STEM ANATOMY and DEVELOPMENT of SUCCESSIVE CAMBIA in the NEOTROPICAL LIANA SECURIDACA RIVINIFOLIA (POLYGALACEAE) Kishore S. Rajp

IAWA Journal, Vol. 33 (4), 2012: 391–402

STEM ANATOMY AND DEVELOPMENT OF SUCCESSIVE CAMBIA IN THE NEOTROPICAL LIANA SECURIDACA RIVINIFOLIA (POLYGALACEAE)

Kishore S. Rajput1,*, Marina B. Fiamengui2 and Carmen R. Marcati2

SUMMARY The pattern of secondary growth and structure of secondary xylem was studied in the stem of the Neotropical liana Securidaca rivinifolia A. St.-Hil. (Polygalaceae). Increase in thickness of the stem was achieved by formation of successive cambia, from which initially two or three successive rings formed complete oval to circular cambia. Thereafter, the successive cambia were always crescent-shaped and never formed a complete cylinder, resulting in dumbbell-shaped cross-sectional outlines of the stems. The first successive cambium originated in the pericyclic parenchyma located outside the crushed protophloem. Prior to the development of cambium, pericyclic parenchyma formed a meristematic band of radially arranged cells. From this band, cells located in the middle of the band became the new ring of cambium. Cells on the inner face of the xylem produced by newly formed cambium differentiated into conjunctive tissue. The first elements to be differentiated from the newly developed cambium were always xylem fibres but differentiation of vessels was also observed occasionally. The xylem was diffuse porous with relatively distinct growth rings and composed of mostly solitary vessels with simple perforation plates, fibres with bordered pits, paratracheal axial parenchyma, and exclusively uniseriate rays. Key words: Cambial variant, Polygalaceae, secondary phloem, secondary xylem, Securidaca, successive cambia.

INTRODUCTION Climbing plants differ from trees and shrubs in a number of characteristics; the most notable is the mechanical properties of the stem (Isnard et al. 2003; Lopes et al. 2008). This variation in the mechanical properties of wood is related with a shift from the self-supporting habit to the climbing habit. The liana habit is often (but not exclusively) associated with the presence of successive cambia, interxylary phloem, dissected or compound xylem, and furrowed xylem (Rajput et al. 2012). These phenomena are often referred to as cambial variants or anomalous secondary growth (Carlquist 2001).

1) Department of Botany, Faculty of Science, The M.S. University of Baroda, Vadodara 390002, India. 2) Depto. Recursos Naturais–Ciências Florestais, Faculdade de Ciências Agronômicas (FCA), Universidade Estadual Paulista (UNESP), Campus de Botucatu, CP237, CEP 18603-970 Botucatu, SP, Brazil. *) Corresponding author [E-mail: [email protected]].

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Variant secondary growth is widespread in lianas and it is considered to increase stem flexibility, to protect the phloem, and to some extent also to increase storage parenchyma (Carlquist 1991, 2001; Patil et al. 2011). It also limits physical disruption of vascular tissues during twisting and bending, and promotes wound healing (Dobbins & Fisher 1986; Fisher & Ewers 1992; Lopes et al. 2008). A supposedly major benefit of the variant secondary growth in lianas is the adaptation to the climbing habit from the self-supporting one. Anomalous secondary thickening is, however, by no means restricted to lianas. In Amaranthaceae (including Chenopodiaceae) and other members of the Caryophyllales successive cambia characterize virtually all, largely erect, self- supporting members of the order (cf. Heklau et al. 2012). Although woody climbers (lianas) are very common in tropical forests, anatomical studies have been rather few (Bamber & Ter Welle 1994; Lopes et al. 2008; Pace et al. 2009, 2011), and very little attention has been given to the cellular composition of xylem in scandent plants (Carlquist 1985; Araujo & Costa 2006; Rajput et al. 2010a; Pace et al. 2011). However, lianas are important subjects for the study of cambial variants, and their development (Obaton 1960; Carlquist 1991; Araujo & Costa 2006; Patil & Rajput 2008; Rajput et al. 2008, 2010a; Pace et al. 2011). The family Polygalaceae comprises herbs, scandent shrubs, shrubs or rarely trees (Xanthophyllum has some large tree species in it) and climbers/lianas (rarely) that occur in both temperate and tropical regions. Secondary xylem of herbs and shrubs in the Polygalaceae is normal, but presence of included phloem of the concentric type is recorded for the different climbing species of the Polygalaceae including Securidaca (Record & Hess 1943; Metcalfe & Chalk 1950) but the development of successive cambia remains to be studied. Characteristically, stems of this species are lobed or dumbbell-shaped as seen in cross-sectional view. The present investigation is, therefore, aimed to elucidate the pattern of secondary growth, the development and structure of xylem and phloem, and correlation of cambial activity with growth ring formation (if any) and stems shape in Securidaca rivinifolia.

