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Wood and bark anatomy of Andriana (Heteromorpheae, Apiaceae) with phylogenetic implications
South African Journal of Botany 115 (2018) 138–142
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Wood and bark anatomy of Andriana (Heteromorpheae, Apiaceae) with phylogenetic implications C. Long a, A. Oskolski a,b,⁎ a b
Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland Park, 2006 Johannesburg, South Africa Komarov Botanical Institute of the Russian Academy of Science, Prof. Popov Str. 2, 197376 St. Petersburg, Russia
a r t i c l e
i n f o
Article history: Received 2 July 2017 Received in revised form 11 January 2018 Accepted 3 February 2018 Available online xxxx Edited by AR Magee Keywords: Apioideae Helical thickenings Heteromorpha clade Phylogenetics Madagascar
a b s t r a c t The wood structure was examined in two species of Andriana, and the bark structure in one species of this genus. The Malagasy endemic genus Andriana is similar to the southern African genera of the tribe Heteromorpheae (Apioideae, Apiaceae) in having axial secretory canals arranged in a single ring in the innermost part of the cortical parenchyma and radial dilatation of secondary phloem. This genus is distinctive, however, from the latter by the common presence of column-shaped to bottle-shaped epidermal cells with very thick cuticle. Helical thickenings on vessel walls have not been found in Andriana; thus this feature can be considered a synapomorphic trait only for the Heteromorpha clade rather than for the tribe Heteromorpheae, as it was previously suggested. Surprisingly, secretory canals in the secondary phloem of Andriana species lack sheaths of specialized axial parenchyma. The absence of these sheaths is thought to be unique within the suborder Apiineae, which comprises the families Araliaceae, Myodocarpaceae, Pittosporaceae, and Apiaceae. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The genus Andriana B.-E. Van Wyk comprises of three shrubby species restricted to the high elevation scrubs in the mountains of northern Madagascar (Van Wyk et al., 2013), i.e. to the Madagascar ericoid thickets sensu Burgess et al. (2004). Along with seven other genera, this genus is placed in the Malagasy clade of the tribe Heteromorpheae M.F. Watson & S.R. Downie, belonging to the family Apiaceae (Downie et al., 2010). The species of Andriana were originally treated by Humbert (1955, 1956) in the genus Heteromorpha, but were subsequently recircumscribed into a new genus based on their more regular leaf shape, serrate leaflet margins, perfectly isodiametric mericarps, and invariable presence of intrajugal oil ducts within the fruit ribs as well as vallecular oil ducts between the ribs (Van Wyk et al., 1999). The genus is named after the Madagascar king Andriantsimitoviaminadriandehibe, where the Malagasy word “andrian” means “noble” (Van Wyk et al., 1999). The tribe Heteromorpheae was described (Downie et al., 2001) to accommodate a group of five African genera of trees, shrubs, suffrutices and perennial herbs (Anginon Raf., Dracosciadium Hilliard, Glia Sond. Heteromorpha Cham. & Schltdl. and Polemannia Eckl. & Zeyh.) that formed a distinct, basally divergent clade in several molecular systematic ⁎ Corresponding author at: Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland Park, 2006 Johannesburg, South Africa. E-mail address: [email protected] (A. Oskolski).
https://doi.org/10.1016/j.sajb.2018.02.003 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
studies of the subfamily Apioideae (Plunkett et al., 1996; Plunkett and Downie, 1999; Downie and Katz-Downie, 1999; Downie et al., 2001). A molecular systematic study by Calviño et al. (2006) suggested that the limits of the tribe should be expanded to include Pseudocarum C.Norman with one species in Africa and one in Madagascar, Oreofraga M.F.Watson & E.L.Barclay with a single species in Socotra, and four genera endemic to Madagascar (i.e. Andriana B.-E.Van Wyk, Anisopoda Baker, Cannaboides B.-E.Van Wyk, Pseudocannaboides B.-E.Van Wyk and Tana B.-E.Van Wyk). The seven latter genera, along with Dracosciadium, were recovered as a clade (the Malagasy clade, Downie et al., 2010) sister to the Heteromorpha clade comprising Anginon, Glia, Heteromorpha and Polemannia. Although the Malagasy clade encompasses two genera from outside Madagascar, in the present paper we will use its name proposed by Downie et al. (2010). Several morphological characters have been uncovered to support the inclusion of the Madagascan genera into the tribe Heteromorpheae. As recent study of fruit structural variation in this tribe (Liu et al., 2016) showed, the fruit epidermis made of bottle shaped cells with very thick cuticle to be a synapomorphic trait for the tribe. Anatomical studies of wood and bark in South African members of Heteromorpheae (Oskolski and Van Wyk, 2008; Kotina et al., 2012) also suggested that the combination of a narrow cortex with a single ring of secretory canals, secretory canals in the secondary phloem and radial dilatation of the secondary phloem, and the presence of helical thickenings on vessel walls is diagnostic for the tribe. The latter feature was also considered as an ancestral character state and a symplesiomorphy for the tribes
C. Long, A. Oskolski / South African Journal of Botany 115 (2018) 138–142
Bupleurieae and Heteromorpheae (Oskolski and Van Wyk, 2008). These suggestions, however, are only provisional, because the stem structure of only one genus (Dracosciadium) belonging to the Malagasy clade of Heteromorpheae has been examined to date. In this study, we aim to describe the anatomical structure of the wood and bark structure in Andriana as a member of the Malagasy clade. On this basis, we seek to interpret the phylogenetic relationships within the tribe Heteromorpheae, to analyse the evolutionary pathways for wood and bark characteristics in this group, and to clarify ecological implications of the stem anatomical traits in Andriana. 2. Material and methods Wood samples of Andriana tsaratananensis (Humbert) B.-E. Van Wyk were obtained from P.P. Lowry II, who collected it on 16.10.2001 on Tsaratanana massif (elevation 2490 m) in northwestern Madagascar. Wood and bark samples of A. marojejyensis (Humbert) B.-E. Van Wyk were collected by the second author with P. Karpunina and M. Nuraliev on 17.10.2015 on the summit of mount Marojejy (elevation 2130 m) in the Marojejy Natural Park, northeastern Madagascar. Herbarium vouchers (P. P. Lowry II # 5363 and P. Karpunina # 244) are deposited in P and TAN. The samples for wood anatomical study were taken from the thickest portions of stems of both species with a secondary xylem radius of more than 5 mm, so that the wood structure may be considered mature. Transverse, radial and tangential sections were made on rotary microtomes (Ernst Leitz GMBH, Wetzlar, Germany and Jung AG Heidelberg, Germany) and stained with a 1:1 alcian blue/safranin mixture. Macerations were made using Jeffrey's solution (Johansen, 1940). Descriptive terminology for wood structure follows recommendations of the IAWA Committee (1989).
