IAWA Journal, Vol. 22 (4), 2001: 355–365 REEXAMINATION OF WOOD ANATOMICAL FEATURES IN PINUS KREMPFII (PINACEAE) by Stefanie M. Ickert-Bond Arizona State University, Department of Plant Biology, Box 871601, Tempe, AZ 85287-1601, U.S.A. SUMMARY Wood anatomy of Pinus krempii Lecomte, a pine endemic to Vietnam, is described using twig and mature wood collections made in 1995. Characteristics of Pinus krempii wood include axial and radial resin canals with 6–8 thin-walled epithelial cells; latewood tracheids with tangential wall pitting; ligniied ray parenchyma with 2–5 pinoid pits per cross-ield; and few to no ray tracheids. Longitudinal tracheid diameter and ray height are smaller in the twig wood than in the mature wood. These features, especially the near absence of ray tracheids, suggest a relationship with Pinus subgenus Strobus section Parrya subsection Balfourianae, which includes P. aristata Engelm. and P. longaeva D. Bailey. Key words: Pinus krempii, Pinus section Parrya, wood anatomy, ray tracheids, conifers. INTRODUCTION Pinus krempii Lecomte is an unusual pine endemic to the Central Highlands of Vietnam. It occurs in dense, mixed montane evergreen forests mainly on ridgetops at 1800 m elevation in humic soils up to 40 cm deep, these often wet and assuring seedling success. The loristic composition of the forest is very diverse (Kha 1967) with large-trunked conifers, such as Dacrycarpus imbricatus (Blume) Laubenfels, D. pilgeri Foxw., Cephalotaxus hainanensis H.L. Li, occasionally Pinus kesiya Royle ex Gordon and Keteleeria evelyiana Mast. and several species of evergreen Fagaceae (Ickert-Bond 1997b). Trees of P. krempii are evergreen and 15–30 m in height, extending high above the general forest canopy (Fig. 1). The trunk is cylindrical, straight, and unbranched for the irst 15–20 m and up to 5.5 m diameter at breast height (dbh) (Fig. 2). The crown is sparse and pyramidal in young trees, becoming umbrella-shaped when older. The branching pattern in the crown is almost dichotomizing and unusual for pines. The bark is persistent, reddish brown, very thin, and slightly scaly with shallow longitudinal issures. Acicular leaves occur in pairs and are arranged in a scissor-like fashion (Rollet 1955; Ickert-Bond 2000). They are oblong-lanceolate, lattened, 4.5–7 cm long and 3.5– 4.1 (up to 5) mm wide, with short, slightly twisted petioles (Fig. 3). Key vegetative features that distinguish this pine from other members of the genus 356 IAWA Journal, Vol. 22 (4), 2001 Fig. 1–3. Habit of Pinus krempii. – 1: Stands with large umbrella-shaped crowns ranging high above the general forest canopy (arrows). – 2: Mature trunk. – 3: Branching pattern with broad needles and ovulate cone (at right). Photographs by Richard Bond. Pinus include the characteristic falcate lat needles, crescent-shaped cross section of the needle, early-deciduous fascicle sheaths, dimorphic mesophyll and the occurrence of dimorphic leaves (Ickert-Bond & Pigg 1996; Ickert-Bond 1997a, 1997b, 2000). Ickert-Bond — Wood anatomy of Pinus krempfii 357 Taxonomically, Pinus krempii has been the subject of much debate. It has been placed in Pinus subgenus Strobus as monotypic in subsection Krempianae Little & Critchield (Little & Critchield 1969; Klaus 1980; Farjon 1984; Price et al. 1998), or in subsection Balfourianae Engelm. (Van der Burgh 1973), or in subgenus Ducampopinus A. Chev. (Ferré 1948). It has even been elevated to a monotypic generic rank (Chevalier 1944; Hudson 1983; Landry 1994) as Ducampopinus krempii (Lecomte) A. Chev. While most morphological features are undisputed, there is conlicting information about characters of the wood anatomy (Buchholz 1951; Budkevich 1958; Greguss 1962; Hudson 1983). Part of the variation reported