Materials and methods

Eight to ten 40–60 mm long segments of the main stems, ranging in diameter from 5–30 mm were collected from two plants of Securidaca rivinifolia A. St.-Hil. (Polygalaceae) growing in Botucatu municipality, in Rio Bonito, São Paulo state, Brazil. Samples were collected from the base (30 cm above the ground), middle (c. 2 metre above ground) and top portion about 8–10 mm thick (5–6 metre above ground) in September 2008 and June 2009. Samples were fixed immediately in FAA70 (Berlyn & Miksche 1976) and transferred to 70% alcohol after 12 hrs of fixation. The samples were cut into smaller pieces (8–10 mm2) and embedded in PEG (30%, 50%, 70%, 90% followed by 2×100% pure PEG) and processed as described by Rupp (1964), modified by Richter (1981) and adapted by Barbosa et al. (2010). Transverse, radial and tangential longitudinal sections of 12–15 µm thickness were obtained with a sliding microtome and stained with a 1% aqueous solution of safranin (Bukatsch 1972) and Astra Blue (Roeser 1972) and mounted permanently in synthetic resin Entelan®.

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Small pieces of secondary xylem adjacent to the outermost cambium ring were macer- ated according to Franklin’s method (Franklin 1945, modified by Kraus & Arduin 1997) at 55 to 60 °C for c. 8 hrs, and stained with an ethanolic solution of safranin (Bukatsch 1972) to study general morphology and dimensional details. Length and width of the sieve tube elements were measured directly from the tangential longitudinal sections. Thirty measurements were chosen randomly to obtain mean and standard deviation for each cell type. Wood anatomical terminology follows the IAWA Committee (1989) and Carlquist (2001).

Results Development of successive cambia The first ring of the vascular cambium remained active for longer than a complete growing season to produce a pencil-thick (8–10 mm in diameter) stem. In the next growing season, when the plants were with new leaves and the apical shoot was growing actively, two small arcs of cambium originated on opposite sides of the stem (Fig. 1, 2A). The pericyclic parenchyma cells in the outer region of the protophloem became meristematic and formed a group of cells arranged in radial rows like cambium (Fig. 2B). From these meristematic bands, cells located in the middle of the band remained meristematic and gave rise to the small segments of the cambium. The cells on the inner face of the newly developed cambial zone formed the conjunctive tissue while cells external to this cambial zone served as site for the origin of future cambium

Figure 1. Overview of Securidaca rivinifolia young stem in cross section. Encircled portion of the stem indicates the position of first successive cambial origin. — Scale bar = 200 µm.

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Figure 2. Transverse view of young and mature stem of Securidaca rivinifolia showing different stages of cambial origin. – A: Enlarged view of Fig. 1 showing newly originated first successive cambium and few xylem cells. Note the parenchymatous derivatives of pericycle on the upper side of newly developing cambium (arrowheads). X = xylem. – B: Enlarged view of newly developing cambium. Note the wide band of parenchyma cells that develops into cambium and conjunctive tissue (arrow). Arrowhead showing newly differentiated phloem element. – C: Enlarged view of newly developing cambium. Arrowheads indicate newly formed sieve tubes. – D: Parenchyma cells located externally to the phloem formed by previous cambium (arrowhead). Parenchyma cells that undergo dedifferentiation and give rise to a new ring of successive cambium. – E: Tan- gential spreading of the newly developed cambium. Note the differentiated fibres on the xylem side (arrowhead). – Scale bars for A = 125 µm; for B–E = 75 µm.