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The bark structure was examined only for A. marojejyensis. Fresh bark samples of this species were cut from branch tips without a visible periderm layer and from other stem parts on which the bark was mature, having more or less a thick periderm. These bark fragments were fixed in 70% ethanol, and then they were embedded in glycol methacrylate (GMA) according to a modification of the Feder and O’Brien (1968) method. Transverse, tangential and radial sections of about 5 μm thick were cut by using a Porter Blum MT-1 ultramicrotome. Bark sections were then stained with Schiff - toluidine blue method before being mounted in Entellan. Measurements of sieve tube members were made in fragments of secondary phloem macerated using Jeffrey's solution (Johansen, 1940). Descriptive bark anatomical terminology follows Angyalossy et al. (2016). 3. Results 3.1. Wood structure (Fig. 1) Samples examined: Andriana marojejyensis: P. Karpunina 244; A. tsaratanonensis: P.P. Lowry II 5363. Growth ring boundaries are absent in A. tsaratanonensis (Fig. 1A), and absent to indistinct in A. marojejyensis, marked by zones of somewhat radially flattened fibres (Fig. 1B). Vessels are angular, rarely rounded in outline, very narrow (tangential diameter b28 μm in A. tsaratanonensis and b 54 μm in A. marojejyensis), few in A. marojejyensis (up to 40 per mm2) and more numerous in A. tsaratanonensis (up to 73 per mm2), mostly in clusters and radial to diagonal multiples groupings up to 10 vessels. Vessel walls of 2.3–8.9 μm thick. Tyloses were not found. Vessel elements (170–) 320–350 (−580) μm long (Table 1). Perforation plates are simple (Fig. 1C, E). Intervessel pits are mostly scalariform, sometimes transitional to alternate, 4.1–8.8 μm in vertical size,
Fig. 1. Wood structure of Andriana. (A) A. tsaratanonensis, transverse section showing the absence of growth rings, vessels in radial multiplies; (B) A. marojejyensis, transverse section showing indistinct growth ring boundaries (white arrowheads), vessels in diagonal groupings; (C) A. marojejyensis, SEM micrograph of vessel element showing simple perforation plate, scalariform to alternate intervessel pitting, and the absence of helical thickenings on vessel wall; (D) A. marojejyensis, tangential section showing 1–4 seriate rays composed mostly of square and upright cells; (E) A. marojejyensis, radial section showing a ray composed mostly of upright cells with few square and procumbent cells, vessel elements with simple perforation plates. Scale bars: A, B, D 200 μm; E 100 μm; C 20 μm.
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C. Long, A. Oskolski / South African Journal of Botany 115 (2018) 138–142
Table 1 Wood and bark anatomical characters of Andriana.
Andriana tsaratananensis [P.P. Lowry 5363] Andriana marojejyensis [P. Karpunina 244]
1
2
3
4
5
6
7
8
9
10
11
12
13
10
327 ± 9.7/ 176–510 348 ± 13.8/ 25–578
6.8 ± 0.13/ 4.1–8.8 6.0 ± 0.16/ 4.9–7.8
59.4/45–73
16.8 ± 0.68/ 8–27 32.3 ± 1.14/ 13–54
3.4/10
9
–
0.83/2.3
3.9/3.3
7.2
–
2.3/10
20
485 ± 8.5/ 381–692 494 ± 10.3/ 362–670
2.6 / 4
0,51/1.3
1.6/1.4
3.1
392 ± 12.5/ 277–606
10
29.4/24–39
1: Radius of wood sample (mm) — 2: Length of vessel elements (mean ± standard error/min-max, μm) — 3: Vertical size of intervessel pits (mean ± standard error/min-max, μm) — 4: Vessel frequency (mean/min-max, per sq. mm) — 5: Tangential diameter of vessels (mean ± standard error/min-max, μm) — 6: Mean/the greatest number of vessels in a vessel group — 7: Percentage of solitary vessels −8: Mean length of libriform fibres (mean ± standard error/min-max, μm) — 9: Width of multiseriate rays (mean/max, cells) — 10: Height of multiseriate rays (mean/max, mm) — 11: Average number of multiseriate/uniseriate rays per mm — 12 Total number of rays per mm — 13: Length of sieve tube members (mean ± standard error/minmax, μm).
mostly with rounded margins and slit-like apertures (Fig. 1C). Vessel-ray and vessel-axial parenchyma pits are similar to intervessel pits in size and shape, mostly with distinct borders and wide lens-like apertures, sometimes with reduced borders. Helical thickenings were not found (Fig. 1C). Vascular tracheids were not found. Fibres libriform, thin- to thick-walled, sometimes very thin-walled, (360–) 480–490 (− 690) μm long (Table 1), fibre walls 2.3–6.2 μm thick in A. tsaratananensis and 2.5–8.9 μm thick in A. marojejgensis, with few simple to minutely bordered pits, with slit-like apertures in radial walls. Septate fibres rarely occur in A. tsaratananensis. Axial parenchyma is scanty paratracheal, mostly in solitary strands (sometimes in uniseriate incomplete sheaths) near the vessels, consist of strands of 2–5 cells A. marojejyensis and of 2–4 cells in A. tsaratananensis. Rays 4–12 per mm, uni- and multiseriate of 2–4 cells in width (Fig. 1D). Ray height commonly b 1 mm, but few rays N 1 mm occur in both species. Multiseriate rays composed mostly of upright and square cells with some procumbent cells mixed throughout the ray (Fig. 1D, E). Uniseriate rays composed mostly of square and upright cells but procumbent cell occur very rarely. Radial canals absent. Crystals not found.
Sieve tube members are 30–37 μm wide, their mean length varies from 277 to 606 μm (Table 1). Sieve plates are compound with 2–6 sieve areas, located on vertical or slightly oblique cross walls (Fig. 2D). Axial parenchyma cells associated with conducting elements occur as single fusiform cells and in strands of 2–4 cells. Axial secretory canals are scattered throughout the secondary phloem. They are lined by a single layer of 6–10 epithelial cells. No parenchyma sheaths near the secretory canals were found (Fig. 2B). The transition from conducting to nonconducting secondary phloem is gradual. Axial parenchyma cells in nonconducting secondary phloem occurs as thin-walled strands (Fig. 2B) and as chambered crystalliferous cells containing druses. Secondary phloem rays are uniseriate and 2–3-seriate (Fig. 2E). Uniseriate rays are composed of mostly square and upright cells (Fig. 2C) while 2- and 3- seriate rays have procumbent cells or also upright and square cells (mostly in uniseriate portions). Dilated rays are extensively enlarged, mostly by tangential expansion and also by anticlinal divisions of ray cells resulting in rays of up to 7 cells wide (Fig. 2F). Many of the ray parenchyma cells contain starch grains. Druses occur in ray cells. No radial secretory canals were found.
