FORMATION of SUCCESSIVE CAMBIA in the MENISPERMUM TREE COCCULUS LAURIFOLIUS (MENISPERMACEAE) Kishore S. Rajput1,* and Sangeeta

Home , Cocculus, Cocculus hirsutus, Menispermaceae, Menispermum, Phytolacca dioica

400 IAWAIAWA Journal Journal 36 (4), 36 2015: (4), 2015 400–408

FORMATION OF SUCCESSIVE CAMBIA IN THE MENISPERMUM TREE COCCULUS LAURIFOLIUS (MENISPERMACEAE)

Kishore S. Rajput1,* and Sangeeta Gupta2 1Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, India 2Wood Anatomy Discipline, Forest Research Institute, Dehradun 248006, India *Corresponding author; e-mail: [email protected]

ABSTRACT Successive cambia are often associated with the climbing or shrub habit, and is less common in trees. We studied formation of successive cambia and structure of secondary xylem in young stems of Cocculus laurifolius DC., a tree species of Menispermaceae. Cell division in the vascular cambium ceased in pencil-thick stems. Subsequently, parenchyma cells located outside the perivascular fibre cap re-differentiated and gave rise to several small segments of meristematic cells, of which the central cells divided repeatedly to initiate the first successive cambium which produces secondary xylem centripetally and phloem centrifugally. Cells located on the inner side of the newly initiated cambium differentiated into conjunctive tissue while cells on the outer side of it divided further and differentiated into sclereids. Xylem was diffuse porous and composed of vessels, fibre tracheids and ray parenchyma cells, and only differed in vessel diameter from wide-vessel climbing relatives. Keywords: Cambial variant, multiple cambia, secondary phloem, xylem, tree habit.

INTRODUCTION Menispermaceae comprise 71 genera and approximately 520 species (Jacques & De Franceschi 2007). Most of them achieve secondary growth by forming successive cambia (Carlquist 2007; Ortiz et al. 2007; Jacques & De Franceschi 2007) while few show normal secondary growth (Tamaio et al. 2010). It is a cosmopolitan family of mainly vines or lianas whereas trees, shrubs or self-supporting herbs are rare (Ortiz et al. 2007). Stem anatomy of the Menispermaceae has been studied extensively in the past (Schenck 1893; Metcalfe & Chalk 1950; Mennega 1982; Carlquist 1996; Rajput & Rao 2003; Jacques & De Franceschi 2007; Tamaio et al. 2009, 2010). Successive cambia have been interpreted as adaptive to the climbing habit (Fisher & Ewers 1991; Carlquist 2001; Patil et al. 2011; Rajput et al. 2012), but have also been reported in a few tree species such as Avicennia marina, Dalbergia paniculata, Gallesia integrifolia, Phyto- lacca dioica, Salvadora persica (Studholme & Philipson 1966; Wheat 1977; Kirchoff & Fahn 1984; Schmitz et al. 2008; Longui et al. 2011; Robert et al. 2011; Rajput et al. 2012). However, in erect shrubs successive cambia can be of common occurrence,

Downloaded from Brill.com10/05/2021 07:09:00AM via free access Rajput & Gupta – Successive cambia in Cocculus 401 e.g. in Amaranthaceae (including the former Chenopodiaceae, cf. Heklau et al. 2012), Combretaceae (Van Vliet 1979) and several other woody families. Successive cambia in different members of the Menispermaceae have been reported to originate from four different types of tissue, viz. cortical parenchyma, the endodermis, the pericycle, and from irregular activity of the vascular cambium itself (Maheu 1902; Jacques & De Franceschi 2007; Tamaio et al. 2009). When studying the wood anatomy of the Menispermaceae, Jacques and De Franceschi (2007) reported that formation of successive cambia in Cocculus laurifolius does not fit into any one of the four origins proposed by Maheu (1902). Therefore, they suggested that other origins for those successive cambia are possible and that careful developmental studies are needed to clarify their precise origins. The present study, therefore, investigates i) the precise origin of successive cambia in C. laurifolius, and ii) any possible differences between the xylem of this self-supporting species and climbing species of the same family.