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(Fig. 2C, E). Inception of the second ring of cambium began as small segments, which gradually extended tangentially and formed crescent-shaped arcs. These arcs ultimately met each other to form a complete cylinder. Functionally this cambium was bidirectional and formed secondary xylem centripetally and secondary phloem including tangential bands of phloem fibres centrifugally. These fibre bands were interrupted at regular intervals by 2–3 parenchyma cells (Fig. 1, 2D, E). The second ring of cambium remained functional for a relatively long period and formed about 2–3 mm of secondary xylem. Thereafter, the next cambial ring originated from the parenchyma cells located outside the phloem produced by the previous cambium. Development of all the successive cambia followed a similar pattern as described above. However, the first 2–3 cambia were able to form a complete cylinder while, subsequently, no complete cylinders are formed, because of loss of cambial activity at two opposed sides of the initially cylindrical cambium. The formation of crescent- shaped cambia only on two opposed sides of the stem in cross section brought about a bilateral flattening of the axis, resulting in the dumbbell-shaped configuration as seen in cross section (Fig. 3A). Cell division and formation of secondary xylem were greater in the median portion of the crescent-sized or “C” shape and declined gradually towards adjacent sides of the segments.

Development and structure of vascular elements With the initiation of secondary xylem development, cells located at the inner face of xylem produced by the newly developed cambium differentiated into conjunctive tissue (Fig. 3B). However, contribution of the newly developed cambium in formation of conjunctive tissue is very little. All the successive cambial segments divided bidirectionally and gave rise to secondary xylem centripetally and secondary phloem centrifugally. The first elements to differentiate on the inner side of each new cambium were always fibres (Fig. 3B) while differentiation of vessels occurred only after the formation of a few xylem fibres (Fig. 2D, E). However, formation of vessel elements at the beginning of the newly developed cambium was also noticed occasionally (Fig. 3C). Growth rings were distinct in the secondary xylem and were delimited by thick- walled and radially flattened latewood fibres (Fig. 4A, B). At the growth ring boundary, rays also showed distinct variation in their radial length, the ray cells were elongated in the earlywood, and more or less isodiametric at the growth ring boundary, resulting in faint ray noding (Fig. 4C). As the stem showed excentric successive cambial rings, the rings were wider in the centre of the crescents, tapering off towards the sides. Xylem was diffuse porous; however, vessels in earlywood were wider than the vessels formed in the latewood, showing a tendency towards vessel dimorphism, i.e. some of the vessel elements were very narrow measuring about 50 to 70 µm in diameter and 590 to 730 µm long including the tails, while wider vessel elements measured about 310 to 620 µm in length and 120 to 380 µm in diameter. The narrow vessel elements were longer than the wider ones and possessed a long tail on both the ends. Perforation plates were simple in oblique end walls of narrow vessel elements and horizontal in wider vessel elements. Vessels were mostly solitary and occasionally in radial or diago-

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Figure 3. Macroscopic (A) and transverse view (B–D) of mature stem of Securidaca rivinifolia showing different stages of cambial origin. – A: Macroscopic view of mature stem showing dumbbell-shaped configuration with eccentric successive rings of cambia (arrowhead) and xylem rings (arrow). Note continuous cambial rings in centre and later-formed arc-shaped successive cambia. – B: Xylem fibres, formed by the newly developed cambium. Arrowhead showing well developed new cambium; arrow indicates parenchyma cells of conjunctive tissue, XY = secondary xylem. – C: Newly developed cambium showing recently formed band of xylem fibres (arrow) and occasionally formation of vessel elements. – D: Macroscopic view of wood showing successive cambial and growth ring markers (arrowheads). —Scale bars for A = 2.5 mm; for B & C = 75 µm; for D = 250 µm.