3.2. Bark structure (Fig. 2)
The genus Andriana shows exclusively simple perforation plates, rather short vessel elements, scanty paratracheal axial parenchyma, the presence of secretory canals in the cortex and secondary phloem, and the absence of the secondary phloem fibers. This set of anatomical characters is typical for the large majority of woody Apiaceae (Metcalfe and Chalk, 1950; Rodriguez, 1957; Oskolski, 2001; Kotina and Oskolski, 2010) examined to date. Only Apiopetalum Baill., Mackinlaya F. Muell. and Centella L., the members of the subfamily Mackinalyoideae, share the occurrence of scalariform perforation plates. Besides, Centella is unique within Apiaceae by the lack of secretory canals in the secondary phloem (Oskolski and Lowry, 2000; Oskolski and Van Wyk, 2010). Despite these similarities, Andriana is very distinctive from other Apiaceae as well as from other members of the suborder Apiineae (i.e. Araliaceae, Myodocarpaceae and Pittosporaceae) by its uniform axial parenchyma in secondary phloem. Unlike Andriana, in other Apiineae the secretory canals in secondary phloem have prominent sheaths of the axial parenchyma, that clearly differs from the axial parenchyma associated with conductive elements by more numerous cells per strand and by common occurrence of crystals and other deposits (Kolalite et al., 2003; Kotina and Oskolski, 2010; Nilova and Oskolski, 2010; Oskolski et al., 2010). In Andriana, however, the sheath axial parenchyma cannot be distinguished from other strands of axial parenchyma occurring in secondary phloem. This feature cannot be considered, however, as an apomorphy for the Malagasy Heteromorpheae because parenchymatous sheaths near secretory canals have been reported in Dracosciadium, also belonging to this clade (Kotina et al., 2012). Andriana is similar to the southern African genera of Heteromorpheae (i.e. Anginon, Dracosciadium, Glia, Heteromorpha and Polemannia) in having axial secretory canals arranged in a single ring in the innermost part of the cortical parenchyma, and radial dilatation of secondary phloem
Samples examined: Andriana marojejyensis: P. Karpunina 244. The epidermis on young parts of stems is composed of a single layer of column-like to bottle-like cells with thin inner walls and thick outer walls covered by prominent cuticle (Fig. 2A). There were no trichomes found. The cortex is narrow (up to 18 cells in width), composed of collenchyma and parenchyma (Fig. 2A). Cortical collenchyma is angularlamellar. Collenchyma cells are 20–50 μm in tangential diameter. There were no crystals found in the cells of the cortical collenchyma. Cortical parenchyma is formed from four to six layers of isodiametric to somewhat vertically elongated, flattened, thin-walled cell of 25–50 μm in tangential diameter occasionally. Druse were observed in some of the cortical parenchyma cells. A single ring of schizogeneous axial secretory canals is present in the cortical parenchyma. These canals are lined with a single of seven to twelve epithelial cells. The lumina of the canals are commonly 60–80 μm in tangential diameter. Dilation of the cortical tissue is affected mostly by tangential stretching of cells and also by anticlinal divisions of the cortical collenchyma and parenchyma cell, thus forming strands of two to four cells. Primary phloem fibers are absent. No chloroplast were observed in cells of cortical collenchyma and parenchyma. Mature bark non-peeling, brittle, with shallow fissured surface. The initiation of first-formed periderm is in the subepidermal layer of cells (Fig. 2A). The phellem (Fig. 2B) is composed of 8–12 layers of isodiametric to somewhat radially flattened cells with thin cell wall (0.3–0.9 μm thick). The phelloderm comprises 4 to 7 layers of isodiametric, thinwalled cells. No crystalliferous cells found in periderm. Secretory canals are absent in the phelloderm. Subsequent periderm are initiated in the outer region of the cortical parenchyma as concentric rings.
4. Discussion
C. Long, A. Oskolski / South African Journal of Botany 115 (2018) 138–142
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Fig. 2. Bark structure of Andriana marojejyensis. (A) transverse section of young bark showing column-like to bottle-like epidermal cells (e), subepidermal initiation of phellogen (ph), cortical collenchyma (cc) and cortical parenchyma (cp) with secretory canals (sc), secondary phloem (sp); (B) transverse section of mature bark showing periderm (p), dilatated cortex (dc) with tangential strands of parenchyma cells (s), secondary phloem (sp) with secretory canals (sc) lacking axial parenchyma sheaths; (C) radial section showing secondary phloem rays composed mostly of upright cells; (D) radial section showing sieve tube members with compound sieve plates (spl) on vertical end walls; (E) tangential section of conductive secondary phloem showing 2–3-seriate phloem rays; (F) tangential section of non-conductive secondary phloem showing dilatated 2–8-seriate phloem rays. Scale bars: A, B, C 100 μm; D 50 μm; E, F 200 μm.