MATERIALS AND METHODS Young stems (3–10 mm thick) of Cocculus laurifolius (Menispermaceae) were collected from three plants growing in the Tropical Botanical Garden Research Institute (TBGRI), Thiruvananthapuram (India). They were fixed in FAA (Berlyn & Miksche 1976) and transferred in 70% alcohol after 12 hrs for further storage and processing. After suitable trimming into smaller pieces (3–4 mm), they were dehydrated through tertiary butyl alcohol (TBA) and processed by routine paraffin embedding (Johansen 1940). Transverse, radial and tangential longitudinal sections of 12–15 µm thickness were cut with the Leica rotary microtome and stained with safranin-fast green combi- nation (Johansen 1940). Subsequently, slides were dehydrated through ethanol xylene series and embedded in DPX. To study the structure of secondary xylem, 15–20 µm thick sections were prepared from wood blocks deposited in the xylarium of the For- est Research Institute (FRI), Dehra Dun, India. These wood blocks were collected from 8-year-old C. laurifolius growing at the Botanical Garden of the Forest Research Institute (Uttarakhand State, Acc. No. DDw 4497, 4643). Vessel lumen diameter and vessel frequency was obtained from transverse sections while dimensional details of ray height, ray width and ray cell diameter was measured from the tangential longitudinal sections. Measurements (50 per feature) were carried out only from the slides prepared from mature stems (xylarium samples) while fresh samples were used only to study the origin of successive cambia. Values in parentheses indicate standard deviation. Important results were micro-photographed with the Leica DME 2000 trinocular research microscope. Wood descriptions follow the IAWA Com- mittee (1989) and Carlquist (2001).

RESULTS Structure of the young stem – In the young stem, the epidermis is composed of thin-walled oval to polygonal cells of varying sizes and covered with a thick cuticle (Fig. 1A, B). The hypodermis is 1–2-layered and poorly differentiated. The cortex is

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4–6 cells wide and composed of thin-walled parenchyma cells. The endodermis is indistinct; the pericycle is composed of dome-shaped pericyclic fibre caps opposite each of the vascular bundles (Fig. 1B), while 3–4 layers of parenchyma cells are present between the protophloem and the fibre caps (Fig. B1 ). As growth progresses, one to two

Figure 1. Transverse view of young stem of Cocculus laurifolius showing initiation of successive cambium. – A: Young stem showing first normal ring of vascular cambium in the early stage of secondary growth. PD = pericyclic derivatives, P = pith. – B: Enlarged view of young stem. Arrowhead indicates pericyclic fibre cap. PD = pericyclic derivatives, C = cortex. – C: Initiation of first successive ring of cambium. Arrow shows one of the vascular bundles formed by the newly initiated successive cambium while arrowhead indicates pericyclic fibre cap. Note the marginal pith cells that differentiate into sclerenchyma (small arrow). P = pith. – D: Enlarged view of young stem showing initiation of first successive ring of cambium (arrowheads). Arrow shows sclereids. PFC = pericyclic fibre cap, P = pericycle cells. — Scale bar for A, B, D = 100 µm; for C = 200 µm.

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Figure 2. Transverse view of young stem of Cocculus laurifolius showing initiation of successive cambium (A–C) and structure of mature secondary xylem (D). – See full legend on next page.

Downloaded from Brill.com10/05/2021 07:09:00AM via free access 404 IAWA Journal 36 (4), 2015 cell layers remain thin-walled while the rest of the derivative cells differentiate into fibres (Fig. 1C, D). The pericyclic fibre caps are interconnected to form a continuous, grooved cylinder (Fig. 1A, B). On the inner side of the pericyclic fibres, medullary rays separate a ring of 11–13 conjoint collateral vascular bundles. The pith is composed of thin-walled parenchyma cells (Fig. 1A) in the early stages of secondary growth but the cells become thick- walled and sclerenchymatous (Fig. 1C) in later stages of development. As the secondary growth progresses, the pericyclic bundle caps become disconnected at the position of the grooves (Fig. 1C).

