaw 1965). from the potting medium, the soil was sterilized. Seeds were sown in 80 cm x 60 cm x 20 cm seedboxes. The seeds Information on the variability of P. indicus can provide the started to germinate after a week of sowing. The seedlings opportunity to develop new and improved cultivars with were then transplanted from seedboxes to polyethylene desirable characteristics and to maintain the diversity of bags a month after sowing to avoid competition among the species, which are significant for genetic conservation planting stocks and were grown for three months. The and management. However, there is limited information seedlings were placed in a partially shaded portion of the on the extent of morphological, anatomical, and genetic greenhouse and were watered twice a week manually. variability and diversity of P. indicus population. The lack of information may hinder the full realization of its Morphological Measurements conservation and management (Delos Reyes et al. 2016; Seven morphological parameters were used to determine Thomson 2006; Hong et al. 2020). the morphological and growth performance of the four Hence, this study aims to characterize the morphological P. indicus variants – namely, shoot length, root length, and leaf anatomical features of the four variants of P. leaf number, leaf area, root collar diameter (RCD), root- indicus; and determine the morphological and anatomical shoot ratio, and seedling biomass. The parameters were parameters that significantly separate the two major measured and subjected to statistical analysis to measure P. indicus variants. The morphological characters, the specific differences between P. indicus variants. In specifically root-shoot biomass, and anatomical both morphological and anatomical characteristics, data characteristics are vital in identifying differences in their were analyzed according to each variant (SN, PNS, PNM, growth performances, while the anatomical characters and PNL) in terms of pooled measurements of all the three can be used for taxonomical, ecological studies, and prickly variants. conservation purposes of the species. The shoot and root length were measured using a ruler (cm). The shoot length was measured from the root collar

278 Philippine Journal of Science Flores et al.: Morphology and Anatomy of P. indicus Vol. 150 No. 1, February 2021 of the seedling up to its terminal bud and was recorded Microscopic examination was done using a compound every second week of the month for three months. On microscope (OptikalSview) of the Forest Product and the other hand, root length was measured from the RCD Paper Sciences Laboratory, CFNR-UPLB, to obtain down to the root tip. It was measured once at the end of photomicrographs of the leaf structure of the four variants the experiment when the seedlings were uprooted. of P. indicus. The anatomical structures of the leaf were identified and the thickness of the upper and lower 2 The leaf number and leaf area (cm ) was observed every epidermis, spongy, and palisade mesophyll plus the size second week of the month from the first month until the of the tissue area (including phloem and xylem) were third month of the seedlings (Sanchez 1967; Sianipar measured. Haupt (1953), Fahn (1967), Bell (2008), and 2015). The third expanded leaf from the apex of the Shipunov (2020) were followed to describe the anatomy seedlings was used for the computation of the leaf area. of the . The leaves were traced in a graphing paper with a 0.5 cm x 0.5 cm grid, and the leaf area was computed using the following formula: Experimental Design and Analysis The experiment used a simple complete randomized (1) design with four treatments having three replicates of 10 seedlings. The experiment included four variants (as RCD measures the increase in girth of the stem using treatments) of P. indicus, namely: a) SN, b) PNS, c) PNM, a Vernier caliper (mm). The RCD of the seedlings was and d) PNL. measured every month until the termination period of the seedlings (three months). The statistical tests used were one-way analysis of variance (ANOVA) and Duncan’s multiple range test Fresh weight (FW) and dry weight (DW) were measured (DMRT), as post hoc. The Pearson’s product-moment in the third month before its termination period. The correlation test was used to explore the relationships FW and DW of each seedling were determined using a between leaf area and anatomical parameters of each P. weighing scale and oven-dried at 80 ˚C until constant indicus variant with pooled prickly variants. All analyses weight. The roots were separated from the shoots. The were performed in R Studio version 4.0. root-shoot ratio and seedling biomass were calculated using the formula below:

(2) RESULTS

(3) Characterization of P. indicus Variants The characterization of the four variants of P. indicus is shown in Table 1. Typically, the fruit contains one to Anatomical Measurements four seeds. The fruit of SN measures 5–7 cm while PNS, The anatomy of seedlings was determined by randomly PNM, and PNL measure about 5–8 cm, 6–7.5 cm, and 6–7 selecting eight seedlings per treatment for fixation. Each cm, respectively. Among the PN variants, PNM contains leaf sample was hand-cut, measuring 1 cm x 1 cm, in the numerous prickles (90–100) of about 5–7 mm in length mid-part including the midrib. Modified Johansen (1940) (Figure 1). histological paraffin technique was used for the anatomical study. The tissues were fixed in 1:1 FAA-A (12 mL 37% formaldehyde, 88 mL 95% ethanol) and FAA-B (10 mL Table 1. Fruit morphological differences among the four variants of glacial acetic acid, 90 mL water) mixture (Hernandez et al. Pterocarpus indicus. 2016) for one week then another week for dehydration in Variant Fruit size Mean length Number of Presence a series of increasing concentration of ethanol (50%, 70%, (cm) of prickles prickles of 95%), absolute ethanol, and tertiary butyl alcohol. The (mm) prickles specimens were then embedded in paraffin wax (melting SN 5–7 0 0 None point, 65 ˚C). Sections with 10-µm thickness was cut using PNS 5–8 6 55–70 Present a rotary microtome. The paraffin ribbons were mounted PNM 6–7.5 7 90–100 Present on a slide using a drop of Haupt's solution (Haupt 1930) and water that functions as an adhesive, air-dried, stained PNL 6–7 9 50–60 Present with safranin (1% aqueous), and counterstained with fast SN – smooth narra, PNS – short prickly narra, PNM – medium prickly narra, PNL – long prickly narra green (0.5% dissolved in 95% ethanol). After staining, the slides containing the specimen were coated with the Entelan® solution.

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Figure 1. Fruits of Narra (Pterocarpus indicus Willd.) collected from Mt. Makiling Forest Reserve (MMFR): (A) smooth narra (Pterocarpus indicus Willd. forma indicus); (B–D) three variants of prickly narra (Pterocarpus indicus Willd. forma echinatus). Bar represents 1 cm.

Morphological characteristics. Figure 2 presents the and root-shoot ratio of prickly (per variant and pooled) morphological characters of the four variants of P. indicus. were consistently lower than the smooth. The shoot length ranges from 10.71–14.92 cm, the root length at 5.03–7.98 cm, the leaf number at 4–6, the leaf Anatomical characteristics. Appendix III presents the area at 15.36–16.88 cm2, the RCD at 1.49–1.72 mm, anatomical characters of the four variants of P. indicus. the root-shoot ratio at 0.1–0.22, and seedling biomass at The upper epidermis ranges from 16.67–23.33 µm, the 3.45–7.89 g. lower epidermis at 10–16.67 µm, the palisade mesophyll at 63.33–80 µm, the spongy mesophyll at 246.67–303.30 All the measured morphological characteristics, except µm, the xylem area at 21.68–49.42 µm2, and the phloem for RCD, were found significantly different between area at 9.58–15.56 µm2. SN and PN, regardless of the variant (Figure 2I and Appendix I). On the other hand, the analysis using pooled The cross-section of a typical dicotyledonous mesomorphic measurements of prickled variants and SN consistently leaf is sheated with an upper and lower epidermis (Hopkins showed significant differentiation of these characters, and Huner 2008), both are uniseriate. The mesophyll, the except shoot length (Figure 2II and Appendix I). RCD was photosynthetic tissues, consists of one to three layers of consistently not significant between smooth and pooled palisade mesophyll cells and spongy mesophyll, which prickled variants. Specifically, the root length, leaf area, is much thicker consisting of cuboidal or oblong cells and seedling biomass of prickly (per variant and pooled) with air spaces. were higher than the smooth. On one hand, the leaf number

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Figure 2. (I) Seven morphological parameters such as root length, shoot length, leaf number, leaf area, RCD, root-shoot ratio, and seedling biomass used for the ANOVA and DMRT analyses using four P. indicus variants; (II) seven morphological parameters analyzed using pooled prickled variants and smooth narra. Means followed by the same letter are not significantly different at 5% level by DMRT. SN – smooth narra, PNS – short prickly narra, PNM – medium prickly narra, PNL – long prickly narra.