(Kotina et al., 2012). Our results support that this combination of traits is diagnostic for Heteromorpheae, as it has been suggested by Kotina et al. (2012). Two Andriana species are distinctive, however, from these genera (with the exception of Dracosciadium whose wood structure remains unknown) by the absence of helical thickenings on vessel walls (Oskolski and Van Wyk, 2008). Thus this feature can be considered as a synapomorphic trait only for the Heteromorpha clade (Calviño et al., 2006; Downie et al., 2010) comprising of Anginon, Glia, Heteromorpha and Polemannia, rather than for the entire Heteromorpheae, as it was previously suggested (Oskolski and Van Wyk, 2008). Andriana differs from the southern African Heteromorpheae by very narrow cortical parenchyma (four to six layers versus six to twenty layers in other genera of this tribe) and by the column- to bottleshaped epidermal cells covered with very thick cuticle. The latter feature occurs also in the mericarps of Anginon and Pseudocannaboides (Liu et al., 2016), but it has not been reported in stem epidermis of any other Heteromorpheae (Kotina et al., 2012). Apparently, these
bark traits can be diagnostic for the genus, but their phylogenetic value is uncertain until the stem structure in other Malagasy genera of Heteromorpheae remains unknown. Unlike the southern African Heteromorpheae, the wood of Andriana almost completely lacks growth rings. Obviously, the cambial activity in these shrubs can persist during the seasonal fluctuations in temperature that have been reported in the mountain scrubs (ericoid thickets) of Madagascar (Burgess et al., 2004). Andriana also resembles Anginon, Glia and Polemannia in having very narrow vessels (less than 54 μm in diameter); it shows, however, lower vessel frequency than the southern African genera (Fig. 3). Narrow vessels is a typical trait for shrubby plants from high elevation habitats (e.g. Outer van der et al., 1981; Noshiro et al., 2010) that can provide hydraulic safety preventing air embolism during temporary arid conditions occurring in the ericoid thickets caused by local fluctuations of temperature, sunlight and precipitations (Burgess et al., 2004). In Andriana, however, the decrease of vessel diameters is not compensated in Andriana by the increase of
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Average vessel diameter, µm
60 50
Andriana
40
Anginon Glia
30
Heteromorpha Polemannia
20 10 0 0
100
200
300
400
500
600
Vessel frequency, per sq.mm Fig. 3. Average tangential diameter of vessels in southern African genera of Heteromorpheae (data taken from Oskolski and Van Wyk (2008)) and in two Andriana species plotted against the average vessel frequency.
vessel frequency, as it commonly occurs (Outer van der et al., 1981). Apparently, few narrow vessels can supply the Andriana shrubs effectively due to great water availability and low transpiration loss that are found in tropical mountains. 5. Conclusions Andriana is similar to other genera of the tribe Heteromorpheae by the axial secretory canals arranged in a single ring in the innermost part of the cortical parenchyma, and the radial dilatation of secondary phloem. This combination of bark traits is diagnostic for the tribe. Andriana is distinctive, however, from other Heteromorpheae by the presence of column- to bottle-shaped epidermal cells covered with very thick cuticle. The absence of sheath axial parenchyma in secondary phloem of Andriana is a unique feature within the suborder Apiineae. As Andriana lacks the helical thickenings on the vessel walls, the presence of these structures may not be considered as a synapomorphic trait for the Heteromorpheae, as it was previously suggested. Acknowledgements We thank the National Research Foundation (incentive grant 109531), the University of Johannesburg and the Russian Foundation of Basic Research (grant 16-04-00725а) for financial support. The second author was also supported by the institutional research project (no. 01201456545) of the Komarov Botanical Institute. The staff of Missouri Botanical Garden's Madagascar Research and Conservation Program, specifically F. Lantoarisoa and P.P. Lowry II are gratefully acknowledged for organization of the fieldwork in Marojejy National Park. We are very indebted to P.D. Karpunina, M.S. Nuraliev, D. Ravelonarivo, V. Razafindrahaja and J.H. Tonkaina for field assistance, and to P.P. Lowry II for giving us the wood sample of A. tsaratanonensis. References Angyalossy, V., Pace, M.R., Evert, R.F., Marcati, C.R., Oskolski, A.A., Terrazas, T., Kotina, E., Lens, F., Mazzoni-Viveiros, S.C., Angeles, G., Machado, S., Crivellaro, A., Rao, K.S., Junikka, L., Nikolaeva, N., Baas, P., 2016. IAWA list of microscopic bark features. IAWA Journal 37, 517–615. Burgess, N., Hales, J.D.A., Underwood, E., Dinerstein, E., Olson, D., Itoua, I., Schipper, J., Ricketts, T., Newman, T., 2004. Terrestrial Ecoregions of Africa and Madagascar: A Conservation Assessment. Island Press, Washington. Calviño, C.I., Tilney, P.M., Van Wyk, B.-E., Downie, S.R., 2006. A molecular phylogenetic study of southern African Apiaceae. American Journal of Botany 93, 1828–1847. Downie, S.R., Katz-Downie, D.S., 1999. Phylogenetic analysis of chloroplast rps16 intron sequences reveals relationships within the woody southern African Apiaceae subfamily Apioideae. Canadian Journal of Botany 77, 1120–1135.
Downie, S.R., Plunkett, G.M., Watson, M.F., Spalik, K., Katz-Downie, D.S., Valiejo-Roman, C.M., Terentieva, E.I., Troitsky, A.V., Lee, B.-Y., Lahham, J., El-Oqlah, A., 2001. Tribes and clades within Apiaceae subfamily Apioideae: the contribution of molecular data. Edinburgh Journal of Botany 58, 301–330. Downie, S.R., Spalik, K., Katz-Downie, D.S., Reduron, J.P., 2010. Major clades within Apiaceae subfamily Apioideae as inferred by phylogenetic analysis of nrDNA ITS sequences. Plant Diversity and Evolution 128, 111–136. Feder, N., O’Brien, T.P., 1968. Plant microtechnique: some principles and new methods. American Journal of Botany 55, 123–142. Humbert, H., 1955. Une merveille de la nature à Madagascar. Première exploration botanique du massif du Marojejy et de ses satellites. Mémoires de l'Institut Scientifique de Madagascar. Série B, Biologie Végétale 6, 1–210. Humbert, H., 1956. Contributions à l'étude de la flore de Madagascar et des Comores. Fascicule 5. Notulae Systematicae (Paris) 15, 113–134. IAWA Committee, 1989. IAWA list of microscopic features for hardwood identification. IAWA Bulletin New Series 10, 219–332. Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York. Kolalite, M.R., Oskolski, A.A., Richter, H.G., Schmitt, U., 2003. Bark anatomy and intercellular canals in the stem of Delarbrea paradoxa (Araliaceae). IAWA Journal 24, 139–154. Kotina, E., Oskolski, A.A., 2010. Survey of the bark anatomy of Araliaceae and selected related taxa. Plant Diversity and Evolution 128, 455–490. Kotina, E.L., Van Wyk, B.-E., Tilney, P.M., Oskolski, A.A., 2012. The systematic significance of bark structure in southern African genera of tribe Heteromorpheae (Apiaceae). Botanical Journal of the Linnean Society 169, 677–691. Liu, M., Van Wyk, B.-E., Tilney, P.M., Plunkett, G.M., Lowry, P.P., Magee, A.R., 2016. The phylogenetic significance of fruit structural variation in the tribe Heteromorpheae (Apiaceae). Pakistan Journal of Botany 48, 201–210. Metcalfe, C.R., Chalk, L., 1950. Anatomy of the dicotyledons. vol. II. Clarendon Press, Oxford. Nilova, M., Oskolski, A.A., 2010. Comparative bark anatomy of Bursaria, Hymenosporum, and Pittosporum (Pittosporaceae). Plant Diversity and Evolution 128, 491–500. Noshiro, S., Ikeda, H., Joshi, L., 2010. Distinct altitudinal trends in the wood structure of Rhododendron arboreum (Ericaceae) in Nepal. IAWA Journal 31, 443–456. Oskolski, A.A., 2001. Phylogenetic relationships within Apiales: evidence from wood anatomy. Edinburgh Journal of Botany 58, 201–206. Oskolski, A.A., Lowry II, P.P., 2000. Wood anatomy of Mackinlaya and Apiopetalum (Araliaceae) and its systematic implications. Annals of the Missouri Botanical Garden 87, 171–182. Oskolski, A.A., Van Wyk, B.-E., 2008. Systematic and phylogenetic value of wood anatomy in Heteromorpheae (Apiaceae, Apioideae). Botanical Journal of the Linnean Society 158, 569–583. Oskolski, A.A., Van Wyk, B.E., 2010. Wood and bark anatomy of Centella: scalariform perforation plates support an affinity with the subfamily Mackinlayoideae (Apiaceae). Plant Systematics and Evolution 289, 127–135. Oskolski, A.A., Rossouw, A.S., Van Wyk, B.E., 2010. Wood and bark anatomy of Steganotaenia and Polemanniopsis (tribe Steganotaenieae, Apiaceae) with notes on phylogenetic implications. Botanical Journal of the Linnean Society 163, 55–69. Outer van der, L., Baas, P., Zandee, M., 1981. Comparative wood anatomy of Symplocus and latitude and altitude of provenance. IAWA Bulletin New Series 2, 3–24. Plunkett, G.M., Downie, S.R., 1999. Major lineages within Apiaceae subfamily Apioideae: a comparison of chloroplast restriction site and DNA sequence data. American Journal of Botany 86, 1014–1026. Plunkett, G.M., Soltis, D.E., Soltis, P.S., 1996. Evolutionary patterns in Apiaceae: inferences based on matK sequence data. Systematic Botany 21, 477–495. Rodriguez, R.L., 1957. Systematic anatomical studies on Myrrhidendron and other woody Umbellales. University of California Publications in Botany 29, 145–318. Van Wyk, B.-E., Tilney, P.M., Winter, P.J.D., 1999. Four new genera of Apiaceae of Madagascar. Taxon 48, 737–745. Van Wyk, B.-E., Tilney, P.M., Magee, A.R., 2013. African Apiaceae. Briza, Pretoria.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Wood and bark anatomy of Andriana (Heteromorpheae, Apiaceae) with phylogenetic implications C. Long a, A. Oskolski a,b,⁎ a b
Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland Park, 2006 Johannesburg, South Africa Komarov Botanical Institute of the Russian Academy of Science, Prof. Popov Str. 2, 197376 St. Petersburg, Russia
a r t i c l e
i n f o
Article history: Received 2 July 2017 Received in revised form 11 January 2018 Accepted 3 February 2018 Available online xxxx Edited by AR Magee Keywords: Apioideae Helical thickenings Heteromorpha clade Phylogenetics Madagascar
a b s t r a c t The wood structure was examined in two species of Andriana, and the bark structure in one species of this genus. The Malagasy endemic genus Andriana is similar to the southern African genera of the tribe Heteromorpheae (Apioideae, Apiaceae) in having axial secretory canals arranged in a single ring in the innermost part of the cortical parenchyma and radial dilatation of secondary phloem. This genus is distinctive, however, from the latter by the common presence of column-shaped to bottle-shaped epidermal cells with very thick cuticle. Helical thickenings on vessel walls have not been found in Andriana; thus this feature can be considered a synapomorphic trait only for the Heteromorpha clade rather than for the tribe Heteromorpheae, as it was previously suggested. Surprisingly, secretory canals in the secondary phloem of Andriana species lack sheaths of specialized axial parenchyma. The absence of these sheaths is thought to be unique within the suborder Apiineae, which comprises the families Araliaceae, Myodocarpaceae, Pittosporaceae, and Apiaceae. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The genus Andriana B.-E. Van Wyk comprises of three shrubby species restricted to the high elevation scrubs in the mountains of northern Madagascar (Van Wyk et al., 2013), i.e. to the Madagascar ericoid thickets sensu Burgess et al. (2004). Along with seven other genera, this genus is placed in the Malagasy clade of the tribe Heteromorpheae M.F. Watson & S.R. Downie, belonging to the family Apiaceae (Downie et al., 2010). The species of Andriana were originally treated by Humbert (1955, 1956) in the genus Heteromorpha, but were subsequently recircumscribed into a new genus based on their more regular leaf shape, serrate leaflet margins, perfectly isodiametric mericarps, and invariable presence of intrajugal oil ducts within the fruit ribs as well as vallecular oil ducts between the ribs (Van Wyk et al., 1999). The genus is named after the Madagascar king Andriantsimitoviaminadriandehibe, where the Malagasy word “andrian” means “noble” (Van Wyk et al., 1999). The tribe Heteromorpheae was described (Downie et al., 2001) to accommodate a group of five African genera of trees, shrubs, suffrutices and perennial herbs (Anginon Raf., Dracosciadium Hilliard, Glia Sond. Heteromorpha Cham. & Schltdl. and Polemannia Eckl. & Zeyh.) that formed a distinct, basally divergent clade in several molecular systematic ⁎ Corresponding author at: Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland Park, 2006 Johannesburg, South Africa. E-mail address: [email protected] (A. Oskolski).