may be related to differences in age of material studied, as some authors have looked at mature stem wood (Budkevich 1958; Hudson 1983) and others at small twigs (Buchholz 1951). Among the differences observed are the presence (Greguss 1962) or absence of ray tracheids (Buchholz 1951; Budkevich 1958; Hudson 1983), the unusual presence (Budkevich 1958) or, more commonly, the absence of axial parenchyma (Greguss 1962), and according to Budkevich (1958) and Hudson (1983) the occurrence of a taxodioid cross-ield pit type unique within Pinus. These characters are important in understanding pine evolution and assigning pines in a classiication system. In order to clarify its structure and variation, this paper describes mature and twig wood anatomy of Pinus krempii based on new collections from Vietnam. Results suggest that P. krempii shares ancestral features with pines in section Parrya and related genera of the Pinaceae. MATERIALS AND METHODS Collections were made of Pinus krempii on May 5 –12, 1995, from Lam Dong Province near Cong Troi, Vietnam. Wood anatomy is described based on both wood from a mature trunk and 16 twigs. A large block (20 cm high and 7 cm wide) of mature wood was obtained from Dr. Tiep of Dalat University, Vietnam, from a tree measuring 5 m dbh. He collected it from a population near BiDoup, Langbian Mountains, Vietnam. The block consists mainly of ine-grained heartwood. Twig wood of 2 cm in diameter was collected from three different populations near Cong Troi, Langbian Mountains (Table 1). The trees varied from 1– 3 m dbh. Three twigs per population sample were examined for a total of nine measured wood samples. Additional slides of Pinus krempii twig wood were available in the Bailey-Wetmore Wood Collection of Harvard University (Table 1). Wood material was rehydrated in water and sectioned on a sliding microtome at 20 μm and subsequently stained with safranin (following Johansen 1940); staining for lignin was accomplished with phloroglucinol (Ruzin 1999). Voucher specimens and wood blocks are deposited in the wood collection housed in the Fossil Plant Collections, Arizona State University Herbarium, Tempe, Arizona (ASU) (Table 1). Measurements of tracheid diameter and ray height were taken from tangential sections, while ray parenchyma diameter was obtained from radial sections. For each of these characters, means, maximum and minimum values were determined. These values were based on 25 measurements per slide. 358 IAWA Journal, Vol. 22 (4), 2001 Table 1. Pinus krempii material examined. Voucher Slide numbers Type of material Locality in Vietnam Herbarium Ickert-Bond 268 268-96-1, 268-96-2, 268-96-3 Twig wood Dalat Flower Garden ASU Ickert-Bond 278 278-96-1, 278-96-2, 278-96-3, 278-96-4, 278-96-5, 278-96-6 Twig wood Công` Trôi-, Dalat ASU Ickert-Bond 300 300-96-1, 300-96-2, 300-96-3 Twig wood Công` Trôi-, Dalat ASU Truong Van Len s. n. A, B Twig wood Boungia / Dalat GH Tiep s. n. T-96-1, T-96-2, T-96-3, T-96-4, T-96-5 Mature wood BiDoup, Lang Bian ASU RESULTS Macroscopic features Heartwood has a reddish brown color. The transition from earlywood to latewood is gradual (Fig. 4). Growth increments are narrow and up to 29 have been counted in the large mature block over 1 cm, whereas twig wood only had 18 growth rings over a 1 cm radius. Microscopic features Axial resin canals are common in the secondary xylem, particularly in the earlywood, and are surrounded by 6– 8 thin-walled epithelial cells (Fig. 4 & 5). Maximum axial resin canal diameter in the large block is 90 μm, and 60 μm in the twig wood. Resin canals of the twig wood are surrounded by axial parenchyma (Fig. 5). Both mature and twig wood have tracheids with radial walls that show uniseriate and biseriate