Downloaded from Brill.com09/27/2021 12:31:54PM via free access Rajput et al. — Successive cambia of Securidaca (Polygalaceae) 397 nal multiples of 2 or 3. The non-septate fibres with distinctly bordered pits measured from 1020 to 1270 µm in length and from 24 to 38 µm in width. The bordered pits measured about 4.8 to 8.2 µm in diameter (Fig. 4E). Axial parenchyma strands were predominantly paratracheal, scanty and limited to a few cells around the vessels. Three to four cells per parenchyma strand. Rays mostly uniseriate while locally biseriate rays were observed rarely. Rays were about 2–16 cells high. However, rays less than four cells in height were composed of upright cells, while rays more than four cells high possessed isodiametric cells (Fig. 4D). Secondary phloem was composed of sieve tube elements, companion cells, axial and ray parenchyma cells. Length and diameter of sieve tube elements produced from the early formed successive cambium measured about 338 to 352 µm and 23 to 27 µm, respectively, while it was 315 to 332 µm and 25 to 28 µm, respectively, for the phloem produced by the outermost cambial ring. Similar to the xylem, phloem rays were mostly uniseriate while biseriate rays were observed occasionally. Each sieve tube element was accompanied by a single companion cell and was characterized by simple sieve plates with well-developed sieve areas on the lateral walls. Older phloem elements formed earlier by the previous successive rings showed heavy accumulation of callose followed by obliteration while sieve tube elements adjacent to each successive ring of the cambium was apparently functional and free from accumulation of callose.

Discussion

Secondary growth in Securidaca rivinifolia is achieved by the formation of excentric successive cambia. The first 2–3 cambia form a complete cylinder but thereafter they become excentric and crescent-shaped resulting in the characteristic stem morphology. Development of successive cambia from the pericyclic parenchyma have been reported in several other species including members of the Menispermaceae (Jacques & De Franceschi 2007; Tamaio et al. 2009) and they are frequent in Caryophyllales (Carlquist 2001). Occurrence of excentric successive cambia has been reported in Menispermaceae, where they result in flattened stems and develop only on one side (Rajput & Rao 2003; Jacques & De Franceschi 2007) but in the present investigation such cambia developed on two opposite sides of the stem thus leading to the dumbbell shape in cross section. The Polygalaceae are considered to be closely related to the Fabaceae, Surianaceae and Quillajaceae (APG website updated July 2009). However, most Fabaceae with anomalous thickening are different and have diffuse interxylary phloem (Metcalfe & Chalk 1983; Rajput 2002) or show complex patterns as in Dolichos (Rajput et al. 2006). To some extent, the stem anatomy of Rhynchosia phaseoloides of Fabaceae and Securidaca are similar. Rhynchosia is well known for its flat ribbon- like structure due to the formation of successive cambia (Schenck 1893; Rajput et al. 2012). Both Rhynchosia phaseoloides of the Fabaceae and Securidaca form excentric and crescent-shaped successive cambia on opposite lateral sides of the stem that result in the flattening of the stem. Prior to the formation of a new cambial cylinder, the pericyclic parenchyma cells undergo repeated periclinal divisions and form a wide band of meristematic cells arranged

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Figure 4. Transverse (A–C) and tangential longitudinal (D, E) view of secondary xylem of Securidaca rivinifolia. – A: Secondary xylem formed by two successive cambial rings showing growth ring markers (arrowheads). – B: Enlarged view of growth ring marker (arrowhead) showing thick-walled fibres in latewood, and wider lumen vessel elements in earlywood. – C: Ray cells in the limit of the growth ring. Note the ray cell dimensions: isodiametric (arrow) in latewood and radially elongate in earlywood (arrowhead). – D: General structure of secondary xylem. Note that rays are mostly uniseriate with 1–2-seriate portions in some rays. – E: Xylem fibres with bordered pits (arrowheads). — Scale bars for A & B = 100 µm; for C & D = 75 µm; for E = 50 µm.