https://doi.org/10.1016/j.sajb.2018.02.003 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
studies of the subfamily Apioideae (Plunkett et al., 1996; Plunkett and Downie, 1999; Downie and Katz-Downie, 1999; Downie et al., 2001). A molecular systematic study by Calviño et al. (2006) suggested that the limits of the tribe should be expanded to include Pseudocarum C.Norman with one species in Africa and one in Madagascar, Oreofraga M.F.Watson & E.L.Barclay with a single species in Socotra, and four genera endemic to Madagascar (i.e. Andriana B.-E.Van Wyk, Anisopoda Baker, Cannaboides B.-E.Van Wyk, Pseudocannaboides B.-E.Van Wyk and Tana B.-E.Van Wyk). The seven latter genera, along with Dracosciadium, were recovered as a clade (the Malagasy clade, Downie et al., 2010) sister to the Heteromorpha clade comprising Anginon, Glia, Heteromorpha and Polemannia. Although the Malagasy clade encompasses two genera from outside Madagascar, in the present paper we will use its name proposed by Downie et al. (2010). Several morphological characters have been uncovered to support the inclusion of the Madagascan genera into the tribe Heteromorpheae. As recent study of fruit structural variation in this tribe (Liu et al., 2016) showed, the fruit epidermis made of bottle shaped cells with very thick cuticle to be a synapomorphic trait for the tribe. Anatomical studies of wood and bark in South African members of Heteromorpheae (Oskolski and Van Wyk, 2008; Kotina et al., 2012) also suggested that the combination of a narrow cortex with a single ring of secretory canals, secretory canals in the secondary phloem and radial dilatation of the secondary phloem, and the presence of helical thickenings on vessel walls is diagnostic for the tribe. The latter feature was also considered as an ancestral character state and a symplesiomorphy for the tribes
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Bupleurieae and Heteromorpheae (Oskolski and Van Wyk, 2008). These suggestions, however, are only provisional, because the stem structure of only one genus (Dracosciadium) belonging to the Malagasy clade of Heteromorpheae has been examined to date. In this study, we aim to describe the anatomical structure of the wood and bark structure in Andriana as a member of the Malagasy clade. On this basis, we seek to interpret the phylogenetic relationships within the tribe Heteromorpheae, to analyse the evolutionary pathways for wood and bark characteristics in this group, and to clarify ecological implications of the stem anatomical traits in Andriana. 2. Material and methods Wood samples of Andriana tsaratananensis (Humbert) B.-E. Van Wyk were obtained from P.P. Lowry II, who collected it on 16.10.2001 on Tsaratanana massif (elevation 2490 m) in northwestern Madagascar. Wood and bark samples of A. marojejyensis (Humbert) B.-E. Van Wyk were collected by the second author with P. Karpunina and M. Nuraliev on 17.10.2015 on the summit of mount Marojejy (elevation 2130 m) in the Marojejy Natural Park, northeastern Madagascar. Herbarium vouchers (P. P. Lowry II # 5363 and P. Karpunina # 244) are deposited in P and TAN. The samples for wood anatomical study were taken from the thickest portions of stems of both species with a secondary xylem radius of more than 5 mm, so that the wood structure may be considered mature. Transverse, radial and tangential sections were made on rotary microtomes (Ernst Leitz GMBH, Wetzlar, Germany and Jung AG Heidelberg, Germany) and stained with a 1:1 alcian blue/safranin mixture. Macerations were made using Jeffrey's solution (Johansen, 1940). Descriptive terminology for wood structure follows recommendations of the IAWA Committee (1989).
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The bark structure was examined only for A. marojejyensis. Fresh bark samples of this species were cut from branch tips without a visible periderm layer and from other stem parts on which the bark was mature, having more or less a thick periderm. These bark fragments were fixed in 70% ethanol, and then they were embedded in glycol methacrylate (GMA) according to a modification of the Feder and O’Brien (1968) method. Transverse, tangential and radial sections of about 5 μm thick were cut by using a Porter Blum MT-1 ultramicrotome. Bark sections were then stained with Schiff - toluidine blue method before being mounted in Entellan. Measurements of sieve tube members were made in fragments of secondary phloem macerated using Jeffrey's solution (Johansen, 1940). Descriptive bark anatomical terminology follows Angyalossy et al. (2016). 3. Results 3.1. Wood structure (Fig. 1) Samples examined: Andriana marojejyensis: P. Karpunina 244; A. tsaratanonensis: P.P. Lowry II 5363. Growth ring boundaries are absent in A. tsaratanonensis (Fig. 1A), and absent to indistinct in A. marojejyensis, marked by zones of somewhat radially flattened fibres (Fig. 1B). Vessels are angular, rarely rounded in outline, very narrow (tangential diameter b28 μm in A. tsaratanonensis and b 54 μm in A. marojejyensis), few in A. marojejyensis (up to 40 per mm2) and more numerous in A. tsaratanonensis (up to 73 per mm2), mostly in clusters and radial to diagonal multiples groupings up to 10 vessels. Vessel walls of 2.3–8.9 μm thick. Tyloses were not found. Vessel elements (170–) 320–350 (−580) μm long (Table 1). Perforation plates are simple (Fig. 1C, E). Intervessel pits are mostly scalariform, sometimes transitional to alternate, 4.1–8.8 μm in vertical size,
Fig. 1. Wood structure of Andriana. (A) A. tsaratanonensis, transverse section showing the absence of growth rings, vessels in radial multiplies; (B) A. marojejyensis, transverse section showing indistinct growth ring boundaries (white arrowheads), vessels in diagonal groupings; (C) A. marojejyensis, SEM micrograph of vessel element showing simple perforation plate, scalariform to alternate intervessel pitting, and the absence of helical thickenings on vessel wall; (D) A. marojejyensis, tangential section showing 1–4 seriate rays composed mostly of square and upright cells; (E) A. marojejyensis, radial section showing a ray composed mostly of upright cells with few square and procumbent cells, vessel elements with simple perforation plates. Scale bars: A, B, D 200 μm; E 100 μm; C 20 μm.
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Table 1 Wood and bark anatomical characters of Andriana.
Andriana tsaratananensis [P.P. Lowry 5363] Andriana marojejyensis [P. Karpunina 244]
1
2
3
4
5
6
7
8
9
10
11
12
13
10
327 ± 9.7/ 176–510 348 ± 13.8/ 25–578
6.8 ± 0.13/ 4.1–8.8 6.0 ± 0.16/ 4.9–7.8
59.4/45–73
16.8 ± 0.68/ 8–27 32.3 ± 1.14/ 13–54
3.4/10
9
–
0.83/2.3
3.9/3.3
7.2
–
2.3/10
20
485 ± 8.5/ 381–692 494 ± 10.3/ 362–670
2.6 / 4
0,51/1.3
1.6/1.4
3.1
392 ± 12.5/ 277–606
10
29.4/24–39
1: Radius of wood sample (mm) — 2: Length of vessel elements (mean ± standard error/min-max, μm) — 3: Vertical size of intervessel pits (mean ± standard error/min-max, μm) — 4: Vessel frequency (mean/min-max, per sq. mm) — 5: Tangential diameter of vessels (mean ± standard error/min-max, μm) — 6: Mean/the greatest number of vessels in a vessel group — 7: Percentage of solitary vessels −8: Mean length of libriform fibres (mean ± standard error/min-max, μm) — 9: Width of multiseriate rays (mean/max, cells) — 10: Height of multiseriate rays (mean/max, mm) — 11: Average number of multiseriate/uniseriate rays per mm — 12 Total number of rays per mm — 13: Length of sieve tube members (mean ± standard error/minmax, μm).