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Of the six leaf anatomical characteristics, significant leaf (Figure 3). In terms of the mean thickness of the differences between prickly (regardless of variant or spongy parenchyma, there was a significant difference pooled) and SN were observed in the thickness of the among variants as well as between pooled prickly and mesophyll tissues (both palisade and spongy) and phloem smooth. The spongy parenchyma thickness of SN was (Appendices II and III). SN has significantly thicker significantly higher than that of the PNS, PNM, and mesophyll but significantly thinner phloem than PN. Two PNL (Appendix III-c, d) as well as when prickly variants of the anatomical parameters (upper and lower epidermis, were pooled. The thickness of the spongy parenchyma and xylem area) were not significantly different among of PNL was distinctly low among the four. The spongy the four variants or between the two main types of narra. mesophyll among prickly variants has more round and compact cells, except for PNS (Appendix V-A to C) 1. Upper and lower epidermis thickness. The mean ranges having few intercellular spaces in the mesophyll cell, of thickness of upper and lower epidermises range from while that of the SN (Appendix V-D) has round to oblong 33.33–43.33 µm and from 23.33–33.33 µm, respectively. cells and with the visible presence of hollow spaces. There were no significant differences in upper and lower epidermis thickness across the four variants and between 3. Vascular bundle. In terms of xylem and phloem area, pooled prickled variants and smooth (Appendices II and there was significant differentiation among the four III 3-a, b). All the leaves from the four variants have variants (Appendix III-e, f), but the analysis of pooled uniseriate upper and lower epidermis (Appendix IV). prickled variants did not differ significantly with the SN in terms of xylem area (Appendix III-e). On the other 2. Palisade and spongy mesophyll thickness. The hand, prickled variants have a significantly higher phloem thickness of palisade and spongy parenchyma differ area than SN (Appendix III-f). The area of xylem and significantly between prickly (per variant and pooled) phloem could have an implication on plants’ ability to and smooth (Appendices II and III-c, d). The mean transport water, minerals, and dissolved organic matters. thicknesses of palisade layers PNS, PNM, and PNL Nonetheless, the results showed no significant difference did not differ not significantly (Appendix III-c) but among the four variants and pooled prickled variants were significantly lower than SN (Appendix III-c). All compared to SN (Figure 4; Appendices II and III). of the variants were mostly made up of two layers of palisade parenchyma located at the upper portion of the

Figure 3. Leaf anatomy of the four variants of P. indicus: A) long/soft prickly narra (PNL), B) medium prickly narra (PNM), C) short/rough prickly narra (PNS), and D) smooth narra (SN) showing the palisade parenchyma (PM) and upper epidermis (UE). Bar represents 100 µm.

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Figure 4. Leaf anatomy of the four variants of P. indicus: A) long/soft prickly narra (PNL), B) medium prickly narra (PNM), C) short/rough prickly narra (PNS), and D) smooth narra (SN) showing the vascular bundle with (XY) as xylem and (PH) as phloem cells. Bar represents 100 µm.

DISCUSSION gradient from the root to the atmosphere and provides a greater area for entry of carbon dioxide (Taiz and A significant difference in mean root length was observed Zeiger 2002). The significantly shorter shoot of the PNL between SN and PN variants (PNS, PNM, and PNL), compared to the two shorter prickly variants is interesting particularly if the mean of prickled variants were pooled but there seems no pattern in other morphological together (Figure 2I). Root length is related to the plants’ characteristics for which it can be attributed to. capacity of the plant to access nutrients and water in the soil; thus, a high root length value suggests good soil The differences in the number of leaves counted among the penetration that aid absorption of more water and mineral narra variants with the same seedling age (three months) resources. As the seedling grows, one to two leaves were suggest the influence of several factors that might have added per month. The significantly longer root of the three been active during the growth of the plant affecting its PN variants than the SN may be related to the capacity to capacity to produce food via photosynthesis. SN has a access nutrients and water in the soil. In general, however, significantly higher mean leaf number compared to the P. indicus is well adapted to a wide range of soil types but three PN variants (Figure 2c). Considerable variation it is well suited in the deep, fertile, loamy, and alluvial between the numbers of leaves during the early growth type of soils. It has a large and extensive spreading root may be due to differences in the genotypes of mother growth habit that enables it to have an excellent potential trees. It could also be due to light intensities. A study for soil stabilization in areas along drainage lines and flood of Agyeman et al. (1999) showed that the leaf number plains (Thomson 2006). of five similarly tropical deciduous species – namely, Afzelia xylocarpa, Angophora costata, Dalbergia On the other hand, shoot length did not differ significantly cochinchinensis, Dipterocarpus alatus, and Hopea between the two narra types was not observed in this odorata were strongly related to the species characteristics study (Figure 2b), except for the significantly lower and their ability to respond to different light intensities. mean shoot length of PNL (Figure 2b). Theoretically, Continuous production of leaves suggests a difference in a high shoot length value suggests sufficient access to yield production due to varying photosynthetic capacity resources that are converted by the leaves to increase in plants. In the Philippines, a growth rate of 12 m3 ha–1 growth and productivity by increasing the photosynthetic yr–1 (172 ft3 ac–1 yr–1) over a 50-year rotation has been rate and increasing the transpiration area. The greater the used for plantation planning purposes (Thomson 2006). transpiration area in plants decreases the water potential