circular-bordered pits (Fig. 7 & 8). Tangential walls of latewood tracheids show numerous circular-bordered pits, typical of soft pines (Fig. 8) (Hudson 1960; Van der Burgh 1973). Tracheids are 32 μm (22– 45) in tangential diameter in the twig wood, 51 μm (39–77) in the mature wood. Uniseriate rays are 2–16 cells and 142–224 μm high (twig wood 77–220 μm, mature wood 154– 418 μm). Fusiform rays are 6–18 cells and 173– 407 μm high (twig wood 110–264 μm, mature wood 198–638 μm). Fusiform rays are bi- or triseriate throughout the central portion with a single transverse resin canal, and taper above and below to uniseriate margins (Fig. 6). Uni- Ickert-Bond — Wood anatomy of Pinus krempfii 359 Fig. 4–6. Pinus krempii wood. – 4 & 5. Cross sections. – 4: Growth rings with gradual transition from earlywood (middle) to latewood (top); note resin canal located in the earlywood. – 5: Resin canal with 7–12 epithelial cells with thin lining (eL) and associated axial parenchyma (aP). – 6: Tangential section showing fusiform ray. — Scale bars = 50 μm. seriate rays are usually homocellular, composed only of parenchyma cells; ray tracheids are nearly always absent (Fig. 7). In more than 400 rays of juvenile and mature wood studied, only two ray tracheids were observed (Fig. 11). Ray parenchyma cells have 1 to 5 pinoid (sensu Greguss 1955) cross-ield pits (Fig. 9 & 10). The cell walls are ligniied as they stain deeply red with phloroglucinol. 360 IAWA Journal, Vol. 22 (4), 2001 Fig. 7–11. Radial sections of Pinus krempii wood. – 7: Biseriate intertracheary pits (at left), and a ray six cells high, composed of only ray parenchyma cells. – 8: Circular bordered pits on radial walls (at left) and also on tangential wall (arrows) of latewood. – 9: Ray parenchyma cells showing nature of wall and cross-ield pits (at arrow). – 10: Cross-ields with 2–4 pinoid pits. – 11: Uniseriate intertracheary pits (at left) and ray composed of ray parenchyma and ray tracheid on top (arrows). — Scale bars = 100 μm for 7; 50 μm for 8 & 9; 30 μm for 10 & 11. Ickert-Bond — Wood anatomy of Pinus krempfii 361 DISCUSSION Main characteristics of the typical wood of Pinus include: 1) Epithelial cells of the resin canals thin-walled (e.g., Esau 1965), 2) Absence of axial parenchyma (Jane 1956), 3) Cross-ield pits pinoid, piceoid or fenestriform (Greguss 1955; Hudson 1960), 4) Typical presence of ray tracheids (Panshin & De Zeeuw 1980). Other genera in the Pinaceae differ from this combination of characters. For example, ray tracheids are absent from Abies, Keteleeria and Pseudolarix (Esau 1965) but are present in Cathaya (Hu & Wang 1984), Larix, Picea, Cedrus, Tsuga and Pseudotsuga (Panshin & De Zeeuw 1980). The absence of resin canals further separates those genera with ray tracheids. Normal resin canals are absent from Cedrus and Tsuga wood, while Cathaya, Larix, Picea and Pseudotsuga have normal resin canals, but are distinguished from Pinus by having thick-walled rather than thin-walled epithelial cells. Taxonomic value of anatomical wood characters Within the genus Pinus, a binary taxonomic division is generally accepted and can be substantiated by characters of the wood anatomy. The dentations of the walls of the ray tracheids (Bailey 1910; Phillips 1941; Hudson 1960) can be used to distinguish between the two subgenera Strobus and Pinus. Ray tracheids have smooth walls to occasionally slightly dentate walls in subgenus Strobus and dentate to heavily dentate walls in subgenus Pinus. In a survey of the wood anatomy of numerous species Hudson (1960) described a scale of 10 different dentation types that are correlated with subgenera. Types 1– 4 correlate with smooth to slightly dentate walls of the ray tracheids and are restricted to subgenus