mostly with rounded margins and slit-like apertures (Fig. 1C). Vessel-ray and vessel-axial parenchyma pits are similar to intervessel pits in size and shape, mostly with distinct borders and wide lens-like apertures, sometimes with reduced borders. Helical thickenings were not found (Fig. 1C). Vascular tracheids were not found. Fibres libriform, thin- to thick-walled, sometimes very thin-walled, (360–) 480–490 (− 690) μm long (Table 1), fibre walls 2.3–6.2 μm thick in A. tsaratananensis and 2.5–8.9 μm thick in A. marojejgensis, with few simple to minutely bordered pits, with slit-like apertures in radial walls. Septate fibres rarely occur in A. tsaratananensis. Axial parenchyma is scanty paratracheal, mostly in solitary strands (sometimes in uniseriate incomplete sheaths) near the vessels, consist of strands of 2–5 cells A. marojejyensis and of 2–4 cells in A. tsaratananensis. Rays 4–12 per mm, uni- and multiseriate of 2–4 cells in width (Fig. 1D). Ray height commonly b 1 mm, but few rays N 1 mm occur in both species. Multiseriate rays composed mostly of upright and square cells with some procumbent cells mixed throughout the ray (Fig. 1D, E). Uniseriate rays composed mostly of square and upright cells but procumbent cell occur very rarely. Radial canals absent. Crystals not found.
Sieve tube members are 30–37 μm wide, their mean length varies from 277 to 606 μm (Table 1). Sieve plates are compound with 2–6 sieve areas, located on vertical or slightly oblique cross walls (Fig. 2D). Axial parenchyma cells associated with conducting elements occur as single fusiform cells and in strands of 2–4 cells. Axial secretory canals are scattered throughout the secondary phloem. They are lined by a single layer of 6–10 epithelial cells. No parenchyma sheaths near the secretory canals were found (Fig. 2B). The transition from conducting to nonconducting secondary phloem is gradual. Axial parenchyma cells in nonconducting secondary phloem occurs as thin-walled strands (Fig. 2B) and as chambered crystalliferous cells containing druses. Secondary phloem rays are uniseriate and 2–3-seriate (Fig. 2E). Uniseriate rays are composed of mostly square and upright cells (Fig. 2C) while 2- and 3- seriate rays have procumbent cells or also upright and square cells (mostly in uniseriate portions). Dilated rays are extensively enlarged, mostly by tangential expansion and also by anticlinal divisions of ray cells resulting in rays of up to 7 cells wide (Fig. 2F). Many of the ray parenchyma cells contain starch grains. Druses occur in ray cells. No radial secretory canals were found.
3.2. Bark structure (Fig. 2)
The genus Andriana shows exclusively simple perforation plates, rather short vessel elements, scanty paratracheal axial parenchyma, the presence of secretory canals in the cortex and secondary phloem, and the absence of the secondary phloem fibers. This set of anatomical characters is typical for the large majority of woody Apiaceae (Metcalfe and Chalk, 1950; Rodriguez, 1957; Oskolski, 2001; Kotina and Oskolski, 2010) examined to date. Only Apiopetalum Baill., Mackinlaya F. Muell. and Centella L., the members of the subfamily Mackinalyoideae, share the occurrence of scalariform perforation plates. Besides, Centella is unique within Apiaceae by the lack of secretory canals in the secondary phloem (Oskolski and Lowry, 2000; Oskolski and Van Wyk, 2010). Despite these similarities, Andriana is very distinctive from other Apiaceae as well as from other members of the suborder Apiineae (i.e. Araliaceae, Myodocarpaceae and Pittosporaceae) by its uniform axial parenchyma in secondary phloem. Unlike Andriana, in other Apiineae the secretory canals in secondary phloem have prominent sheaths of the axial parenchyma, that clearly differs from the axial parenchyma associated with conductive elements by more numerous cells per strand and by common occurrence of crystals and other deposits (Kolalite et al., 2003; Kotina and Oskolski, 2010; Nilova and Oskolski, 2010; Oskolski et al., 2010). In Andriana, however, the sheath axial parenchyma cannot be distinguished from other strands of axial parenchyma occurring in secondary phloem. This feature cannot be considered, however, as an apomorphy for the Malagasy Heteromorpheae because parenchymatous sheaths near secretory canals have been reported in Dracosciadium, also belonging to this clade (Kotina et al., 2012). Andriana is similar to the southern African genera of Heteromorpheae (i.e. Anginon, Dracosciadium, Glia, Heteromorpha and Polemannia) in having axial secretory canals arranged in a single ring in the innermost part of the cortical parenchyma, and radial dilatation of secondary phloem
Samples examined: Andriana marojejyensis: P. Karpunina 244. The epidermis on young parts of stems is composed of a single layer of column-like to bottle-like cells with thin inner walls and thick outer walls covered by prominent cuticle (Fig. 2A). There were no trichomes found. The cortex is narrow (up to 18 cells in width), composed of collenchyma and parenchyma (Fig. 2A). Cortical collenchyma is angularlamellar. Collenchyma cells are 20–50 μm in tangential diameter. There were no crystals found in the cells of the cortical collenchyma. Cortical parenchyma is formed from four to six layers of isodiametric to somewhat vertically elongated, flattened, thin-walled cell of 25–50 μm in tangential diameter occasionally. Druse were observed in some of the cortical parenchyma cells. A single ring of schizogeneous axial secretory canals is present in the cortical parenchyma. These canals are lined with a single of seven to twelve epithelial cells. The lumina of the canals are commonly 60–80 μm in tangential diameter. Dilation of the cortical tissue is affected mostly by tangential stretching of cells and also by anticlinal divisions of the cortical collenchyma and parenchyma cell, thus forming strands of two to four cells. Primary phloem fibers are absent. No chloroplast were observed in cells of cortical collenchyma and parenchyma. Mature bark non-peeling, brittle, with shallow fissured surface. The initiation of first-formed periderm is in the subepidermal layer of cells (Fig. 2A). The phellem (Fig. 2B) is composed of 8–12 layers of isodiametric to somewhat radially flattened cells with thin cell wall (0.3–0.9 μm thick). The phelloderm comprises 4 to 7 layers of isodiametric, thinwalled cells. No crystalliferous cells found in periderm. Secretory canals are absent in the phelloderm. Subsequent periderm are initiated in the outer region of the cortical parenchyma as concentric rings.