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In contrast, as leaf number was higher in SN than in characteristics. Previous studies revealed that the RCD prickled variants, SN had a significantly smaller mean varies directly with the rate of root elongation (Lecompte leaf area compared to prickled variants (Figure 2d). This et al. 2001). However, in P. indicus this tendency was not trade-off, however, follows the general pattern in plants evident as SN has significantly smaller mean root length in relation to the equitable distribution of photosynthates. than PN variants, but RCD did not vary between types This result supports the observation of Rojo (1972) that and among prickled variants. two forms of P. indicus are differentiated in terms of their leaf structure. SN has an ovate leaf while PN has a A study conducted by Mukherjee (2004) among southern lanceolate leaf. The shape of the leaf of PNS, PNM, and seedlings shows that plants with a higher grade in PNL has long tapering end at the leaf apex, with PNS RCD have better growth and survival performance than having the longest leaf taper. The variation in leaf area lower grade plants. The trees of P. indicus can tolerate may be caused by anatomical differences as an adaptation steady to storm type of wind because of its large buttress to environmental stress. Broad leaves often suggest a large and sturdy stem. Seedlings with a high value of RCD surface area for light absorbance, resulting in maximum are preferred to be planted in areas with a marginal growth rate in plants (Wilson et al. 1999). However, this environment such as unstable soil. All variants have high relationship was not generally observed in the case of RCD, suggesting that the species can be planted in areas narra. A significant correlation was found between the with a harsh environmental condition such as strong winds leaf area of PN and some anatomical characteristics. and high light intensity based on its silvical characteristics. PNL leaf area was significantly positively correlated to A high value of the root-shoot ratio in plants indicates upper epidermis (R2 = 0.9996, P = 0.0185), while both high absorption and storage capacity for water. In PNM and PNS leaf area were significantly negatively this study, SN showed significantly higher root-shoot correlated to lower epidermis (R2 = 0.9996, P = 0.01396 biomass than the PN (Figure 2f). A high root-shoot ratio and R2 = 0.9995, P = 0.01396, respectively) and to palisade is advantageous in the environmental condition where mesophyll (R2 = 0.9995, P = 0.01396 and R2 = 0.9995, water is limiting in the soil (Silva et al. 2012). The measure P = 0.01396, respectively). The leaf area of pooled PN of root-shoot ratio in plants shows the balance between variants was significantly positively correlated to palisade the transpiration rate in shoot and the water-absorbing mesophyll (R2 = 0.5143, P = 0.02966). In the study of Xu capacity of the root to compensate for the loss of water et al. (2012), they found out that the mesophyll volume in plants (Beikircher and Mayr 2009). Variation in plant's of Eucalyptus saligna is correlated with leaf area. Under measure of the root-shoot ratio can be related to adaptive full-sun conditions, the photosynthetic capacity per unit response developed by plants to avoid environmental leaf area increases, thus resulting in an increased thickness stress. The allocation of relative carbon to either shoot or in leaves, which is accompanied by the production of an root growth depends on the structural part of a plant that additional layer of palisade cells (Pompelli et al. 2010). experiences environmental resource limitation (Kozlowski The light-induced chloroplast movements in the palisade and Pallardy 2002). Furthermore, the seedling growth mesophylls are essential for efficient leaf photosynthesis and performance in terms of root-shoot ratio may also and light utilization in leaf cells (Gotoh et al. 2018). be influenced by other factors such as genetic makeup, In several experimental studies, the most frequently used ontogenetic stage, and seed sources. measure of morphological traits in plants is stem diameter However, there was a contrasting pattern in terms of to produce a more reliable measure of seedling quality seedling biomass, with PN showing significantly higher (Duryea and Dougherty 1991). The stem diameter is one biomass than the SN (Figure 2g). The seedling biomass in factor that is usually considered in evaluating transplanted plants is a good indicator of performance under local and seedlings from the nursery area to the field condition to marginal environments (Lebrija-Trejos et al. 2011). The measure seedling adaptability and performance rate in its seedling biomass affects the capability of plants to capture new environmental condition. RCD is associated with the light, nutrients, water, and CO₂ needed for sustenance trees’ capacity of trees to acclimate to stresses found in of various processes and functions. Higher seedling the environment and adapt to those changes through the biomass indicates the greater capacity of the variant to development of different characteristics, thereby allowing store photosynthate materials. The variation in biomass the tree to persist in the resource limiting environment allocation of the seedlings may be either linked to the (Sala et al. 2010). However, our result showed no geographic location of species along different gradients significant difference in mean RCD between narra or water availability in plants that is a key functional trait types and among the PN variants, except that PNM has in the carbon balance (Poorter 2001). significantly higher RCD than either long or short prickled variants (Figure 2e). Similar to shoot length, this could not be attributed to a distinct pattern with other morphological