Strobus, while Types 5 –10, with increasingly pronounced dentations, deine subgenus Pinus. In the present study the few ray tracheids that were observed showed smooth walls of Type 1 similar to those in P. cembra (Hudson 1960) or P. aristata (Süss 1989) of subgenus Strobus (Table 2). Other characters that are frequently used in assigning species to sections within Pinus include cross-ield pitting (Greguss 1962; Van der Burgh 1973), circular bordered pits on tangential walls of latewood (Hudson 1960; Van der Burgh 1973), and ligniication of ray parenchyma cells (Van der Burgh 1973). Both Hudson (1960) and Van der Burgh (1973) showed in their surveys of wood anatomy of numerous pine species that the frequency of circular bordered pits on tangential walls of the latewood corresponds to the binary division of the genus Pinus. In subgenus Strobus, namely section Strobi and section Parrya, numerous circular bordered pits occur in the latewood tangential walls, while in subgenus Pinus relatively few pits occur in the latewood tangential walls. An exception is found in subsection Lumholtzii of subgenus Pinus, in which numerous circular bordered pits occur in the latewood. In the present study, numerous circular bordered pits were found on the tangential walls of the latewood of P. krempii, afiliating the wood with subgenus Strobus. Van der Burgh (1973) assumes the directionality for this character to be from numerous to few circular bordered pits as realized in all sections of subgenus Pinus. Van der Burgh (1973), in his survey of the wood anatomy of 67 pine species, distinguished between two general types of cross-ield pitting: (ʻType aʼ) those with 1– 3 362 IAWA Journal, Vol. 22 (4), 2001 Table 2. Comparison of selected character states of Pinus wood anatomy between subgenus Pinus, subgenus Strobus (subsections Strobi and Parrya), and Pinus krempii. Characters Subgenus Pinus Section Pinus All subsections Subgenus Strobus Section Strobi Section Parrya Subsection Strobi Subsection Balfourianae Walls of the ray tracheids Heavily dentate (H. sc. 7–11) Smooth to slightly dentate (H. sc. 1–4) Smooth to slightly dentate (H. sc. 1–6) Smooth, if present (H. sc. 2) Cross-ield pitting / cross-ield 1–3 large fenestriform pits 1–3 large fenestriform pits 3–5 small pinoid pits 1–4 small pinoid pits Circular bordered pits on tangential walls of latewood None or few Numerous Numerous Numerous Ray parenchyma cells Non-ligniied Ligniied Ligniied Ligniied Presence of ray tracheids Frequent Frequent Infrequent Very rare to absent Pinus krempii H. sc. = Hudson scale. large fenestriform pits and (ʻType bʼ) those with numerous small pinoid pits per crossield. ʻType aʼ occurs in both section Strobi (subgenus Strobus) and section Pinus (subgenus Pinus), while all other sections are characterized by numerous small pinoid pits per cross-ield of ʻType bʼ. Specimens of P. krempii showed 2–5 pinoid pits per cross-ield pit; in subgenus Strobus this character state is conined to section Parrya. Süss (1989) showed 3–5 small “pinoid or piceoid” pits for 25-year-old branch wood of P. aristata, which is also of section Parrya. Van der Burgh (1973) hypothesized that the ancestral state is that of section Parrya because this character state is also found in all branch wood of other sections of Pinus; and other genera of the Pinaceae show numerous small well developed “pinoid or piceoid” cross-ield pits as well. The ligniication of ray parenchyma cells has also been used by Bailey (1910) and later by Van der Burgh (1973) to distinguish among sections of the genus Pinus. Two categories are recognized, one with ligniied ray parenchyma cell walls as in sections Strobus, Parrya, Pinea and Sula, the other with either unligniied and heavily