Origin of successive cambia – As the stem reaches a diameter of 4–5 mm, the vascular cambium ceases to divide and a first ring of successive cambium is initiated from the cortical parenchyma cells situated outside to the perivascular fibre cap (Fig. C1 , D). These parenchyma cells divide repeatedly after de-differentiation and form 4–6 cell layers of radially arranged cells (Fig. 2A). Cells situated on the inner side differentiate into conjunctive tissue while cells on the outer side serve as site for initiation of further successive cambia. The cells located in the middle of these layers give rise to the first successive cambium (Fig. 2B). From the newly originated cambium, several small alternate segments of it begin to differentiate into xylem fibres internally and phloem elements externally (Fig. 2C) while cells from the rest of the alternate segments of the cambium undergo radial enlargement and differentiate into rays (Fig. 1D, 2C). Formation of further successive cambia follows a similar pattern.

Structure of secondary xylem – Thick stems of C. laurifolius are composed of thick concentric rings of secondary xylem alternating with the thin secondary phloem rings (Fig. 2D). Secondary xylem is diffuse-porous with distinct growth rings (Fig. 2D, 3A) and composed of vessels, fibre tracheids, axial and ray parenchyma cells. Very wide and high heterocellular medullary and secondary rays separate the axial elements of secondary xylem and phloem (Fig. 2D, 3B). Rays are 298 (± 10) µm wide and 2287 (± 36) µm tall. All ray cells are square and/or upright in radial view while oval to polygonal in tangential view (Fig. 3C). Acicular to styloid crystals are abundant in ray cells of all the samples investigated (Fig. 3C). The axial parenchyma is diffuse to

← Figure 2. Transverse view of young stem of Cocculus laurifolius showing initiation of successive cambium (A–C) and structure of mature secondary xylem (D). – A: Initiation of cell division and formation of first successive cambium (arrowhead). Note differentiating xylem elements from the newly formed cambium (arrow). Small arrow showing crushed protophloem. PFC = pericyclic fibre cap. – B: Enlarged view of Fig. 2A showing initiation of cambium. Note that central cells in the meristematic band give rise to cambium (arrowhead). Arrow indicates pericyclic derivative differentiating into sclerenchyma fibre. CP = cortical parenchyma, E = endodermis, PFC = pericyclic fibre cap, P = pericyclic derivative. – C: Differentiation of xylem internally and phloem externally from newly formed cambium. Note the distinct pattern of vascular bundles (arrow) separated by wide interfascicular rays (arrowhead). – D: Structure of secondary xylem in a sample from Dehra Dun. Note the variations in the structure of xylem and vessel arrangement. — Scale bar for A, C = 100 µm; for B = 75 µm; for D = 250 µm.

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Figure 3. Transverse (A, B, D) and tangential longitudinal (C, E, F) view of stem of Cocculus laurifolius showing structure of secondary xylem. – A: Xylem showing distinct growth ring boundary (arrowhead) in Dehra Dun samples. Note the diffuse porous nature of the xylem. – B: Oval to polygonal ray cells of varying sizes. R = ray. – C: Crystals in the ray cells (arrowheads). – D: Structure of secondary xylem showing an arrangement of axial and radial elements. – E: Narrow vessel showing simple perforation plate (V) on lateral wall. – F: Large intervessel pits on the lateral walls. Note scalariform vessel-to-axial parenchyma pits with strongly reduced borders (arrowhead). — Scale bar for A, B = 200 µm; for C, E, F = 50 µm; for D = 100 µm.