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Anatomical Characteristics temperature in the environment (Ma et al. 2012). A good Greater thickness upper epidermis thickness indicates proportion of the palisade and spongy mesophyll in the better protection to avoid damaging mechanism in the leaves indicates a good physiological function of the plant internal tissues of the leaves due to excessive irradiance in photosynthesis, transpiration, and nitrogen-fixing in more than what plant can process for photosynthesis plants that will increase the growth, development, and (Terashima et al. 2001). However, in some plants that productivity of plants. These, however, were not visible grow in extreme environmental conditions, the epidermis yet from the morphological characteristics between narra may be several layers thick to protect against excessive types, except for the significant number of leaves and loss from transpiration. In this study, P. indicus variants are higher root-shoot ratio in SN than in prickled variants. characterized by the uniseriate upper and lower epidermis, In a typical leaf structure, the intercellular air spaces and the mean thickness of both at these epidermises was account for as much as 70% of the leaf’s volume that the same across the variants (Appendices III-a,b and IV). helps in the facilitation of the exchange of gases and Generally, mesophytic plants have a single thin layer of the water vapors (Ma et al. 2012). An increase in spongy upper epidermis (Wang et al. 2016; Clements 1905). The mesophyll was observed by Craven et al. (2010) in Acacia thicker epidermal layer in plants decreases the transpiration koa with increasing light intensity. The alteration in the of water and protects the internal tissues from high irradiance leaf structure of plants is a result of an adaptive change from the sun. Furthermore, the large epidermal thickness in plant development to tolerate environmental stress and improves the photosynthetic rate and enhances the growth decrease the vulnerability of plants to climate change. and development of the variant. The thicker lower epidermal cells in P. indicus variants contain numerous stomata in the Vascular tissues (phloem and xylem) play an essential role abaxial surface to reduce transpiration and for further gas in the continuum of plant processes and functions. It is exchange in the leaves, thereby providing cooling the plants. generally known that the xylem facilitates the transport of The epidermal cells are transparent,permitting most of the water and nutrients from the roots to the leaves to sustain light to pass through to the underlying cells. the plant during evapotranspiration and photosynthesis, while the phloem is concerned for the translocation of The epidermal characteristics of P. indicus contrast well sugar and nutrients produced during photosynthesis to with other documented plants in the country, albeit very facilitate respiration and the growth of plant tissues. As few. Bitayan et al. (2020) documented a biseriate upper the leaves of the plant lose water through transpiration, epidermis (though could be uniseriate plus hypodermis) the water potential in the leaf cell becomes negative that in Rhododendron subsessile in the harsh conditions of the causes water to move from xylem cells gradually. Xylem Cordillera central range in northern Philippines. Rayos and is the specialized tissue of vascular plants that transports Hadsall (2016), on the other hand, observed multiseriate water and nutrients from the plant-soil interface to stems upper epidermis in epiphytic Medinilla magnifica, M. and leaves (Myburg et al. 2013). In this study, only the miniata, M. pendula, and M. teysmannii. difference in the phloem area was evident with PN variants generally having a larger area of phloem than SN (Figure All variants were made up of two to three layers of palisade 4 and Appendix III-f). mesophyll at the upper portion of the leaf following the general characteristics of mesophytic plants. The number of palisade layers is directly related to high light intensity in the environment. However, significant differences in CONCLUSIONS the thickness of palisade and spongy mesophyll between PN and SN were evident. SN had significantly thicker Among the seven morphological parameters used, the mesophyll tissues compared to PN variants (Appendices mean root length, mean leaf number, and mean leaf area III-c,d and V; Figure 3). Since most chloroplasts are strictly separated the two major P. indicus forms. SN has located in the palisade mesophyll, some are in the spongy a significantly lower mean root length and mean lower mesophyll, thicker palisade suggests a higher amount of leaf area but has a significantly higher mean leaf area chloroplast (Shi et al. 2010) and, hence, may indicate the than the prickled variants. Root-shoot ratio and biomass higher photosynthetic rate in SN compared to the prickled can also be used to clearly show the difference in the variants. Although only early growth was considered only growth performance of the narra types, although these in this study, this result may explain the significantly two parameters seem to be inversely related in P. indicus. higher leaf number in SN as the photosynthate is used Anatomical features particularly the mesophyll tissues by the plants for growth and development. On the other phloem show also between the two narra types. However, hand, thicker spongy mesophyll suggests more efficient a clear pattern in variability among prickly variants in gas exchange and reduced evapotranspiration in leaf terms of morphological and anatomical characteristics as a result of a sufficient supply of light intensity and were not detected. Variability in morphological and