ligniied walls occurs in all other sections. Ray parenchyma cells in Pinus krempii are ligniied as in sections Strobus and Parrya. Position of P. krempii within the genus Pinus In summary, the wood of P. krempii has characteristics typical of the genus Pinus, including thin-walled epithelial cells, axial parenchyma restricted to near the resin canals, and both vertical and horizontal resin canals. Afinity with the soft pines (Pinus subgenus Strobus and, in particular, section Parrya) is indicated by the occurrence of Ickert-Bond — Wood anatomy of Pinus krempfii 363 numerous circular-bordered pits on tangential walls of latewood tracheids (Hudson 1960; Van der Burgh 1973; Table 2), ligniied ray parenchyma cells, and pinoid crossield pitting. These features occur within subgenus Strobus section Parrya, in such species as Pinus gerardiana Wall., P. bungeana Zucc. (Van der Burgh 1973), and P. longaeva (Baas et al. 1986; Süss 1989) and in some species of subgenus Pinus section Pinus subsection Sylvestres. Pinoid pits also occur in wood of section Pinaster subsection Australes of subgenus Pinus, e.g., Pinus pungens Lamb. ex Michx. and P. rigida Mill. (Hudson 1960). My indings of the near absence of ray tracheids in wood of Pinus krempii concordant with results from several authors (Buchholz 1951; Budkevich 1958; Hudson 1983) are in contrast to Gregussʼ (1962) reports that ray tracheids are commonly present. This plesiomorphic character state is shared with pines of subgenus Strobus section Parrya subsection Balfourianae, e.g., P. aristata and P. longaeva (Van der Burgh 1973; Baas et al. 1986; Süss 1989) and by the Abietoideae. Van der Burgh (1973) documented that all species of subgenus Strobus section Strobi and section Parrya lack ray tracheids in the irst three growth rings. Slow-growing branches can lack them for up to eight years. Baas et al. (1986) showed that P. longaeva of section Parrya lacks ray tracheids even in some mature stem wood. Additionally, Süss (1989) remarked on the rare occurrence of ray tracheids in a 25-year-old branch sample of P. aristata of section Parrya. In contrast, all species of subgenus Pinus show welldeveloped ray tracheids in the latewood of the irst growth ring and in the following growth rings in both earlywood and latewood (Van der Burgh 1973). Based on wood anatomy Mirov (1967) also concluded that P. krempii, P. aristata and P. longaeva and Cretaceous pine fossils are more basal within Pinus, linking them to other genera within the Pinaceae. Other fossil woods with Picea- and Pinus-afinity have been reported to lack ray tracheids (Kräusel 1919, 1949; Watari 1941). Outgroup comparison of Pinus with the genera Abies, Keteleeria and Pseudolarix also establishes the plesiomorphic condition of this character state (Hart 1987; Wang & Szmidt 1993; Price et al. 1998; Liston et al. 1999; Wang et al. 1999). In addition to wood anatomical features, a combination of characters, including ovulate cone morphology, biogeography, paleobotany and molecular systematics support a basal position for Pinus section Parrya (Bailey 1910; Van der Burgh 1973; Klaus 1980; Millar 1998; Wang et al. 1999, 2000). The present study of Pinus krempii wood thus clariies the character states as conforming with subgenus Strobus section Parrya, a section believed to be basal within the genus as supported by recent molecular data (Wang et al. 1999, 2000). ACKNOWLEDGEMENTS I thank T. D. Ly, N.T. Hiê. p, N. D. Khôi, National Centre for Science and Technology of Vietnam and Phan Kê´ Lô. c, University of Hanoi, for help in obtaining specimens and ield assistance. Special thanks to Kathleen B. Pigg for her helpful discussions and advice on the manuscript. 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