Downloaded from Brill.com10/05/2021 07:09:00AM via free access 406 IAWA Journal 36 (4), 2015 diffuse-in-aggregates, arranged in small tangential lines of few cells that do not extend to the whole distance between two broad rays (Fig. 3D). Vessels are mostly solitary, round to oval, rarely in radial multiples of 2–3. Vessel elements with simple perforation plates in transverse to oblique end walls, rarely in lateral walls (Fig. 3E). Intervessel pits alternate, 6 to 9 µm in diameter. Vessel-to-axial parenchyma cells are large and simple and scalariform in narrow vessels (Fig. 3F). In each xylem plate, narrow and wide vessels are intermixed and 50–153 (± 21) µm in diameter, and 232–287 (± 9) µm in element length. Tyloses frequent and usually lignified. Fibre-tracheids are non-septate with numerous small bordered pits in radial and tangential walls. Vasicentric tracheids infrequent and of equal length as vessel elements.

DISCUSSION

In an earlier study by Jacques and De Franceschi (2007) they doubted about the origin of successive cambia in C. laurifolius from four different types of tissue as reported by Maheu (1902). Our observations are in agreement with Maheu (1902) and confirm that the first successive cambium initiates from the inner cortical layers. The present study shows that the first successive cambium initiates from the cortical parenchyma cells situated outside the pericyclic fibre bundle caps after cessation of cell division in the first vascular cambium. Carlquist (1996) also recorded initiation of successive cambia in Menispermaceae from the innermost layers of the cortex while our previ- ous study reported a similar origin of the first successive cambium in C. hirsutus, a climbing member of the Menispermaceae (Rajput & Rao 2003). Plants have evolved different modes of secondary thickening for mechanical support and safety of vessels for hydraulic conductivity (Carlquist 2001; Rajput et al. 2008; Robert et al. 2011). In the tree habit, the stem performs the main function of mechanical support while in stems of climbers tensile stresses dominate and the crown loads are usually transferred onto the host (Givnish 1995). Therefore, anatomical characteristics of climbing plants differ from self-supporting plants and show remarkable changes in the structure of secondary xylem (Caballé 1993; Rowe & Speck 1996; Isnard et al. 2003a, b). Comparison of secondary xylem within genera having both arboreal and climbing species will help to understand the structural alterations induced by the shift from the self-supporting to the climbing habit. Tamaio and Brandes (2010) carried out a similar comparison in Abuta grandifolia when comparing the tree and liana habit in Menispermaceae. According to them no significant differences exist between the xylem structure of climbing and erect members, except for the presence of diffuse axial parenchyma in lianas as contrasted with diffuse to diffuse-in-aggregates parenchyma in shrubs. The present study is in agreement with Tamaio and Brandes (2010) and finds little difference between the xylem of arborescent (C. laurifolius) and the climbing species (C. hirsutus) as described by Rajput and Rao (2003). Overall, the gross structure of secondary xylem is similar to other species of Menispermaceae (Maheu 1902; Metcalfe & Chalk 1950; Obaton 1960; Mennega 1982; Carlquist 1996; Rajput & Rao 2003; Jacques & De Franceschi 2007; Tamaio et al. 2009, 2010; Tamaio & Brandes 2010).

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One of the apparent dissimilarities between the arborescent and vining habit of Abuta grandifolia is the difference in vessel diameter (Mennega 1982). In C. laurifolius vessel diameter is also less than in the climbing species and similar observations have been reported by earlier workers (Mennega 1982; Rajput & Rao 2003; Jacques & De Franceschi 2007). Tamaio and Brandes (2010) reported a similar dis- similarity in vessel diameter in Abuta grandifolia when comparing shrub and liana habit in the Menispermaceae.

ACKNOWLEDGEMENTS

The authors are thankful to the Department of Science and Technology (SERB) for financial support. Thanks are also due to Dr. Santosh Kumar, Scientist, TBGRI and Dr. P.S. Nagar, Department of Botany, the Maharaja Sayajirao University of Baroda, for providing the fresh samples and to the Director, Forest Research Institute (FRI), Dehra Dun for the administrative support. The authors are thankful to anonymous reviewers and Prof. Baas for their critical suggestions on earlier versions of the manuscript.

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Accepted: 24 August 2015

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