285 Philippine Journal of Science Flores et al.: Morphology and Anatomy of P. indicus Vol. 150 No. 1, February 2021 anatomical features in relation to the prickle size of CLEMENTS ES. 1905. The Relation of Leaf Structure the PN (taxonomically as P. indicus forma echinatus) to Physical Factors. Transactions of the American Mi- may be more visible if plants were exposed to distinct croscopical Society, Vol. 26. Twenty-Seventh Annual environmental conditions. Molecular markers are powerful Meeting (Dec. 1905). Wiley. 89p. to discriminate species, conspecific populations, or possibly CRAVENS D, GULAMHUSSEIN S, BERLYN GP. 2010. individuals. However, in the absence of molecular data, Physiological and anatomical responses of Acacia koa the morphological and anatomical features observed in (Gray) seedlings to varying light and drought condi- this study show promising contributions to detecting tions. Botany 69(2): 205–213. differentiation between the two major forms of P. indicus. DELOS REYES MA, MAGPANTAY, GD, CAGALA- WAN A, LAPIS A, CALINAWAN NM. 2016. Assess- ment of genetic diversity of narra (Pterocarpus indicus ACKNOWLEDGMENTS Willd.) populations from various seed sources in the Philippines using RAPD. Journal of Environmental The authors wish to thank the DFBS and MCME-CFNR, Science and Management 192(2): 54–63. UPLB for allowing the use of its facilities for the conduct of this study. This study is supported partly by the project DURYEA ML, DOUGHERTY PM. 1991. Forest Regen- funded by the Department of Science and Technology eration Manual, 1st Ed. Netherlands: Springer. 433p. through the Philippine Council for Agriculture, Aquatic doi: 10.1007/978-94-011-3800-0 and Natural Resources Research and Development FAHN A. 1964. Some anatomical adaptations of desert “Germplasm Conservation of Selected Indigenous Forest plants. Phytomorphology 14: 93–102. Trees in Mount Makiling Forest Reserve (MMFR).” FAHN A. 1967. Plant Anatomy. Oxford: Pergamon Press Ltd. 541p. GOTOH E, SUETSUGU N, HIGA T, MATSUSHITA T, NOTE ON APPENDICES TSUKAYA H, WADA M. 2018. Palisade cell shape The complete appendices section of the study is accessible affects the light-induced chloroplast movement and at http://philjournsci.dost.gov.ph leaf photosynthesis. Scientific Reports 8(1472): 1–9. HAUPT AW. 1930. A gelatin fixative for paraffin sections. Stain Technology 5(3): 97–98. REFERENCES HAUPT AW. 1953. Plant Morphology. McGraw-Hill, University of Michigan, USA. 464p. AGYEMAN VK, SWAINE MD, THOMPTION J. 1999. Responses of tropical forest tree seedlings to irradiance HERNANDEZ JO, FERNANDO ES, MALABRIGO JR. and the derivation of light response index. Journal of PL, QUIMADO MO, MALDIA LSJ. 2016. Xerophytic Ecology 87: 815–827. characteristics of philippinensis Benth. & Hook. f. Philippine Journal of Science 145(3): 259–269. BEIKIRCHER B, MAYR S. 2009. Intraspecific differ- ences in drought tolerance and acclimation in hydrau- HONG Z, WU, Z, ZHAO K, YANG Z, ZHANG N, lics of Ligustrum vulgare and Viburnum lantana. Tree GUO J, TEMBROCK LR, XU D. 2020. Comparative Physiol 29: 765–775. analysis of five complete chloroplast genomes from the Pterocarpus (Fabaceae). International Journal of BELL AD. 2008. Plant Form: An Illustrated Guide to Molecular Sciences 21(3758): 1–18. Morphology. Oxford University Press: London. 393p. HOPKINS WG, HÜNER PA. 2008. Introduction to Plant Physiology, Fourth edition. Hoboken, NJ: John Wiley BITAYAN MGV, CERVANTES SS, NAPALDET JT. & Sons, Inc. 523p. 2020. Morpho‑anatomical characterization of Rho- dodendron subsessile Rendle, an endangered species JOHANSEN DA. 1940. Plant Microtechnique. New York: of the Cordillera Central Range, Philippines. Journal McGraw-Hill. 523p. of Forestry Research https://doi.org/10.1007/s11676- KOZLOWSKI TT, PALLARDY SG. 2002. Acclimation 019-01087-5 and adaptive responses of woody plants to environ- BRADSHAW AD. 1965. Evolutionary significance of mental stresses. Bot Rev 68: 270–334. phenotypic plasticity in plants. Advances in Genetics LEBRIJA-TREJOS E, PÉREZ-GARCÍA EA, MEAVE 13: 115–155. JA, POORTER L, BONGERS F. 2011. Environmen-