4. Discussion
C. Long, A. Oskolski / South African Journal of Botany 115 (2018) 138–142
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Fig. 2. Bark structure of Andriana marojejyensis. (A) transverse section of young bark showing column-like to bottle-like epidermal cells (e), subepidermal initiation of phellogen (ph), cortical collenchyma (cc) and cortical parenchyma (cp) with secretory canals (sc), secondary phloem (sp); (B) transverse section of mature bark showing periderm (p), dilatated cortex (dc) with tangential strands of parenchyma cells (s), secondary phloem (sp) with secretory canals (sc) lacking axial parenchyma sheaths; (C) radial section showing secondary phloem rays composed mostly of upright cells; (D) radial section showing sieve tube members with compound sieve plates (spl) on vertical end walls; (E) tangential section of conductive secondary phloem showing 2–3-seriate phloem rays; (F) tangential section of non-conductive secondary phloem showing dilatated 2–8-seriate phloem rays. Scale bars: A, B, C 100 μm; D 50 μm; E, F 200 μm.
(Kotina et al., 2012). Our results support that this combination of traits is diagnostic for Heteromorpheae, as it has been suggested by Kotina et al. (2012). Two Andriana species are distinctive, however, from these genera (with the exception of Dracosciadium whose wood structure remains unknown) by the absence of helical thickenings on vessel walls (Oskolski and Van Wyk, 2008). Thus this feature can be considered as a synapomorphic trait only for the Heteromorpha clade (Calviño et al., 2006; Downie et al., 2010) comprising of Anginon, Glia, Heteromorpha and Polemannia, rather than for the entire Heteromorpheae, as it was previously suggested (Oskolski and Van Wyk, 2008). Andriana differs from the southern African Heteromorpheae by very narrow cortical parenchyma (four to six layers versus six to twenty layers in other genera of this tribe) and by the column- to bottleshaped epidermal cells covered with very thick cuticle. The latter feature occurs also in the mericarps of Anginon and Pseudocannaboides (Liu et al., 2016), but it has not been reported in stem epidermis of any other Heteromorpheae (Kotina et al., 2012). Apparently, these
bark traits can be diagnostic for the genus, but their phylogenetic value is uncertain until the stem structure in other Malagasy genera of Heteromorpheae remains unknown. Unlike the southern African Heteromorpheae, the wood of Andriana almost completely lacks growth rings. Obviously, the cambial activity in these shrubs can persist during the seasonal fluctuations in temperature that have been reported in the mountain scrubs (ericoid thickets) of Madagascar (Burgess et al., 2004). Andriana also resembles Anginon, Glia and Polemannia in having very narrow vessels (less than 54 μm in diameter); it shows, however, lower vessel frequency than the southern African genera (Fig. 3). Narrow vessels is a typical trait for shrubby plants from high elevation habitats (e.g. Outer van der et al., 1981; Noshiro et al., 2010) that can provide hydraulic safety preventing air embolism during temporary arid conditions occurring in the ericoid thickets caused by local fluctuations of temperature, sunlight and precipitations (Burgess et al., 2004). In Andriana, however, the decrease of vessel diameters is not compensated in Andriana by the increase of
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Average vessel diameter, µm
60 50
Andriana
40
Anginon Glia
30
Heteromorpha Polemannia
20 10 0 0
100
200
300
400
500
600
Vessel frequency, per sq.mm Fig. 3. Average tangential diameter of vessels in southern African genera of Heteromorpheae (data taken from Oskolski and Van Wyk (2008)) and in two Andriana species plotted against the average vessel frequency.
vessel frequency, as it commonly occurs (Outer van der et al., 1981). Apparently, few narrow vessels can supply the Andriana shrubs effectively due to great water availability and low transpiration loss that are found in tropical mountains. 5. Conclusions Andriana is similar to other genera of the tribe Heteromorpheae by the axial secretory canals arranged in a single ring in the innermost part of the cortical parenchyma, and the radial dilatation of secondary phloem. This combination of bark traits is diagnostic for the tribe. Andriana is distinctive, however, from other Heteromorpheae by the presence of column- to bottle-shaped epidermal cells covered with very thick cuticle. The absence of sheath axial parenchyma in secondary phloem of Andriana is a unique feature within the suborder Apiineae. As Andriana lacks the helical thickenings on the vessel walls, the presence of these structures may not be considered as a synapomorphic trait for the Heteromorpheae, as it was previously suggested. Acknowledgements We thank the National Research Foundation (incentive grant 109531), the University of Johannesburg and the Russian Foundation of Basic Research (grant 16-04-00725а) for financial support. The second author was also supported by the institutional research project (no. 01201456545) of the Komarov Botanical Institute. The staff of Missouri Botanical Garden's Madagascar Research and Conservation Program, specifically F. Lantoarisoa and P.P. Lowry II are gratefully acknowledged for organization of the fieldwork in Marojejy National Park. We are very indebted to P.D. Karpunina, M.S. Nuraliev, D. Ravelonarivo, V. Razafindrahaja and J.H. Tonkaina for field assistance, and to P.P. Lowry II for giving us the wood sample of A. tsaratanonensis. References Angyalossy, V., Pace, M.R., Evert, R.F., Marcati, C.R., Oskolski, A.A., Terrazas, T., Kotina, E., Lens, F., Mazzoni-Viveiros, S.C., Angeles, G., Machado, S., Crivellaro, A., Rao, K.S., Junikka, L., Nikolaeva, N., Baas, P., 2016. IAWA list of microscopic bark features. IAWA Journal 37, 517–615. Burgess, N., Hales, J.D.A., Underwood, E., Dinerstein, E., Olson, D., Itoua, I., Schipper, J., Ricketts, T., Newman, T., 2004. Terrestrial Ecoregions of Africa and Madagascar: A Conservation Assessment. Island Press, Washington. Calviño, C.I., Tilney, P.M., Van Wyk, B.-E., Downie, S.R., 2006. A molecular phylogenetic study of southern African Apiaceae. American Journal of Botany 93, 1828–1847. Downie, S.R., Katz-Downie, D.S., 1999. Phylogenetic analysis of chloroplast rps16 intron sequences reveals relationships within the woody southern African Apiaceae subfamily Apioideae. Canadian Journal of Botany 77, 1120–1135.
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