Downloaded from Brill.com09/27/2021 12:31:54PM via free access Rajput et al. — Successive cambia of Securidaca (Polygalaceae) 399 in radial files. From this meristematic band, cells in the middle differentiated into the cambial segment that formed secondary xylem internally and phloem externally. Cells on the inner side of the newly formed cambium gave rise to conjunctive tissue, and outer cells act as a site for the future cambium. This developmental pattern is similar to that in various members of the Caryophyllales studied so far (Rajput 2001, 2002; Carlquist 2003, 2007). In the Caryophyllales, the band of parenchyma cells that differentiate into a new cambial ring is distinct and multi-layered. However, in Securidaca before the development of a distinct cambial zone, differentiation of secondary xylem and phloem start concomitant with divisions in the cambial cells. Therefore, a distinct cambial zone with radially arranged cells may be seen only after the differentiation of 10–15 xylem derivatives. There is not much information available on the seasonal behaviour of the vascular cambium of climbing species. Thus it becomes of interest to discuss the number of cambia formed annually in plants with successive cambia. Formation of additional cambial rings appears to be associated with the plant habit. In annual herbaceous species there may be more than one, as in some species of Ipomoea (Rajput et al. 2008). In Spinacia there are several cambia active simultaneously (Rajput et al. 2010b). Similarly Heklau (1992) and Heklau et al. (2012) reported that in annuals, each successive cambium remains active for a few weeks, and much longer in subshrubs. In perennial woody lianas like Cocculus cambia may be active for one or more growing seasons (Rajput & Rao 2003). On the other hand, Heklau et al. (2012) reported up to 24 cambia formed within one growing season in Atriplex sagittata (Chenopodiaceae). Securidaca, being a perennial scandent shrub/woody liana, formed a single cambium in one growing season. With the onset of the growing season, the previous cambium ring reactivates and forms the secondary xylem and a new ring of cambium is formed only after formation of 20–28 elements of xylem. It is evident from Figure 3A–C that the xylem formed at the end of cambial activity is characterized by thick-walled elements while newly formed elements possess thin walls and wider lumina. In the classification proposed by Carlquist (2001) the growth rings of Securidaca would fit into growth ring type 1C. Structurally, secondary xylem of climbing species is often characterized by abundant parenchyma, tall and wide multiseriate rays, and exceptionally wide vessels, often co-occurring with narrow vessels. In Securidaca with its narrow rays and scanty axial parenchyma this liana syndrome applies only to its two vessel size classes. In angio- sperms vessels vary considerably in diameter, ranging from (20–)40–300(–700) µm (Lösch 2003). Nearly 41% of dicotyledons have vessel diameters between 40 and 79 µm (Metcalfe & Chalk 1983; Wheeler et al. 2007; Heklau et al. 2012). However, the presence of two size classes of vessels is in line with several floristic comparisons (Europe, the Middle East and tropical Queensland) suggesting a dual strategy for hy- draulic efficiency and safety in climbers (cf. Baaset al. 1983; Baas & Schweingruber 1987; Bamber & Ter Welle 1994). In Securidaca, some of the vessel elements are notably narrow in diameter and are different from the fibriform vessels but may be perform- ing analogous functions. We are in agreement with Heklau et al. (2012) in relation to the role of these very narrow vessels in cavitation resistance that may be important to survive long and extreme dry conditions during the dryer part of the year.

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ACKNOWLEDGEMENTS

One of the authors (KSR) is grateful to CAPES-PVE for financial assistance under a Visiting Profes- sor Fellowship and to CNPq for grants to Fiamengui MB (Proc. 830858/1999-7-Master Grants) and Marcati CR (Proc. 301352/2008-9-Researcher Grants). We are also thankful to anonymous reviewers, Prof. Pieter Baas and Prof. Silvia Machado, for their valuable suggestions.

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