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APPENDICES

Appendix I. Summary of the analysis of variance for different Appendix II. Summary of the analysis of variance for different morphological parameters measured. anatomical parameters measured. Parameter P-valuea P-valueb P-valuec Anatomical measurement P-valuea P-valueb P-valuec Root length (cm) 0.0012 0.0000 0.8290 Upper epidermis 0.7010 0.2440 0.9210 Shoot length (cm) 0.0000 0.2500 0.0000 Lower epidermis 0.3490 0.8160 0.0983 Leaf number 0.0000 0.0000 0.2890 Palisade mesophyll 0.0257 0.0022 0.6560 Leaf area (cm²) 0.0006 0.0000 0.9960 Spongy mesophyll 0.0000 0.0325 0.0009 Root collar diameter (mm) 0.0093 0.0624 0.0133 Xylem area 0.0240 0.8850 0.0178 Root-shoot ratio 0.0068 0.0031 0.0965 Phloem area 0.0266 0.0145 0.1650 Seedling biomass (g) 0.0117 0.0011 0.8590 SN – smooth narra, PNS – short prickly narra, PNM – medium prickly narra, PNL – long prickly narra; aANOVA analysis using the four variants, bANOVA a b ANOVA analysis using the four variants, ANOVA analysis for the pooled prickled analysis for the pooled prickled variants and Smooth Narra, and cANOVA analysis c variants and Smooth Narra, and ANOVA analysis for the prickly variants only. for the prickly variants only.

Appendix III. (I) Six anatomical parameters such as upper epidermis (a), lower epidermis (b), palisade mesophyll (c), spongy mesophyll (d), xylem area (e), and phloem area (f) used for the ANOVA and DMRT analyses using four P. indicus variants; (II) six anatomical parameters analyzed using pooled prickled variants and smooth narra. Means followed by the same letter are not significantly different at 5% level by DMRT; SN – smooth narra, PNS – short prickly narra, PNM – medium prickly narra, PNL – long prickly narra.

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Appendix IV. Leaf anatomy of the four variants of P. indicus: A) long/soft prickly narra (PNL), B) medium prickly narra (PNM), C) short/rough prickly narra (PNS), and D) smooth narra (SN) showing the upper epidermis (UE) and lower epidermis (LE). Bar represents 100 µm.

Appendix V. Leaf anatomy of the four variants of P. indicus: A) long/soft prickly narra (PNL), B) medium prickly narra (PNM), C) short/rough prickly narra (PNS), and D) smooth narra (SN) showing the spongy mesophyll (SM) and lower epidermis (LE). Bar represents 100 µm.

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